Two-stage evolution of mammalian adipose tissue thermogenesis.

Brown adipose tissue (BAT) is a heater organ that expresses thermogenic uncoupling protein 1 (UCP1) to maintain high body temperatures during cold stress. BAT thermogenesis is considered an overarching mammalian trait, but its evolutionary origin is unknown. We show that adipose tissue of marsupials, which diverged from eutherian mammals ~150 million years ago, expresses a nonthermogenic UCP1 variant governed by a partial transcriptomic BAT signature similar to that found in eutherian beige adipose tissue. We found that the reconstructed UCP1 sequence of the common eutherian ancestor displayed typical thermogenic activity, whereas therian ancestor UCP1 is nonthermogenic. Thus, mammalian adipose tissue thermogenesis may have evolved in two distinct stages, with a prethermogenic stage in the common therian ancestor linking UCP1 expression to adipose tissue and thermal stress. We propose that in a second stage, UCP1 acquired its thermogenic function specifically in eutherians, such that the onset of mammalian BAT thermogenesis occurred only after the divergence from marsupials.

High prevalence of PRDM9-independent recombination hotspots in placental mammals.

In many mammals, recombination events are concentrated in hotspots directed by a sequence-specific DNA-binding protein named PRDM9. Intriguingly, PRDM9 has been lost several times in vertebrates, and notably among mammals, it has been pseudogenized in the ancestor of canids. In the absence of PRDM9, recombination hotspots tend to occur in promoter-like features such as CpG islands. It has thus been proposed that one role of PRDM9 could be to direct recombination away from PRDM9-independent hotspots. However, the ability of PRDM9 to direct recombination hotspots has been assessed in only a handful of species, and a clear picture of how much recombination occurs outside of PRDM9-directed hotspots in mammals is still lacking. In this study, we derived an estimator of past recombination activity based on signatures of GC-biased gene conversion in substitution patterns. We quantified recombination activity in PRDM9-independent hotspots in 52 species of boreoeutherian mammals. We observe a wide range of recombination rates at these loci: several species (such as mice, humans, some felids, or cetaceans) show a deficit of recombination, while a majority of mammals display a clear peak of recombination. Our results demonstrate that PRDM9-directed and PRDM9-independent hotspots can coexist in mammals and that their coexistence appears to be the rule rather than the exception. Additionally, we show that the location of PRDM9-independent hotspots is relatively more stable than that of PRDM9-directed hotspots, but that PRDM9-independent hotspots nevertheless evolve slowly in concert with DNA hypomethylation.

The Diverse Evolutionary Histories of Domesticated Metaviral Capsid Genes in Mammals.

Selfish genetic elements comprise significant fractions of mammalian genomes. In rare instances, host genomes domesticate segments of these elements for function. Using a complete human genome assembly and 25 additional vertebrate genomes, we re-analyzed the evolutionary trajectories and functional potential of capsid (CA) genes domesticated from Metaviridae, a lineage of retrovirus-like retrotransposons. Our study expands on previous analyses to unearth several new insights about the evolutionary histories of these ancient genes. We find that at least five independent domestication events occurred from diverse Metaviridae, giving rise to three universally retained single-copy genes evolving under purifying selection and two gene families unique to placental mammals, with multiple members showing evidence of rapid evolution. In the SIRH/RTL family, we find diverse amino-terminal domains, widespread loss of protein-coding capacity in RTL10 despite its retention in several mammalian lineages, and differential utilization of an ancient programmed ribosomal frameshift in RTL3 between the domesticated CA and protease domains. Our analyses also reveal that most members of the PNMA family in mammalian genomes encode a conserved putative amino-terminal RNA-binding domain (RBD) both adjoining and independent from domesticated CA domains. Our analyses lead to a significant correction of previous annotations of the essential CCDC8 gene. We show that this putative RBD is also present in several extant Metaviridae, revealing a novel protein domain configuration in retrotransposons. Collectively, our study reveals the divergent outcomes of multiple domestication events from diverse Metaviridae in the common ancestor of placental mammals.

Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements.

Vocal production learning ("vocal learning") is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat (Rousettus aegyptiacus) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution.

Marsupials have monoallelic MEST expression with a conserved antisense lncRNA but MEST is not imprinted.

