Prospecting for Zoonotic Pathogens by Using Targeted DNA Enrichment.

More than 60 zoonoses are linked to small mammals, including some of the most devastating pathogens in human history. Millions of museum-archived tissues are available to understand natural history of those pathogens. Our goal was to maximize the value of museum collections for pathogen-based research by using targeted sequence capture. We generated a probe panel that includes 39,916 80-bp RNA probes targeting 32 pathogen groups, including bacteria, helminths, fungi, and protozoans. Laboratory-generated, mock-control samples showed that we are capable of enriching targeted loci from pathogen DNA 2,882‒6,746-fold. We identified bacterial species in museum-archived samples, including Bartonella, a known human zoonosis. These results showed that probe-based enrichment of pathogens is a highly customizable and efficient method for identifying pathogens from museum-archived tissues.

Comparison of genetic variation between rare and common congeners of Dipodomys with estimates of contemporary and historical effective population size.

Species with low effective population sizes are at greater risk of extinction because of reduced genetic diversity. Such species are more vulnerable to chance events that decrease population sizes (e.g. demographic stochasticity). Dipodomys elator, (Texas kangaroo rat) is a kangaroo rat that is classified as threatened in Texas and field surveys from the past 50 years indicate that the distribution of this species has decreased. This suggests geographic range reductions that could have caused population fluctuations, potentially impacting effective population size. Conversely, the more common and widespread D. ordii (Ord's kangaroo rat) is thought to exhibit relative geographic and demographic stability. We assessed the genetic variation of D. elator and D. ordii samples using 3RAD, a modified restriction site associated sequencing approach. We hypothesized that D. elator would show lower levels of nucleotide diversity, observed heterozygosity, and effective population size when compared to D. ordii. We were also interested in identifying population structure within contemporary samples of D. elator and detecting genetic variation between temporal samples to understand demographic dynamics. We analyzed up to 61,000 single nucleotide polymorphisms. We found that genetic variability and effective population size in contemporary D. elator populations is lower than that of D. ordii. There is slight, if any, population structure within contemporary D. elator samples, and we found low genetic differentiation between spatial or temporal historical samples. This indicates little change in nuclear genetic diversity over 30 years. Results suggest that genetic diversity of D. elator has remained stable despite reduced population size and/or abundance, which may indicate a metapopulation-like system, whose fluctuations might counteract species extinction.

Rapid divergence of a gamete recognition gene promoted macroevolution of Eutheria.

BACKGROUND: Speciation genes contribute disproportionately to species divergence, but few examples exist, especially in vertebrates. Here we test whether Zan, which encodes the sperm acrosomal protein zonadhesin that mediates species-specific adhesion to the egg's zona pellucida, is a speciation gene in placental mammals. RESULTS: Genomic ontogeny reveals that Zan arose by repurposing of a stem vertebrate gene that was lost in multiple lineages but retained in Eutheria on acquiring a function in egg recognition. A 112-species Zan sequence phylogeny, representing 17 of 19 placental Orders, resolves all species into monophyletic groups corresponding to recognized Orders and Suborders, with <5% unsupported nodes. Three other rapidly evolving germ cell genes (Adam2, Zp2, and Prm1), a paralogous somatic cell gene (TectA), and a mitochondrial gene commonly used for phylogenetic analyses (Cytb) all yield trees with poorer resolution than the Zan tree and inferior topologies relative to a widely accepted mammalian supertree. Zan divergence by intense positive selection produces dramatic species differences in the protein's properties, with ordinal divergence rates generally reflecting species richness of placental Orders consistent with expectations for a speciation gene that acts across a wide range of taxa. Furthermore, Zan's combined phylogenetic utility and divergence exceeds those of all other genes known to have evolved in Eutheria by positive selection, including the only other mammalian speciation gene, Prdm9. CONCLUSIONS: Species-specific egg recognition conferred by Zan's functional divergence served as a mode of prezygotic reproductive isolation that promoted the extraordinary adaptive radiation and success of Eutheria.

