Color vision evolution in egg-laying mammals: insights from visual photoreceptors and daily activities of Australian echidnas.

Egg-laying mammals (monotremes) are considered "primitive" due to traits such as oviparity, cloaca, and incomplete homeothermy, all of which they share with reptiles. Two groups of monotremes, the terrestrial echidna (Tachyglossidae) and semiaquatic platypus (Ornithorhynchidae), have evolved highly divergent characters since their emergence in the Cenozoic era. These evolutionary differences, notably including distinct electrosensory and chemosensory systems, result from adaptations to species-specific habitat conditions. To date, very few studies have examined the visual adaptation of echidna and platypus. In the present study, we show that echidna and platypus have different light absorption spectra in their dichromatic visual sensory systems at the molecular level. We analyzed absorption spectra of monotreme color opsins, long-wavelength sensitive opsin (LWS) and short-wavelength sensitive opsin 2 (SWS2). The wavelength of maximum absorbance (λmax) in LWS was 570.2 in short-beaked echidna (Tachyglossus aculeatus) and 560.6 nm in platypus (Ornithorhynchus anatinus); in SWS2, λmax was 451.7 and 442.6 nm, respectively. Thus, the spectral range in echidna color vision is ~ 10 nm longer overall than in platypus. Natural selection analysis showed that the molecular evolution of monotreme color opsins is generally functionally conserved, suggesting that these taxa rely on species-specific color vision. In order to understand the usage of color vision in monotremes, we made 24-h behavioral observations of captive echidnas at warm temperatures and analyzed the resultant ethograms. Echidnas showed cathemeral activity and various behavioral repertoires such as feeding, traveling, digging, and self-grooming without light/dark environment selectivity. Halting (careful) behavior is more frequent in dark conditions, which suggests that echidnas may be more dependent on vision during the day and olfaction at night. Color vision functions have contributed to dynamic adaptations and dramatic ecological changes during the ~ 60 million years of divergent monotreme evolution. The ethogram of various day and night behaviors in captive echidnas also contributes information relevant to habitat conservation and animal welfare in this iconic species, which is locally endangered.

Enhanced mitotic arrest and chromosome resolution for cytogenetic analysis in the eastern mosquitofish, Gambusia holbrooki.

Maximising the number of cells arrested at metaphase and their resolution is fundamentally important for molecular cytogenetic investigations, particularly in fish, which typically yield low mitotic index and have highly condensed chromosomes. To overcome these limitations, fish were injected with a mitotic stimulator (the yeast, Saccharomyces cerevisiae) to improve the mitotic index, and the intercalating agent ethidium bromide to produce elongated chromosomes. Specifically, adults were injected with activated yeast and then Colcemid (0.025 µg/µl solution, 10 µl per 1 g of body weight) at 24-96 h post yeast injections, followed by chromosome preparations from multiple tissues. Results showed that gill tissue had the highest number of dividing cells at 72 h post yeast exposure with no significant (p > 0.05) differences between the sexes. Nonetheless, sex-specific differences in the mitotic index were observed in spleen, kidney, and liver, which may be attributed to sex-specific differences in immune responses. For elongation of mitotic chromosomes, individuals (both sexes) were first injected with activated yeast and after 48 h with ethidium bromide (2 or 4 µg/ml) and Colcemid (0.05 µg/µl solution, 10 µl per 1 g of body weight). Following which, animals were sampled at three time points (1, 4 and 8 h) for chromosome preparations. The results show that the optimum elongation of metaphase chromosomes of males and females was achieved by using 2 µg/ml and 4 µg/ml, respectively, for 1 h. Interestingly, the average mitotic chromosome length (µm) of males and females post-ethidium bromide exposure was significantly different (p < 0.05) for both concentrations, except at 1 h exposure for 2 µg/ml EtBr. Such differences can be attributed to overall chromosomal condensation differences between sexes. Regardless, the increased mitotic index and chromosome resolution could benefit cytogenetic studies in other fish species.