The imprinted isoform of the Mest gene in mice is involved in key mammalian traits such as placental and fetal growth, maternal care and mammary gland maturation. The imprinted isoform has a distinct differentially methylated region (DMR) at its promoter in eutherian mammals but in marsupials, there are no differentially methylated CpG islands between the parental alleles. Here, we examined similarities and differences in the MEST gene locus across mammals using a marsupial, the tammar wallaby, a monotreme, the platypus, and a eutherian, the mouse, to investigate how imprinting of this gene evolved in mammals. By confirming the presence of the short isoform in all mammalian groups (which is imprinted in eutherians), this study suggests that an alternative promoter for the short isoform evolved at the MEST gene locus in the common ancestor of mammals. In the tammar, the short isoform of MEST shared the putative promoter CpG island with an antisense lncRNA previously identified in humans and an isoform of a neighbouring gene CEP41. The antisense lncRNA was expressed in tammar sperm, as seen in humans. This suggested that the conserved lncRNA might be important in the establishment of MEST imprinting in therian mammals, but it was not imprinted in the tammar. In contrast to previous studies, this study shows that MEST is not imprinted in marsupials. MEST imprinting in eutherians, therefore must have occurred after the marsupial-eutherian split with the acquisition of a key epigenetic imprinting control region, the differentially methylated CpG islands between the parental alleles.

Progressive Exaptation of Endogenous Retroviruses in Placental Evolution in Cattle.

Viviparity is made possible by the placenta, a structure acquired relatively recently in the evolutionary history of eutherian mammals. Compared to oviparity, it increases the survival rate of the fetus, owing to the eutherian placenta. Questions such as "How was the placenta acquired?" and "Why is there diversity in placental morphology among mammalian species?" remain largely unsolved. Our present understanding of the molecules regulating placental development remains unclear, owing in no small part to the persistent obscurity surrounding the molecular mechanisms underlying placental acquisition. Numerous genes associated with the development of eutherian placental morphology likely evolved to function at the fetal-maternal interface in conjunction with those participating in embryogenesis. Therefore, identifying these genes, how they were acquired, and how they came to be expressed specifically at the fetal-maternal interface will shed light on some crucial molecular mechanisms underlying placental evolution. Exhaustive studies support the hypothesis that endogenous retroviruses (ERVs) could be evolutional driving forces for trophoblast cell fusion and placental structure in mammalian placentas including those of the bovine species. This review focuses on bovine ERVs (BERVs) and their expression and function in the placenta.

Genome sequence analysis of Malayan pangolin (Manis javanica) forensic samples reveals the presence of Paraburkholderia fungorum sequences.

Background: The Malayan pangolin (Manis javanica) is a placental mammal and is listed as Critically Endangered on the IUCN Red List of Threatened Species. Most previous attempts to breed pangolins in captivity have met with little success because of dietary issues, infections, and other complications, although a previous study reported breeding pangolins in captivity to the third generation. In our previous pangolin genome sequencing data analysis, we obtained a considerable amount of bacterial DNA from a pregnant female Malayan pangolin (named "UM3"), which was likely infected by Paraburkholderia fungorum-an agent of biodegradation and bioremediation in agriculture. Methodology: Here, we further confirmed and characterized this bacterial species using PCR, histological staining, whole-genome sequencing, and bioinformatics approaches. PCR assays with in-house designed primer sets and 16S universal primers showed clear positive bands in the cerebrum, cerebellum, lung, and blood of UM3 suggesting that UM3 might have developed septicaemia. Histological staining showed the presence of Gram-negative rod-shaped bacteria in the pangolin brain and lungs, indicating the colonization of the bacteria in these two organs. In addition, PCR screening of UM3's fetal tissues revealed the presence of P. fungorum in the gastrocnemius muscle, but not in other tissues that we examined. We also sequenced and reconstructed the genome of pangolin P. fungorum, which has a genome size of 7.7 Mbps. Conclusion: Our study is the first to present detailed evidence of the presence of P. fungorum in a pangolin and her fetus (although preliminary results were presented in our previous article). Here, we raise the concern that P. fungorum may potentially infect humans, especially YOPI (young, old, pregnant, and immunocompromised) people. Therefore, caution should be exercised when using this bacterial species as biodegradation or bioremediation agents in agriculture.

Retrovirus-Derived RTL/SIRH: Their Diverse Roles in the Current Eutherian Developmental System and Contribution to Eutherian Evolution.