SINE-Based Phylogenomics Reveal Extensive Introgression and Incomplete Lineage Sorting in Myotis.

Using presence/absence data from over 10,000 Ves SINE insertions, we reconstructed a phylogeny for 11 Myotis species. With nearly one-third of individual Ves gene trees discordant with the overall species tree, phylogenetic conflict appears to be rampant in this genus. From the observed conflict, we infer that ILS is likely a major contributor to the discordance. Much of the discordance can be attributed to the hypothesized split between the Old World and New World Myotis clades and with the first radiation of Myotis within the New World. Quartet asymmetry tests reveal signs of introgression between Old and New World taxa that may have persisted until approximately 8 MYA. Our introgression tests also revealed evidence of both historic and more recent, perhaps even contemporary, gene flow among Myotis species of the New World. Our findings suggest that hybridization likely played an important role in the evolutionary history of Myotis and may still be happening in areas of sympatry. Despite limitations arising from extreme discordance, our SINE-based phylogeny better resolved deeper relationships (particularly the positioning of M. brandtii) and was able to identify potential introgression pathways among the Myotis species sampled.

Genomic analysis of a parasite invasion: Colonization of the Americas by the blood fluke Schistosoma mansoni.

Schistosoma mansoni, a snail-borne, blood fluke that infects humans, was introduced into the Americas from Africa during the Trans-Atlantic slave trade. As this parasite shows strong specificity to the snail intermediate host, we expected that adaptation to South American Biomphalaria spp. snails would result in population bottlenecks and strong signatures of selection. We scored 475,081 single nucleotide variants in 143 S. mansoni from the Americas (Brazil, Guadeloupe and Puerto Rico) and Africa (Cameroon, Niger, Senegal, Tanzania, and Uganda), and used these data to ask: (i) Was there a population bottleneck during colonization? (ii) Can we identify signatures of selection associated with colonization? (iii) What were the source populations for colonizing parasites? We found a 2.4- to 2.9-fold reduction in diversity and much slower decay in linkage disequilibrium (LD) in parasites from East to West Africa. However, we observed similar nuclear diversity and LD in West Africa and Brazil, suggesting no strong bottlenecks and limited barriers to colonization. We identified five genome regions showing selection in the Americas, compared with three in West Africa and none in East Africa, which we speculate may reflect adaptation during colonization. Finally, we infer that unsampled populations from central African regions between Benin and Angola, with contributions from Niger, are probably the major source(s) for Brazilian S. mansoni. The absence of a bottleneck suggests that this is a rare case of a serendipitous invasion, where S. mansoni parasites were pre-adapted to the Americas and able to establish with relative ease.

Urogenital schistosomiasis in Nigeria post receipt of the largest single praziquantel donation in Africa.

Schistosomiasis control efforts in Nigeria received a boost in 2016 when Merck Group made the largest single donation of praziquantel to an African country. We examined urine samples from 2,023 school age children from 15 locations in 10 states and an Internally Displaced Person's (IDP) camp in Nigeria. We recorded an overall Schistosoma haematobium prevalence of 10.4% in the 10 states that ranged between 6 - 37%, while prevalence in the IDP camp was 2.9%. The highest infection prevalence (37%) recorded was from the population in Wasai Dam area in Minjibir (Kano State), while five locations had no positive urine samples. We observed heavy intensity of infection (≥ 50 eggs/10 ml urine) in 87.9% of infected samples and co-occurrence of the eggs of S. haematobium and S. mansoni in urine for two participants. The overall prevalence we recorded is slightly above the national average (9.5%) reported in 2015. Our findings indicate that despite the ongoing administration of praziquantel in Nigeria, urogenital schistosomiasis is still prevalent with heavy intensity of infection. Large-scale epidemiological monitoring is required to monitor the efficacy of schistosomiasis control in Nigeria.

Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies.

Identification and characterization of microRNAs (miRNAs) and their transposable element origins in the saltwater crocodile, Crocodylus porosus.

