Phased nanopore assembly with Shasta and modular graph phasing with GFAse.

Reference-free genome phasing is vital for understanding allele inheritance and the impact of single-molecule DNA variation on phenotypes. To achieve thorough phasing across homozygous or repetitive regions of the genome, long-read sequencing technologies are often used to perform phased de novo assembly. As a step toward reducing the cost and complexity of this type of analysis, we describe new methods for accurately phasing Oxford Nanopore Technologies (ONT) sequence data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of ONT PromethION sequencing, including those using proximity ligation, and show that newer, higher accuracy ONT reads substantially improve assembly quality.

Analysis and benchmarking of small and large genomic variants across tandem repeats.

Tandem repeats (TRs) are highly polymorphic in the human genome, have thousands of associated molecular traits and are linked to over 60 disease phenotypes. However, they are often excluded from at-scale studies because of challenges with variant calling and representation, as well as a lack of a genome-wide standard. Here, to promote the development of TR methods, we created a catalog of TR regions and explored TR properties across 86 haplotype-resolved long-read human assemblies. We curated variants from the Genome in a Bottle (GIAB) HG002 individual to create a TR dataset to benchmark existing and future TR analysis methods. We also present an improved variant comparison method that handles variants greater than 4 bp in length and varying allelic representation. The 8.1% of the genome covered by the TR catalog holds ~24.9% of variants per individual, including 124,728 small and 17,988 large variants for the GIAB HG002 'truth-set' TR benchmark. We demonstrate the utility of this pipeline across short-read and long-read technologies.

Genome sequences of 31 mycobacteriophages isolated on Mycobacterium smegmatis mc2155 at room temperature.

We report the genome sequences of 31 mycobacteriophages isolated on Mycobacterium smegmatis mc2155 at room temperature. The genomes add to the diversity of Clusters A, B, C, G, and K. Collectively, the genomes include 70 novel protein-coding genes that have no close relatives among the actinobacteriophages.

Benchmarking of small and large variants across tandem repeats.

Tandem repeats (TRs) are highly polymorphic in the human genome, have thousands of associated molecular traits, and are linked to over 60 disease phenotypes. However, their complexity often excludes them from at-scale studies due to challenges with variant calling, representation, and lack of a genome-wide standard. To promote TR methods development, we create a comprehensive catalog of TR regions and explore its properties across 86 samples. We then curate variants from the GIAB HG002 individual to create a tandem repeat benchmark. We also present a variant comparison method that handles small and large alleles and varying allelic representation. The 8.1% of the genome covered by the TR catalog holds ∼24.9% of variants per individual, including 124,728 small and 17,988 large variants for the GIAB HG002 TR benchmark. We work with the GIAB community to demonstrate the utility of this benchmark across short and long read technologies.

Social isolation shortens lifespan through oxidative stress in ants.

Social isolation negatively affects health, induces detrimental behaviors, and shortens lifespan in social species. Little is known about the mechanisms underpinning these effects because model species are typically short-lived and non-social. Using colonies of the carpenter ant Camponotus fellah, we show that social isolation induces hyperactivity, alters space-use, and reduces lifespan via changes in the expression of genes with key roles in oxidation-reduction and an associated accumulation of reactive oxygen species. These physiological effects are localized to the fat body and oenocytes, which perform liver-like functions in insects. We use pharmacological manipulations to demonstrate that the oxidation-reduction pathway causally underpins the detrimental effects of social isolation on behavior and lifespan. These findings have important implications for our understanding of how social isolation affects behavior and lifespan in general.

Social network position is a major predictor of ant behavior, microbiota composition, and brain gene expression.

The physiology and behavior of social organisms correlate with their social environments. However, because social environments are typically confounded by age and physical environments (i.e., spatial location and associated abiotic factors), these correlations are usually difficult to interpret. For example, associations between an individual's social environment and its gene expression patterns may result from both factors being driven by age or behavior. Simultaneous measurement of pertinent variables and quantification of the correlations between these variables can indicate whether relationships are direct (and possibly causal) or indirect. Here, we combine demographic and automated behavioral tracking with a multiomic approach to dissect the correlation structure among the social and physical environment, age, behavior, brain gene expression, and microbiota composition in the carpenter ant Camponotus fellah. Variations in physiology and behavior were most strongly correlated with the social environment. Moreover, seemingly strong correlations between brain gene expression and microbiota composition, physical environment, age, and behavior became weak when controlling for the social environment. Consistent with this, a machine learning analysis revealed that from brain gene expression data, an individual's social environment can be more accurately predicted than any other behavioral metric. These results indicate that social environment is a key regulator of behavior and physiology.