The molecular evolution of spermatogenesis across mammals.

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1-6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

Characterising the Gut Microbiomes in Wild and Captive Short-Beaked Echidnas Reveals Diet-Associated Changes.

The gut microbiome plays a vital role in health and wellbeing of animals, and an increasing number of studies are investigating microbiome changes in wild and managed populations to improve conservation and welfare. The short-beaked echidna (Tachyglossus aculeatus) is an iconic Australian species, the most widespread native mammal, and commonly held in zoos. Echidnas are cryptic animals, and much is still unknown about many aspects of their biology. Furthermore, some wild echidna populations are under threat, while echidnas held in captivity can have severe gastric health problems. Here, we used citizen science and zoos to collect echidna scats from across Australia to perform the largest gut microbiome study on any native Australian animal. Using 16S rRNA gene metabarcoding of scat samples, we characterised and compared the gut microbiomes of echidnas in wild (n = 159) and managed (n = 44) populations, which were fed four different diets. Wild echidna samples were highly variable, yet commonly dominated by soil and plant-fermenting bacteria, while echidnas in captivity were dominated by gut commensals and plant-fermenting bacteria, suggesting plant matter may play a significant role in echidna diet. This work demonstrates significant differences between zoo held and wild echidnas, as well as managed animals on different diets, revealing that diet is important in shaping the gut microbiomes in echidnas. This first analysis of echidna gut microbiome highlights extensive microbial diversity in wild echidnas and changes in microbiome composition in managed populations. This is a first step towards using microbiome analysis to better understand diet, gastrointestinal biology, and improve management in these iconic animals.

Functional Diversity and Evolution of Bitter Taste Receptors in Egg-Laying Mammals.

Egg-laying mammals (monotremes) are a sister clade of therians (placental mammals and marsupials) and a key clade to understand mammalian evolution. They are classified into platypus and echidna, which exhibit distinct ecological features such as habitats and diet. Chemosensory genes, which encode sensory receptors for taste and smell, are believed to adapt to the individual habitats and diet of each mammal. In this study, we focused on the molecular evolution of bitter taste receptors (TAS2Rs) in monotremes. The sense of bitter taste is important to detect potentially harmful substances. We comprehensively surveyed agonists of all TAS2Rs in platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus) and compared their functions with orthologous TAS2Rs of marsupial and placental mammals (i.e., therians). As results, the agonist screening revealed that the deorphanized monotreme receptors were functionally diversified. Platypus TAS2Rs had broader receptive ranges of agonists than those of echidna TAS2Rs. While platypus consumes a variety of aquatic invertebrates, echidna mainly consumes subterranean social insects (ants and termites) as well as other invertebrates. This result indicates that receptive ranges of TAS2Rs could be associated with feeding habits in monotremes. Furthermore, some orthologous receptors in monotremes and therians responded to ß-glucosides, which are feeding deterrents in plants and insects. These results suggest that the ability to detect ß-glucosides and other substances might be shared and ancestral among mammals.

COVID restrictions impact wildlife monitoring in Australia.

The global COVID-19 pandemic has imposed restrictions on people's movement, work and access to places at multiple international, national and sub-national scales. We need a better understanding of how the varied restrictions have impacted wildlife monitoring as gaps in data continuity caused by these disruptions may limit future data use and analysis. To assess the effect of different levels of COVID-19 restrictions on both citizen science and traditional wildlife monitoring, we analyse observational records of a widespread and iconic monotreme, the Australian short-beaked echidna (Tachyglossus aculeatus), in three states of Australia. We compare citizen science to observations from biodiversity data repositories across the three states by analysing numbers of observations, coverage in protected areas, and geographic distribution using an index of remoteness and accessibility. We analyse the effect of restriction levels by comparing these data from each restriction level in 2020 with corresponding periods in 2018-2019. Our results indicate that stricter and longer restrictions reduced numbers of scientific observations while citizen science showed few effects, though there is much variation due to differences in restriction levels in each state. Geographic distribution and coverage of protected and non-protected areas were also reduced for scientific monitoring while citizen science observations were little affected. This study shows that citizen science can continue to record accurate and widely distributed species observational data, despite pandemic restrictions, and thus demonstrates the potential value of citizen science to other researchers who require reliable data during periods of disruption.