MicroRNAs (miRNAs) are 18-24 nucleotide regulatory RNAs. They are involved in the regulation of genetic and biological pathways through post transcriptional gene silencing and/or translational repression. Data suggests a slow evolutionary rate for the saltwater crocodile (Crocodylus porosus) over the past several million years when compared to birds, the closest extant relatives of crocodilians. Understanding gene regulation in the saltwater crocodile in the context of relatively slow genomic change thus holds potential for the investigation of genomics, evolution, and adaptation. Utilizing eleven tissue types and sixteen small RNA libraries, we report 644 miRNAs in the saltwater crocodile with >78% of miRNAs being novel to crocodilians. We also identified potential targets for the miRNAs and analyzed the relationship of the miRNA repertoire to transposable elements (TEs). Results suggest an increased association of DNA transposons with miRNAs when compared to retrotransposons. This work reports the first comprehensive analysis of miRNAs in Crocodylus porosus and addresses the potential impacts of miRNAs in regulating the genome in the saltwater crocodile. In addition, the data suggests a supporting role of TEs as a source for miRNAs, adding to the increasing evidence that TEs play a significant role in the evolution of gene regulation.

Differential Expression in Testis and Liver Transcriptomes from Four Species of Peromyscus (Rodentia: Cricetidae).

The genus Peromyscus represents a rapidly diverged clade of Cricetid rodents that contains multiple cryptic species and has a propensity for morphologic conservation across its members. The unresolved relationships in previously proposed phylogenies reflect a suspected rapid adaptive radiation. To identify functional groups of genes that may be important in reproductive isolation in a reoccurring fashion across the Peromyscus phylogeny, liver and testis transcriptomes from four species (P. attwateri, P. boylii, P. leucopus, and P. maniculatus) were generated and differential expression (DE) tests were conducted. Taxa were selected to represent members diverged from a common ancestor: P. attwateri + P. boylii (clade A), and P. leucopus + P. maniculatus (clade B). Comparison of clades (A vs. B) suggested that 252 transcripts had significant DE in the liver data set, whereas significant DE was identified for 657 transcripts in the testis data set. Further, 45 genes had DE isoforms in the 657 testis transcripts and most of these functioned in major reproductive roles such as acrosome assembly, spermatogenesis, and cell cycle processes (meiosis). DE transcripts in the liver mapped to more broad gene ontology terms (metabolic processes, catabolic processes, response to chemical, and regulatory processes), and DE transcripts in the testis mapped to gene ontology terms associated with reproductive processes, such as meiosis, sperm motility, acrosome assembly, and sperm-egg fusion. These results suggest that a suite of genes that conduct similar functions in the testes may be responsible for the adaptive radiation events and potential reoccurring speciation of Peromyscus in terms of reproduction through varying expression levels.

Ancient Hybridization and Adaptive Introgression of an Invadolysin Gene in Schistosome Parasites.

Introgression among parasite species has the potential to transfer traits of biomedical importance across species boundaries. The parasitic blood fluke Schistosoma haematobium causes urogenital schistosomiasis in humans across sub-Saharan Africa. Hybridization with other schistosome species is assumed to occur commonly, because genetic crosses between S. haematobium and livestock schistosomes, including S. bovis, can be staged in the laboratory, and sequencing of mtDNA and rDNA amplified from microscopic miracidia larvae frequently reveals markers from different species. However, the frequency, direction, age, and genomic consequences of hybridization are unknown. We hatched miracidia from eggs and sequenced the exomes from 96 individual S. haematobium miracidia from infected patients from Niger and the Zanzibar archipelago. These data revealed no evidence for contemporary hybridization between S. bovis and S. haematobium in our samples. However, all Nigerien S. haematobium genomes sampled show hybrid ancestry, with 3.3-8.2% of their nuclear genomes derived from S. bovis, providing evidence of an ancient introgression event that occurred at least 108-613 generations ago. Some S. bovis-derived alleles have spread to high frequency or reached fixation and show strong signatures of directional selection; the strongest signal spans a single gene in the invadolysin gene family (Chr. 4). Our results suggest that S. bovis/S. haematobium hybridization occurs rarely but demonstrate profound consequences of ancient introgression from a livestock parasite into the genome of S. haematobium, the most prevalent schistosome species infecting humans.