A caste differentiation mutant elucidates the evolution of socially parasitic ants.

Most ant species have two distinct female castes-queens and workers-yet the developmental and genetic mechanisms that produce these alternative phenotypes remain poorly understood. Working with a clonal ant, we discovered a variant strain that expresses queen-like traits in individuals that would normally become workers. The variants show changes in morphology, behavior, and fitness that cause them to rely on workers in wild-type (WT) colonies for survival. Overall, they resemble the queens of many obligately parasitic ants that have evolutionarily lost the worker caste and live inside colonies of closely related hosts. The prevailing theory for the evolution of these workerless social parasites is that they evolve from reproductively isolated populations of facultative intermediates that acquire parasitic phenotypes in a stepwise fashion. However, empirical evidence for such facultative ancestors remains weak, and it is unclear how reproductive isolation could gradually arise in sympatry. In contrast, we isolated these variants just a few generations after they arose within their WT parent colony, implying that the complex phenotype reported here was induced in a single genetic step. This suggests that a single genetic module can decouple the coordinated mechanisms of caste development, allowing an obligately parasitic variant to arise directly from a free-living ancestor. Consistent with this hypothesis, the variants have lost one of the two alleles of a putative supergene that is heterozygous in WTs. These findings provide a plausible explanation for the evolution of ant social parasites and implicate new candidate molecular mechanisms for ant caste differentiation.

Phased nanopore assembly with Shasta and modular graph phasing with GFAse.

As a step towards simplifying and reducing the cost of haplotype resolved de novo assembly, we describe new methods for accurately phasing nanopore data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of Oxford Nanopore Technologies' (ONT) PromethION sequencing, including those using proximity ligation and show that newer, higher accuracy ONT reads substantially improve assembly quality.

The genomic basis of army ant chemosensory adaptations.

The evolution of mass raiding has allowed army ants to become dominant arthropod predators in the tropics. Although a century of research has led to many discoveries about behavioural, morphological and physiological adaptations in army ants, almost nothing is known about the molecular basis of army ant biology. Here we report the genome of the iconic New World army ant Eciton burchellii, and show that it is unusually compact, with a reduced gene complement relative to other ants. In contrast to this overall reduction, a particular gene subfamily (9-exon ORs) expressed predominantly in female antennae is expanded. This subfamily has previously been linked to the recognition of hydrocarbons, key olfactory cues used in insect communication and prey discrimination. Confocal microscopy of the brain showed a corresponding expansion in a putative hydrocarbon response centre within the antennal lobe, while scanning electron microscopy of the antenna revealed a particularly high density of hydrocarbon-sensitive sensory hairs. E. burchellii shares these features with its predatory and more cryptic relative, the clonal raider ant. By integrating genomic, transcriptomic and anatomical analyses in a comparative context, our work thus provides evidence that army ants and their relatives possess a suite of modifications in the chemosensory system that may be involved in behavioural coordination and prey selection during social predation. It also lays the groundwork for future studies of army ant biology at the molecular level.

Comparative Development of the Ant Chemosensory System.

The insect antennal lobe (AL) contains the first synapses of the olfactory system, where olfactory sensory neurons (OSNs) contact second-order projection neurons (PNs). In Drosophila melanogaster, OSNs expressing specific receptor genes send stereotyped projections to one or two of about 50 morphologically defined glomeruli [1-3]. The mechanisms for this precise matching between OSNs and PNs have been studied extensively in D. melanogaster, where development is deterministic and independent of neural activity [4-6]. However, a number of insect lineages, most notably the ants, have receptor gene repertoires many times larger than D. melanogaster and exhibit more structurally complex antennal lobes [7-12]. Moreover, perturbation of OSN function via knockout of the odorant receptor (OR) co-receptor, Orco, results in drastic AL reductions in ants [13, 14], but not in Drosophila [15]. Here, we characterize AL development in the clonal raider ant, Ooceraea biroi. We find that, unlike in Drosophila, ORs and Orco are expressed before the onset of glomerulus formation, and Orco protein is trafficked to developing axon terminals, raising the possibility that ORs play a role during ant AL development. Additionally, ablating ant antennae at the onset of pupation results in AL defects that recapitulate the Orco mutant phenotype. Thus, early loss of functional OSN innervation reveals latent structure in the AL that develops independently of peripheral input, suggesting that the AL is initially pre-patterned and later refined in an OSN-dependent manner. This two-step process might increase developmental flexibility and thereby facilitate the rapid evolution and expansion of the ant chemosensory system.

Complete, high-quality genomes from long-read metagenomic sequencing of two wolf lichen thalli reveals enigmatic genome architecture.