EchidnaCSI: Engaging the public in research and conservation of the short-beaked echidna.

The short-beaked echidna is an iconic Australian animal and the most-widespread native mammal, inhabiting diverse environments. The cryptic nature of echidnas has limited research into their ecology in most areas; however, from the well-researched and endangered Kangaroo Island echidna population, we understand that the threats include habitat loss, roads, and invasive species. To obtain more information about echidnas Australia-wide, we established the Echidna Conservation Science Initiative (EchidnaCSI) citizen science project. EchidnaCSI calls on members of the public to submit photographs of wild echidnas and learn to identify and collect echidna scats for molecular analysis. To facilitate participation, we developed a smartphone application as well as ongoing social and traditional media activities and community events. In 3 y, more than 9,000 members of the public have downloaded the EchidnaCSI app, collecting 400 scats and submitting over 8,000 sightings of echidnas from across Australia. A subset of submitted scat samples were subjected to DNA extraction and PCR, which validated the approach of using citizen science for scat collection and viability for molecular analysis. To assess the impact of the project through public participation, we surveyed our participants (n = 944) to understand their demographics and motivations for engagement. Survey results also revealed that EchidnaCSI served as a gateway into citizen science more generally for many participants. EchidnaCSI demonstrates the potential for using citizen science approaches to collect high-quality data and material from a cryptic species over a very large geographic area and the considerable engagement value of citizen science research.

Microchromosomes are building blocks of bird, reptile, and mammal chromosomes.

Microchromosomes, once considered unimportant shreds of the chicken genome, are gene-rich elements with a high GC content and few transposable elements. Their origin has been debated for decades. We used cytological and whole-genome sequence comparisons, and chromosome conformation capture, to trace their origin and fate in genomes of reptiles, birds, and mammals. We find that microchromosomes as well as macrochromosomes are highly conserved across birds and share synteny with single small chromosomes of the chordate amphioxus, attesting to their origin as elements of an ancient animal genome. Turtles and squamates (snakes and lizards) share different subsets of ancestral microchromosomes, having independently lost microchromosomes by fusion with other microchromosomes or macrochromosomes. Patterns of fusions were quite different in different lineages. Cytological observations show that microchromosomes in all lineages are spatially separated into a central compartment at interphase and during mitosis and meiosis. This reflects higher interaction between microchromosomes than with macrochromosomes, as observed by chromosome conformation capture, and suggests some functional coherence. In highly rearranged genomes fused microchromosomes retain most ancestral characteristics, but these may erode over evolutionary time; surprisingly, de novo microchromosomes have rapidly adopted high interaction. Some chromosomes of early-branching monotreme mammals align to several bird microchromosomes, suggesting multiple microchromosome fusions in a mammalian ancestor. Subsequently, multiple rearrangements fueled the extraordinary karyotypic diversity of therian mammals. Thus, microchromosomes, far from being aberrant genetic elements, represent fundamental building blocks of amniote chromosomes, and it is mammals, rather than reptiles and birds, that are atypical.

The Evolution of Viviparity in Vertebrates.