A genomic resource for the sedentary semi-endoparasitic reniform nematode, Rotylenchulus reniformis Linford & Oliveira.

The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasitic species that is pathogenic on many row crops, fruits, and vegetables. Here, the authors present a draft genome assembly of R. reniformis using small- and large-insert libraries sequenced on the Illumina GAIIx and MiSeq platforms.

Mammalian transposable elements and their impacts on genome evolution.

Transposable elements (TEs) are genetic elements with the ability to mobilize and replicate themselves in a genome. Mammalian genomes are dominated by TEs, which can reach copy numbers in the hundreds of thousands. As a result, TEs have had significant impacts on mammalian evolution. Here we summarize the current understanding of TE content in mammal genomes and find that, with a few exceptions, most fall within a predictable range of observations. First, one third to one half of the genome is derived from TEs. Second, most mammalian genomes are dominated by LINE and SINE retrotransposons, more limited LTR retrotransposons, and minimal DNA transposon accumulation. Third, most mammal genome contains at least one family of actively accumulating retrotransposon. Finally, horizontal transfer of TEs among lineages is rare. TE exaptation events are being recognized with increasing frequency. Despite these beneficial aspects of TE content and activity, the majority of TE insertions are neutral or deleterious. To limit the deleterious effects of TE proliferation, the genome has evolved several defense mechanisms that act at the epigenetic, transcriptional, and post-transcriptional levels. The interaction between TEs and these defense mechanisms has led to an evolutionary arms race where TEs are suppressed, evolve to escape suppression, then are suppressed again as the defense mechanisms undergo compensatory change. The result is complex and constantly evolving interactions between TEs and host genomes.

Conflicting Evolutionary Histories of the Mitochondrial and Nuclear Genomes in New World Myotis Bats.

The rapid diversification of Myotis bats into more than 100 species is one of the most extensive mammalian radiations available for study. Efforts to understand relationships within Myotis have primarily utilized mitochondrial markers and trees inferred from nuclear markers lacked resolution. Our current understanding of relationships within Myotis is therefore biased towards a set of phylogenetic markers that may not reflect the history of the nuclear genome. To resolve this, we sequenced the full mitochondrial genomes of 37 representative Myotis, primarily from the New World, in conjunction with targeted sequencing of 3648 ultraconserved elements (UCEs). We inferred the phylogeny and explored the effects of concatenation and summary phylogenetic methods, as well as combinations of markers based on informativeness or levels of missing data, on our results. Of the 294 phylogenies generated from the nuclear UCE data, all are significantly different from phylogenies inferred using mitochondrial genomes. Even within the nuclear data, quartet frequencies indicate that around half of all UCE loci conflict with the estimated species tree. Several factors can drive such conflict, including incomplete lineage sorting, introgressive hybridization, or even phylogenetic error. Despite the degree of discordance between nuclear UCE loci and the mitochondrial genome and among UCE loci themselves, the most common nuclear topology is recovered in one quarter of all analyses with strong nodal support. Based on these results, we re-examine the evolutionary history of Myotis to better understand the phenomena driving their unique nuclear, mitochondrial, and biogeographic histories.

Evolution and Diversity of Transposable Elements in Vertebrate Genomes.