Fungal genomes display incredible levels of complexity and diversity, and are exceptional study systems for genome evolution. Here we used the Oxford Nanopore MinION sequencing platform to generate high-quality fungal genomes from complex metagenomic samples of lichen thalli. We sequenced two wolf lichens using one flow cell per sample, generating 17.1 Gbps for Letharia lupina and 14.3 Gbps for Letharia columbiana. The resulting L. lupina genome is one of the most contiguous lichen genomes available to date, with 49.2 Mbp contained on 31 contigs. The L. columbiana genome, while less contiguous, is still relatively high quality, with 52.3 Mbp on a total of 161 contigs. Each thallus for both species contained multiple distinct haplotypes, a phenomenon that has rarely been empirically demonstrated. The Oxford Nanopore sequencing technologies are robust and effective when applied to complex symbioses, and have the potential to fundamentally transform our understanding of fungal genetics.

Globally invasive populations of the clonal raider ant are derived from Bangladesh.

Identifying the native range of invasive species is useful to understand their evolution and natural history, as well as to develop new methods to control potentially harmful introduced organisms. The clonal raider ant, Ooceraea biroi, is an introduced species and an increasingly important social insect model organism, but its native range remains unknown. Here, we report a new series of O. biroi collections from Bangladesh, Singapore, Vietnam and China. We use a molecular phylogeny constructed with five gene fragments from 27 samples to determine that invasive lineages of O. biroi originated in Bangladesh. These lineages may have spread from Bangladesh via the historically significant Bay of Bengal shipping ports. Ooceraea biroi shares multiple features of its biology with other introduced ants, including parthenogenesis, retention of heterozygosity and presence of multiple egg-layers in the colony. Using laboratory rearing and microsatellite markers, we show that colonies collected from disturbed habitat in Bangladesh have these traits in common with colonies from the invasive range. Ancestral populations with sexual reproduction in primary habitats either remain to be discovered or have gone extinct. Our findings advance our understanding of the global spread of the clonal raider ant and highlight a suite of general traits that make certain ants prone to becoming invasive.

Autonomic cardiac regulation facilitates acute heat tolerance in rainbow trout: in situ and in vivo support.

Acute warming in fish increases heart rate (fH) and cardiac output to peak values, after which performance plateaus or declines and arrhythmia may occur. This cardiac response can place a convective limitation on systemic oxygen delivery at high temperatures. To test the hypothesis that autonomic cardiac regulation protects cardiac performance in rainbow trout during acute warming, we investigated adrenergic and cholinergic regulation during the onset and progression of cardiac limitations. We explored the direct effects of adrenergic stimulation by acutely warming an in situ working perfused heart until arrhythmia occurred, cooling the heart to restore rhythmicity and rewarming with increasing adrenergic stimulation. Adrenergic stimulation produced a clear, dose-dependent increase in the temperature and peak fH achieved prior to the onset of arrhythmia. To examine how this adrenergic protection functions in conjunction with cholinergic vagal inhibition in vivo, rainbow trout fitted with ECG electrodes were acutely warmed in a respirometer until they lost equilibrium (CTmax) with and without muscarinic (atropine) and ß-adrenergic (sotalol) antagonists. Trout exhibited roughly equal and opposing cholinergic and adrenergic tone on fH that persisted up to critical temperatures. ß-Adrenergic blockade significantly lowered peak fH by 14-17%, while muscarinic blockade significantly lowered the temperature for peak fH by 2.0°C. Moreover, muscarinic and ß-adrenergic blockers injected individually or together significantly reduced CTmax by up to 3°C, indicating for the first time that cardiac adrenergic stimulation and cholinergic inhibition can enhance acute heat tolerance in rainbow trout at the level of the heart and the whole animal.

The genomic architecture and molecular evolution of ant odorant receptors.

The massive expansions of odorant receptor (OR) genes in ant genomes are notable examples of rapid genome evolution and adaptive gene duplication. However, the molecular mechanisms leading to gene family expansion remain poorly understood, partly because available ant genomes are fragmentary. Here, we present a highly contiguous, chromosome-level assembly of the clonal raider ant genome, revealing the largest known OR repertoire in an insect. While most ant ORs originate via local tandem duplication, we also observe several cases of dispersed duplication followed by tandem duplication in the most rapidly evolving OR clades. We found that areas of unusually high transposable element density (TE islands) were depauperate in ORs in the clonal raider ant, and found no evidence for retrotransposition of ORs. However, OR loci were enriched for transposons relative to the genome as a whole, potentially facilitating tandem duplication by unequal crossing over. We also found that ant OR genes are highly AT-rich compared to other genes. In contrast, in flies, OR genes are dispersed and largely isolated within the genome, and we find that fly ORs are not AT-rich. The genomic architecture and composition of ant ORs thus show convergence with the unrelated vertebrate ORs rather than the related fly ORs. This might be related to the greater gene numbers and/or potential similarities in gene regulation between ants and vertebrates as compared to flies.