In the vertebrate tree of life, viviparity or live birth has independently evolved many times, resulting in a rich diversity of reproductive strategies. Viviparity is believed to be a mode of reproduction that evolved from the ancestral condition of oviparity or egg laying, where most of the fetal development occurs outside the body. Today, there is not a simple model of parity transition to explain this species-specific divergence in modes of reproduction. Most evidence points to a gradual series of evolutionary adaptations that account for this phenomenon of reproduction, elegantly displayed by various viviparous squamates that exhibit placentae formed by the appositions of maternal and embryonic tissues, which share significant homology with the tissues that form the placenta in therian mammals. In an era where the genomes of many vertebrate species are becoming available, studies are now exploring the molecular basis of this transition from oviparity to viviparity, and in some rare instances its possible reversibility, such as the Australian three-toed skink (Saiphos equalis). In contrast to the parity diversity in squamates, mammals are viviparous with the notable exception of the egg-laying monotremes. Advancing computational tools coupled with increasing genome availability across species that utilize different reproductive strategies promise to reveal the molecular underpinnings of the ancestral transition of oviparity to viviparity. As a result, the dramatic changes in reproductive physiology and anatomy that accompany these parity changes can be reinterpreted. This chapter will briefly explore the vertebrate modes of reproduction using a phylogenetic framework and where possible highlight the role of potential candidate genes that may help explain the polygenic origins of live birth.

Evolution, Expression and Meiotic Behavior of Genes Involved in Chromosome Segregation of Monotremes.

Chromosome segregation at mitosis and meiosis is a highly dynamic and tightly regulated process that involves a large number of components. Due to the fundamental nature of chromosome segregation, many genes involved in this process are evolutionarily highly conserved, but duplications and functional diversification has occurred in various lineages. In order to better understand the evolution of genes involved in chromosome segregation in mammals, we analyzed some of the key components in the basal mammalian lineage of egg-laying mammals. The chromosome passenger complex is a multiprotein complex central to chromosome segregation during both mitosis and meiosis. It consists of survivin, borealin, inner centromere protein, and Aurora kinase B or C. We confirm the absence of Aurora kinase C in marsupials and show its absence in both platypus and echidna, which supports the current model of the evolution of Aurora kinases. High expression of AURKBC, an ancestor of AURKB and AURKC present in monotremes, suggests that this gene is performing all necessary meiotic functions in monotremes. Other genes of the chromosome passenger complex complex are present and conserved in monotremes, suggesting that their function has been preserved in mammals. Cohesins are another family of genes that are of vital importance for chromosome cohesion and segregation at mitosis and meiosis. Previous work has demonstrated an accumulation and differential loading of structural maintenance of chromosomes 3 (SMC3) on the platypus sex chromosome complex at meiotic prophase I. We investigated if a similar accumulation occurs in the echidna during meiosis I. In contrast to platypus, SMC3 was only found on the synaptonemal complex in echidna. This indicates that the specific distribution of SMC3 on the sex chromosome complex may have evolved specifically in platypus.

Flavors of Non-Random Meiotic Segregation of Autosomes and Sex Chromosomes.

Segregation of chromosomes is a multistep process occurring both at mitosis and meiosis to ensure that daughter cells receive a complete set of genetic information. Critical components in the chromosome segregation include centromeres, kinetochores, components of sister chromatid and homologous chromosomes cohesion, microtubule organizing centres, and spindles. Based on the cytological work in the grasshopper Brachystola, it has been accepted for decades that segregation of homologs at meiosis is fundamentally random. This ensures that alleles on chromosomes have equal chance to be transmitted to progeny. At the same time mechanisms of meiotic drive and an increasing number of other examples of non-random segregation of autosomes and sex chromosomes provide insights into the underlying mechanisms of chromosome segregation but also question the textbook dogma of random chromosome segregation. Recent advances provide a better understanding of meiotic drive as a prominent force where cellular and chromosomal changes allow autosomes to bias their segregation. Less understood are mechanisms explaining observations that autosomal heteromorphism may cause biased segregation and regulate alternating segregation of multiple sex chromosome systems or translocation heterozygotes as an extreme case of non-random segregation. We speculate that molecular and cytological mechanisms of non-random segregation might be common in these cases and that there might be a continuous transition between random and non-random segregation which may play a role in the evolution of sexually antagonistic genes and sex chromosome evolution.

Towards complete and error-free genome assemblies of all vertebrate species.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

Restriction of an intron size en route to endothermy.