Transposable elements (TEs) are selfish genetic elements that mobilize in genomes via transposition or retrotransposition and often make up large fractions of vertebrate genomes. Here, we review the current understanding of vertebrate TE diversity and evolution in the context of recent advances in genome sequencing and assembly techniques. TEs make up 4-60% of assembled vertebrate genomes, and deeply branching lineages such as ray-finned fishes and amphibians generally exhibit a higher TE diversity than the more recent radiations of birds and mammals. Furthermore, the list of taxa with exceptional TE landscapes is growing. We emphasize that the current bottleneck in genome analyses lies in the proper annotation of TEs and provide examples where superficial analyses led to misleading conclusions about genome evolution. Finally, recent advances in long-read sequencing will soon permit access to TE-rich genomic regions that previously resisted assembly including the gigantic, TE-rich genomes of salamanders and lungfishes.

Improved genome assembly of American alligator genome reveals conserved architecture of estrogen signaling.

The American alligator, Alligator mississippiensis, like all crocodilians, has temperature-dependent sex determination, in which the sex of an embryo is determined by the incubation temperature of the egg during a critical period of development. The lack of genetic differences between male and female alligators leaves open the question of how the genes responsible for sex determination and differentiation are regulated. Insight into this question comes from the fact that exposing an embryo incubated at male-producing temperature to estrogen causes it to develop ovaries. Because estrogen response elements are known to regulate genes over long distances, a contiguous genome assembly is crucial for predicting and understanding their impact. We present an improved assembly of the American alligator genome, scaffolded with in vitro proximity ligation (Chicago) data. We use this assembly to scaffold two other crocodilian genomes based on synteny. We perform RNA sequencing of tissues from American alligator embryos to find genes that are differentially expressed between embryos incubated at male- versus female-producing temperature. Finally, we use the improved contiguity of our assembly along with the current model of CTCF-mediated chromatin looping to predict regions of the genome likely to contain estrogen-responsive genes. We find that these regions are significantly enriched for genes with female-biased expression in developing gonads after the critical period during which sex is determined by incubation temperature. We thus conclude that estrogen signaling is a major driver of female-biased gene expression in the post-temperature sensitive period gonads.

Genome sequence of Phormia regina Meigen (Diptera: Calliphoridae): implications for medical, veterinary and forensic research.

BACKGROUND: Blow flies (Diptera: Calliphoridae) are important medical, veterinary and forensic insects encompassing 8 % of the species diversity observed in the calyptrate insects. Few genomic resources exist to understand the diversity and evolution of this group. RESULTS: We present the hybrid (short and long reads) draft assemblies of the male and female genomes of the common North American blow fly, Phormia regina (Diptera: Calliphoridae). The 550 and 534 Mb draft assemblies contained 8312 and 9490 predicted genes in the female and male genomes, respectively; including > 93 % conserved eukaryotic genes. Putative X and Y chromosomes (21 and 14 Mb, respectively) were assembled and annotated. The P. regina genomes appear to contain few mobile genetic elements, an almost complete absence of SINEs, and most of the repetitive landscape consists of simple repetitive sequences. Candidate gene approaches were undertaken to annotate insecticide resistance, sex-determining, chemoreceptors, and antimicrobial peptides. CONCLUSIONS: This work yielded a robust, reliable reference calliphorid genome from a species located in the middle of a calliphorid phylogeny. By adding an additional blow fly genome, the ability to tease apart what might be true of general calliphorids vs. what is specific of two distinct lineages now exists. This resource will provide a strong foundation for future studies into the evolution, population structure, behavior, and physiology of all blow flies.

Pinpointing the vesper bat transposon revolution using the Miniopterus natalensis genome.