Eutherians have 11 retrotransposon Gag-like (RTL)/sushi-ichi retrotransposon homolog (SIRH) genes presumably derived from a certain retrovirus. Accumulating evidence indicates that the RTL/SIRH genes play a variety of roles in the current mammalian developmental system, such as in the placenta, brain, and innate immune system, in a eutherian-specific manner. It has been shown that the functional role of Paternally Expressed 10 (PEG10) in placental formation is unique to the therian mammals, as are the eutherian-specific roles of PEG10 and PEG11/RTL1 in maintaining the fetal capillary network and the endocrine regulation of RTL7/SIRH7 (aka Leucine Zipper Down-Regulated in Cancer 1 (LDOCK1)) in the placenta. In the brain, PEG11/RTL1 is expressed in the corticospinal tract and hippocampal commissure, mammalian-specific structures, and in the corpus callosum, a eutherian-specific structure. Unexpectedly, at least three RTL/SIRH genes, RTL5/SIRH8, RTL6/SIRH3, and RTL9/SIRH10, play important roles in combating a variety of pathogens, namely viruses, bacteria, and fungi, respectively, suggesting that the innate immunity system of the brain in eutherians has been enhanced by the emergence of these new components. In this review, we will summarize the function of 10 out of the 11 RTL/SIRH genes and discuss their roles in eutherian development and evolution.

Retrovirus-Derived RTL9 Plays an Important Role in Innate Antifungal Immunity in the Eutherian Brain.

Retrotransposon Gag-like (RTL) genes play a variety of essential and important roles in the eutherian placenta and brain. It has recently been demonstrated that RTL5 and RTL6 (also known as sushi-ichi retrotransposon homolog 8 (SIRH8) and SIRH3) are microglial genes that play important roles in the brain's innate immunity against viruses and bacteria through their removal of double-stranded RNA and lipopolysaccharide, respectively. In this work, we addressed the function of RTL9 (also known as SIRH10). Using knock-in mice that produce RTL9-mCherry fusion protein, we examined RTL9 expression in the brain and its reaction to fungal zymosan. Here, we demonstrate that RTL9 plays an important role, degrading zymosan in the brain. The RTL9 protein is localized in the microglial lysosomes where incorporated zymosan is digested. Furthermore, in Rtl9 knockout mice expressing RTL9ΔC protein lacking the C-terminus retroviral GAG-like region, the zymosan degrading activity was lost. Thus, RTL9 is essentially engaged in this reaction, presumably via its GAG-like region. Together with our previous study, this result highlights the importance of three retrovirus-derived microglial RTL genes as eutherian-specific constituents of the current brain innate immune system: RTL9, RTL5 and RTL6, responding to fungi, viruses and bacteria, respectively.

Ecoevolutionary processes structure milk microbiomes across the mammalian tree of life.

Milk production is an ancient adaptation that unites all mammals. Milk contains a microbiome that can contribute to offspring health and microbial-immunological development. We generated a comprehensive milk microbiome dataset (16S rRNA gene) for the class Mammalia, representing 47 species from all placental superorders, to determine processes structuring milk microbiomes. We show that across Mammalia, milk exposes offspring to maternal bacterial and archaeal symbionts throughout lactation. Deterministic processes of environmental selection accounted for 20% of milk microbiome assembly processes; milk microbiomes were similar from mammals with the same host superorder (Afrotheria, Laurasiathera, Euarchontoglires, and Xenarthra: 6%), environment (marine captive, marine wild, terrestrial captive, and terrestrial wild: 6%), diet (carnivore, omnivore, herbivore, and insectivore: 5%), and milk nutrient content (sugar, fat, and protein: 3%). We found that diet directly and indirectly impacted milk microbiomes, with indirect effects being mediated by milk sugar content. Stochastic processes, such as ecological drift, accounted for 80% of milk microbiome assembly processes, which was high compared to mammalian gut and mammalian skin microbiomes (69% and 45%, respectively). Even amid high stochasticity and indirect effects, our results of direct dietary effects on milk microbiomes provide support for enteromammary trafficking, representing a mechanism by which bacteria are transferred from the mother's gut to mammary gland and then to offspring postnatally. The microbial species present in milk reflect both selective pressures and stochastic processes at the host level, exemplifying various ecological and evolutionary factors acting on milk microbiomes, which, in turn, set the stage for offspring health and development.

A burst of genomic innovation at the origin of placental mammals mediated embryo implantation.

The origin of embryo implantation in mammals ~148 million years ago was a dramatic shift in reproductive strategy, yet the molecular changes that established mammal implantation are largely unknown. Although progesterone receptor signalling predates the origin of mammals and is highly conserved in, and critical for, successful mammal pregnancy, it alone cannot explain the origin and subsequent diversity of implantation strategies throughout the placental mammal radiation. MiRNAs are known to be flexible and dynamic regulators with a well-established role in the pathophysiology of mammal placenta. We propose that a dynamic core microRNA (miRNA) network originated early in placental mammal evolution, responds to conserved mammal pregnancy cues (e.g. progesterone), and facilitates species-specific responses. Here we identify 13 miRNA gene families that arose at the origin of placental mammals and were subsequently retained in all descendent lineages. The expression of these miRNAs in response to early pregnancy molecules is regulated in a species-specific manner in endometrial epithelia of species with extreme implantation strategies (i.e. bovine and human). Furthermore, this set of miRNAs preferentially target proteins under positive selective pressure on the ancestral eutherian lineage. Discovery of this core embryo implantation toolkit and specifically adapted proteins helps explain the origin and evolution of implantation in mammals.