Ca2+-insensitive and -sensitive E1 subunits of the 2-oxoglutarate dehydrogenase complex (OGDHC) regulate tissue-specific NADH and ATP supply by mutually exclusive OGDH exons 4a and 4b. Here we show that their splicing is enforced by distant lariat branch points (dBPs) located near the 5' splice site of the intervening intron. dBPs restrict the intron length and prevent transposon insertions, which can introduce or eliminate dBP competitors. The size restriction was imposed by a single dominant dBP in anamniotes that expanded into a conserved constellation of four dBP adenines in amniotes. The amniote clusters exhibit taxon-specific usage of individual dBPs, reflecting accessibility of their extended motifs within a stable RNA hairpin rather than U2 snRNA:dBP base-pairing. The dBP expansion took place in early terrestrial species and was followed by a uridine enrichment of large downstream polypyrimidine tracts in mammals. The dBP-protected megatracts permit reciprocal regulation of exon 4a and 4b by uridine-binding proteins, including TIA-1/TIAR and PUF60, which promote U1 and U2 snRNP recruitment to the 5' splice site and BP, respectively, but do not significantly alter the relative dBP usage. We further show that codons for residues critically contributing to protein binding sites for Ca2+ and other divalent metals confer the exon inclusion order that mirrors the Irving-Williams affinity series, linking the evolution of auxiliary splicing motifs in exons to metallome constraints. Finally, we hypothesize that the dBP-driven selection for Ca2+-dependent ATP provision by E1 facilitated evolution of endothermy by optimizing the aerobic scope in target tissues.

Platypus and echidna genomes reveal mammalian biology and evolution.

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.

The emergence of the brain non-CpG methylation system in vertebrates.

Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett syndrome. However, it is unclear whether the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distantly related animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. We also found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole-genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests that the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage.

Reduced Gonadotrophin Receptor Expression Is Associated with a More Aggressive Ovarian Cancer Phenotype.

Follicle-stimulating hormone (FSH) and luteinising hormone (LH) play important roles in regulating cell growth and proliferation in the ovary. However, few studies have explored the expression of FSH and LH receptors (FSHR and LHCGR) in ovarian cancer, and their functional roles in cancer progression remain inconclusive. This study investigated the potential impact of both mRNA (FSHR, LHCGR) and protein (FSHR, LHCGR) expression on ovarian cancer progression using publicly available online databases, qRT-PCR (high grade serous ovarian cancers, HGSOC, n = 29 and benign ovarian tumors, n = 17) and immunohistochemistry (HGSOC, n = 144). In addition, we investigated the effect of FSHR and LHCGR siRNA knockdown on the pro-metastatic behavior of serous ovarian cancer cells in vitro. High FSHR or high LHCGR expression in patients with all subtypes of high-grade ovarian cancer was significantly associated with longer progression-free survival (PFS) and overall survival (OS). High FSHR protein expression was associated with increased PFS (p = 0.050) and OS (p = 0.025). HGSOC patients with both high FSHR and high LHCGR protein levels had the best survival outcome, whilst both low FSHR and low LHCGR expression was associated with poorest survival (p = 0.019). Knockdown of FSHR significantly increased the invasion of serous ovarian cancer cells (OVCAR3 and COV362) in vitro. LHCGR knockdown also promoted invasion of COV362 cells. This study highlights that lower FSHR and LHCGR expression is associated with a more aggressive epithelial ovarian cancer phenotype and promotes pro-metastatic behaviour.

A Comprehensive Molecular and Clinical Analysis of the piRNA Pathway Genes in Ovarian Cancer.