BACKGROUND: Around 40 million years ago DNA transposons began accumulating in an ancestor of bats in the family Vespertilionidae. Since that time, Class II transposons have been continuously reinvading and accumulating in vespertilionid genomes at a rate that is unprecedented in mammals. Miniopterus (Miniopteridae), a genus of long-fingered bats that was recently elevated from Vespertilionidae, is the sister taxon to the vespertilionids and is often used as an outgroup when studying transposable elements in vesper bats. Previous wet-lab techniques failed to identify Helitrons, TcMariners, or hAT transposons in Miniopterus. Limitations of those methods and ambiguous results regarding the distribution of piggyBac transposons left some questions as to the distribution of Class II elements in this group. The recent release of the Miniopterus natalensis genome allows for transposable element discovery with a higher degree of precision. RESULTS: Here we analyze the transposable element content of M. natalensis to pinpoint with greater accuracy the taxonomic distribution of Class II transposable elements in bats. These efforts demonstrate that, compared to the vespertilionids, Class II TEs are highly mutated and comprise only a small portion of the M. natalensis genome. Despite the limited Class II content, M. natalensis possesses a limited number of lineage-specific, low copy number piggyBacs and shares several TcMariner families with vespertilionid bats. Multiple efforts to identify Helitrons, one of the major TE components of vesper bat genomes, using de novo repeat identification and structural based searches failed. CONCLUSIONS: These observations combined with previous results inform our understanding of the events leading to the unique Class II element acquisition that characterizes vespertilionids. While it appears that a small number of TcMariner and piggyBac elements were deposited in the ancestral Miniopterus + vespertilionid genome, these elements are not present in M. natalensis genome at high copy number. Instead, this work indicates that the vesper bats alone experienced the expansion of TEs ranging from Helitrons to piggyBacs to hATs.

Transposable Element Targeting by piRNAs in Laurasiatherians with Distinct Transposable Element Histories.

PIWI proteins and PIWI-interacting RNAs (piRNAs) are part of a cellular pathway that has evolved to protect genomes against the proliferation of transposable elements (TEs). PIWIs and piRNAs assemble into complexes that are involved in epigenetic and post-transcriptional repression of TEs. Most of our understanding of the mechanisms of piRNA-mediated TE silencing comes from fruit fly and mouse models. However, even in these well-studied animals it is unclear how piRNA responses relate to variable TE expression and whether the strength of the piRNA response affects TE content over time. Here, we assessed the evolutionary interactions between TE and piRNAs in a statistical framework using three nonmodel laurasiatherian mammals as a study system: dog, horse, and a vesper bat. These three species diverged ∼80 million years ago and have distinct genomic TE contents. By comparing species with distinct TE landscapes, we aimed to identify clear relationships among TE content, expression, and piRNAs. We found that the TE subfamilies that are the most transcribed appear to elicit the strongest "ping-pong" response. This was most evident among long interspersed elements, but the relationships between expression and ping-pong pilRNA (piRNA-like) expression were more complex among SINEs. SINE transcripts were equally abundant in the dog and horse yet new SINE insertions were relatively rare in the horse genome, where we identified a stronger piRNA response. Our analyses suggest that the piRNA response can have a strong impact on the TE composition of a genome. However, our results also suggest that the presence of a robust piRNA response is apparently not sufficient to stop TE mobilization and accumulation.

Accurate Transposable Element Annotation Is Vital When Analyzing New Genome Assemblies.

Transposable elements (TEs) are mobile genetic elements with the ability to replicate themselves throughout the host genome. In some taxa TEs reach copy numbers in hundreds of thousands and can occupy more than half of the genome. The increasing number of reference genomes from nonmodel species has begun to outpace efforts to identify and annotate TE content and methods that are used vary significantly between projects. Here, we demonstrate variation that arises in TE annotations when less than optimal methods are used. We found that across a variety of taxa, the ability to accurately identify TEs based solely on homology decreased as the phylogenetic distance between the queried genome and a reference increased. Next we annotated repeats using homology alone, as is often the case in new genome analyses, and a combination of homology and de novo methods as well as an additional manual curation step. Reannotation using these methods identified a substantial number of new TE subfamilies in previously characterized genomes, recognized a higher proportion of the genome as repetitive, and decreased the average genetic distance within TE families, implying recent TE accumulation. Finally, these finding-increased recognition of younger TEs-were confirmed via an analysis of the postman butterfly (Heliconius melpomene). These observations imply that complete TE annotation relies on a combination of homology and de novo-based repeat identification, manual curation, and classification and that relying on simple, homology-based methods is insufficient to accurately describe the TE landscape of a newly sequenced genome.