Insights into mammalian TE diversity through the curation of 248 genome assemblies.

We examined transposable element (TE) content of 248 placental mammal genome assemblies, the largest de novo TE curation effort in eukaryotes to date. We found that although mammals resemble one another in total TE content and diversity, they show substantial differences with regard to recent TE accumulation. This includes multiple recent expansion and quiescence events across the mammalian tree. Young TEs, particularly long interspersed elements, drive increases in genome size, whereas DNA transposons are associated with smaller genomes. Mammals tend to accumulate only a few types of TEs at any given time, with one TE type dominating. We also found association between dietary habit and the presence of DNA transposon invasions. These detailed annotations will serve as a benchmark for future comparative TE analyses among placental mammals.

The contribution of historical processes to contemporary extinction risk in placental mammals.

Species persistence can be influenced by the amount, type, and distribution of diversity across the genome, suggesting a potential relationship between historical demography and resilience. In this study, we surveyed genetic variation across single genomes of 240 mammals that compose the Zoonomia alignment to evaluate how historical effective population size (Ne) affects heterozygosity and deleterious genetic load and how these factors may contribute to extinction risk. We find that species with smaller historical Ne carry a proportionally larger burden of deleterious alleles owing to long-term accumulation and fixation of genetic load and have a higher risk of extinction. This suggests that historical demography can inform contemporary resilience. Models that included genomic data were predictive of species' conservation status, suggesting that, in the absence of adequate census or ecological data, genomic information may provide an initial risk assessment.

A genomic timescale for placental mammal evolution.

The precise pattern and timing of speciation events that gave rise to all living placental mammals remain controversial. We provide a comprehensive phylogenetic analysis of genetic variation across an alignment of 241 placental mammal genome assemblies, addressing prior concerns regarding limited genomic sampling across species. We compared neutral genome-wide phylogenomic signals using concatenation and coalescent-based approaches, interrogated phylogenetic variation across chromosomes, and analyzed extensive catalogs of structural variants. Interordinal relationships exhibit relatively low rates of phylogenomic conflict across diverse datasets and analytical methods. Conversely, X-chromosome versus autosome conflicts characterize multiple independent clades that radiated during the Cenozoic. Genomic time trees reveal an accumulation of cladogenic events before and immediately after the Cretaceous-Paleogene (K-Pg) boundary, implying important roles for Cretaceous continental vicariance and the K-Pg extinction in the placental radiation.

Integrating gene annotation with orthology inference at scale.

Annotating coding genes and inferring orthologs are two classical challenges in genomics and evolutionary biology that have traditionally been approached separately, limiting scalability. We present TOGA (Tool to infer Orthologs from Genome Alignments), a method that integrates structural gene annotation and orthology inference. TOGA implements a different paradigm to infer orthologous loci, improves ortholog detection and annotation of conserved genes compared with state-of-the-art methods, and handles even highly fragmented assemblies. TOGA scales to hundreds of genomes, which we demonstrate by applying it to 488 placental mammal and 501 bird assemblies, creating the largest comparative gene resources so far. Additionally, TOGA detects gene losses, enables selection screens, and automatically provides a superior measure of mammalian genome quality. TOGA is a powerful and scalable method to annotate and compare genes in the genomic era.

Evolutionary constraint and innovation across hundreds of placental mammals.

Zoonomia is the largest comparative genomics resource for mammals produced to date. By aligning genomes for 240 species, we identify bases that, when mutated, are likely to affect fitness and alter disease risk. At least 332 million bases (~10.7%) in the human genome are unusually conserved across species (evolutionarily constrained) relative to neutrally evolving repeats, and 4552 ultraconserved elements are nearly perfectly conserved. Of 101 million significantly constrained single bases, 80% are outside protein-coding exons and half have no functional annotations in the Encyclopedia of DNA Elements (ENCODE) resource. Changes in genes and regulatory elements are associated with exceptional mammalian traits, such as hibernation, that could inform therapeutic development. Earth's vast and imperiled biodiversity offers distinctive power for identifying genetic variants that affect genome function and organismal phenotypes.

Origin and Evolution of Marsupial-specific Imprinting Clusters Through Lineage-specific Gene Duplications and Acquisition of Promoter Differential Methylation.