Ovarian cancer (OC) is one of the most lethal gynecological malignancies, yet molecular mechanisms underlying its origin and progression remain poorly understood. With increasing reports of piRNA pathway deregulation in various cancers, we aimed to better understand its role in OC through a comprehensive analysis of key genes: PIWIL1-4, DDX4, HENMT1, MAEL, PLD6, TDRD1,9 and mutants of PIWIL1 (P1∆17) and PIWIL2 (PL2L60). High-throughput qRT-PCR (n = 45) and CSIOVDB (n = 3431) showed differential gene expression when comparing benign ovarian tumors, low grade OC and high grade serous OC (HGSOC). Significant correlation of disparate piRNA pathway gene expression levels with better progression free, post-progression free and overall survival suggests a complex role of this pathway in OC. We discovered PIWIL3 expression in chemosensitive but not chemoresistant primary HGSOC cells, providing a potential target against chemoresistant disease. As a first, we revealed that follicle stimulating hormone increased PIWIL2 expression in OV-90 cells. PIWIL1, P1∆17, PIWIL2, PL2L60 and MAEL overexpression in vitro and in vivo decreased motility and invasion of OVCAR-3 and OV-90 cells. Interestingly, P1∆17 and PL2L60, induced increased motility and invasion compared to PIWIL1 and PIWIL2. Our results in HGSOC highlight the intricate role piRNA pathway genes play in the development of malignant neoplasms.

Transcriptome and translatome co-evolution in mammals.

Gene-expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer1. Here we report an analysis of the co-evolution of translatomes and transcriptomes using ribosome-profiling and matched RNA-sequencing data for three organs (brain, liver and testis) in five mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the different organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifically counterbalanced global dosage reductions during the evolution of sex chromosomes and the effects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of buffering, some genes evolved faster at the translatome layer-potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is reflected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs.

Chromosomics: Bridging the Gap between Genomes and Chromosomes.

The recent advances in DNA sequencing technology are enabling a rapid increase in the number of genomes being sequenced. However, many fundamental questions in genome biology remain unanswered, because sequence data alone is unable to provide insight into how the genome is organised into chromosomes, the position and interaction of those chromosomes in the cell, and how chromosomes and their interactions with each other change in response to environmental stimuli or over time. The intimate relationship between DNA sequence and chromosome structure and function highlights the need to integrate genomic and cytogenetic data to more comprehensively understand the role genome architecture plays in genome plasticity. We propose adoption of the term 'chromosomics' as an approach encompassing genome sequencing, cytogenetics and cell biology, and present examples of where chromosomics has already led to novel discoveries, such as the sex-determining gene in eutherian mammals. More importantly, we look to the future and the questions that could be answered as we enter into the chromosomics revolution, such as the role of chromosome rearrangements in speciation and the role more rapidly evolving regions of the genome, like centromeres, play in genome plasticity. However, for chromosomics to reach its full potential, we need to address several challenges, particularly the training of a new generation of cytogeneticists, and the commitment to a closer union among the research areas of genomics, cytogenetics, cell biology and bioinformatics. Overcoming these challenges will lead to ground-breaking discoveries in understanding genome evolution and function.

Non-invasive genetic sexing technique for analysis of short-beaked echidna (Tachyglossus aculeatus) populations.

Identifying male and female echidnas is challenging due to the lack of external genitalia or any other differing morphological features. This limits studies of wild populations and is a major problem for echidna captive management and breeding. Non-invasive genetic approaches to determine sex minimise the need for handling animals and are used extensively in other mammals. However, currently available approaches cannot be applied to monotremes because their sex chromosomes share no homology with sex chromosomes in other mammals. In this study we used recently identified X and Y chromosome-specific sequences to establish a non-invasive polymerase chain reaction-based technique to determine the sex of echidnas. Genomic DNA was extracted from echidna hair follicles followed by amplification of two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametolog (CRSPY) and anti-Müllerian hormone Y-gametolog (AMHY)) and the X chromosome gene (anti-Müllerian hormone X-gametolog (AMHX)). Using this technique, we identified the sex of 10 juvenile echidnas born at Perth Zoo, revealing that eight of the 10 echidnas were female. Future use of the genetic sexing technique in echidnas will inform captive management, continue breeding success and can be used to investigate sex ratios and population dynamics in wild populations.