Genomic imprinting is a parent-of-origin-specific expression phenomenon that plays fundamental roles in many biological processes. In animals, imprinting is only observed in therian mammals, with ∼200 imprinted genes known in humans and mice. The imprinting pattern in marsupials has been minimally investigated by examining orthologs to known eutherian imprinted genes. To identify marsupial-specific imprinting in an unbiased way, we performed RNA-seq studies on samples of fetal brain and placenta from the reciprocal cross progeny of two laboratory opossum stocks. We inferred allele-specific expression for >3,000 expressed genes and discovered/validated 13 imprinted genes, including three previously known imprinted genes, Igf2r, Peg10, and H19. We estimate that marsupials imprint ∼60 autosomal genes, which is a much smaller set compared with eutherians. Among the nine novel imprinted genes, three noncoding RNAs have no known homologs in eutherian mammals, while the remaining genes have important functions in pluripotency, transcription regulation, nucleolar homeostasis, and neural differentiation. Methylation analyses at promoter CpG islands revealed differentially methylated regions in five of these marsupial-specific imprinted genes, suggesting that differential methylation is a common mechanism in the epigenetic regulation of marsupial imprinting. Clustering and co-regulation were observed at marsupial imprinting loci Pou5f3-Npdc1 and Nkrfl-Ipncr2, but eutherian-type multi-gene imprinting clusters were not detected. Also differing from eutherian mammals, the brain and placenta imprinting profiles are remarkably similar in opossums, presumably due to the shared origin of these organs from the trophectoderm. Our results contribute to a fuller understanding of the origin, evolution, and mechanisms of genomic imprinting in therian mammals.

Methodological challenges in the genomic analysis of an endangered mammal population with low genetic diversity.

Recently, populations of various species with very low genetic diversity have been discovered. Some of these persist in the long term, but others could face extinction due to accelerated loss of fitness. In this work, we characterize 45 individuals of one of these populations, belonging to the Iberian desman (Galemys pyrenaicus). For this, we used the ddRADseq technique, which generated 1421 SNPs. The heterozygosity values of the analyzed individuals were among the lowest recorded for mammals, ranging from 26 to 91 SNPs/Mb. Furthermore, the individuals from one of the localities, highly isolated due to strong barriers, presented extremely high inbreeding coefficients, with values above 0.7. Under this scenario of low genetic diversity and elevated inbreeding levels, some individuals appeared to be almost genetically identical. We used different methods and simulations to determine if genetic identification and parentage analysis were possible in this population. Only one of the methods, which does not assume population homogeneity, was able to identify all individuals correctly. Therefore, genetically impoverished populations pose a great methodological challenge for their genetic study. However, these populations are of primary scientific and conservation interest, so it is essential to characterize them genetically and improve genomic methodologies for their research.

Super-enhancers conserved within placental mammals maintain stem cell pluripotency.

Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share an evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolution in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that displayed remarkable conservation in placental mammals and were sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR-Cas9 approach severely impaired stem cell pluripotency. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals.

Evolution of the ancestral mammalian karyotype and syntenic regions.

Decrypting the rearrangements that drive mammalian chromosome evolution is critical to understanding the molecular bases of speciation, adaptation, and disease susceptibility. Using 8 scaffolded and 26 chromosome-scale genome assemblies representing 23/26 mammal orders, we computationally reconstructed ancestral karyotypes and syntenic relationships at 16 nodes along the mammalian phylogeny. Three different reference genomes (human, sloth, and cattle) representing phylogenetically distinct mammalian superorders were used to assess reference bias in the reconstructed ancestral karyotypes and to expand the number of clades with reconstructed genomes. The mammalian ancestor likely had 19 pairs of autosomes, with nine of the smallest chromosomes shared with the common ancestor of all amniotes (three still conserved in extant mammals), demonstrating a striking conservation of synteny for ∼320 My of vertebrate evolution. The numbers and types of chromosome rearrangements were classified for transitions between the ancestral mammalian karyotype, descendent ancestors, and extant species. For example, 94 inversions, 16 fissions, and 14 fusions that occurred over 53 My differentiated the therian from the descendent eutherian ancestor. The highest breakpoint rate was observed between the mammalian and therian ancestors (3.9 breakpoints/My). Reconstructed mammalian ancestor chromosomes were found to have distinct evolutionary histories reflected in their rates and types of rearrangements. The distributions of genes, repetitive elements, topologically associating domains, and actively transcribed regions in multispecies homologous synteny blocks and evolutionary breakpoint regions indicate that purifying selection acted over millions of years of vertebrate evolution to maintain syntenic relationships of developmentally important genes and regulatory landscapes of gene-dense chromosomes.