First genome-wide analysis of the endangered, endemic lichen Cetradonia linearis reveals isolation by distance and strong population structure.

PREMISE OF THE STUDY: Lichenized fungi are evolutionarily diverse and ecologically important, but little is known about the processes that drive their diversification and genetic differentiation. Distributions are often assumed to be wholly shaped by ecological requirements rather than dispersal limitations. Furthermore, although asexual and sexual reproductive structures are observable, the lack of information about recombination rates makes inferences about reproductive strategies difficult. We investigated the population genomics of Cetradonia linearis, a federally endangered lichen in the southern Appalachians of eastern North America, to test the relative contributions of environmental and geographic distance in shaping genetic structure, and to characterize the mating system and genome-wide recombination. METHODS: Whole-genome shotgun sequencing was conducted to generate data for 32 individuals of C. linearis. A reference genome was assembled, and reads from all samples were aligned to generate a set of single-nucleotide polymorphisms for further analyses. KEY RESULTS: We found evidence for low rates of recombination and for isolation by distance, but not for isolation by environment. The species is putatively unisexual, given that only one mating-type locus was found. Hindcast species distribution models and the distribution of genetic diversity support C. linearis having a larger range during the Last Glacial Maximum in the southern portion of its current extent. CONCLUSIONS: Our findings contribute to the understanding of factors that shape genetic diversity in C. linearis and in fungi more broadly. Because all populations are highly genetically differentiated, the extirpation of any population would mean the loss of unique genetic diversity; therefore, our results support the continued conservation of this species.

DNA barcoding Brooklyn (New York): A first assessment of biodiversity in Marine Park by citizen scientists.

DNA barcoding is both an important research and science education tool. The technique allows for quick and accurate species identification using only minimal amounts of tissue samples taken from any organism at any developmental phase. DNA barcoding has many practical applications including furthering the study of taxonomy and monitoring biodiversity. In addition to these uses, DNA barcoding is a powerful tool to empower, engage, and educate students in the scientific method while conducting productive and creative research. The study presented here provides the first assessment of Marine Park (Brooklyn, New York, USA) biodiversity using DNA barcoding. New York City citizen scientists (high school students and their teachers) were trained to identify species using DNA barcoding during a two-week long institute. By performing NCBI GenBank BLAST searches, students taxonomically identified 187 samples (1 fungus, 70 animals and 116 plants) and also published 12 novel DNA barcodes on GenBank. Students also identified 7 ant species and demonstrated the potential of DNA barcoding for identification of this especially diverse group when coupled with traditional taxonomy using morphology. Here we outline how DNA barcoding allows citizen scientists to make preliminary taxonomic identifications and contribute to modern biodiversity research.

Social regulation of insulin signaling and the evolution of eusociality in ants.

Queens and workers of eusocial Hymenoptera are considered homologous to the reproductive and brood care phases of an ancestral subsocial life cycle. However, the molecular mechanisms underlying the evolution of reproductive division of labor remain obscure. Using a brain transcriptomics screen, we identified a single gene, insulin-like peptide 2 (ilp2), which is always up-regulated in ant reproductives, likely because they are better nourished than their nonreproductive nestmates. In clonal raider ants (Ooceraea biroi), larval signals inhibit adult reproduction by suppressing ilp2, thus producing a colony reproductive cycle reminiscent of ancestral subsociality. However, increasing ILP2 peptide levels overrides larval suppression, thereby breaking the colony cycle and inducing a stable division of labor. These findings suggest a simple model for the origin of ant eusociality via nutritionally determined reproductive asymmetries potentially amplified by larval signals.

orco Mutagenesis Causes Loss of Antennal Lobe Glomeruli and Impaired Social Behavior in Ants.

Life inside ant colonies is orchestrated with diverse pheromones, but it is not clear how ants perceive these social signals. It has been proposed that pheromone perception in ants evolved via expansions in the numbers of odorant receptors (ORs) and antennal lobe glomeruli. Here, we generate the first mutant lines in the clonal raider ant, Ooceraea biroi, by disrupting orco, a gene required for the function of all ORs. We find that orco mutants exhibit severe deficiencies in social behavior and fitness, suggesting they are unable to perceive pheromones. Surprisingly, unlike in Drosophila melanogaster, orco mutant ants also lack most of the ∼500 antennal lobe glomeruli found in wild-type ants. These results illustrate that ORs are essential for ant social organization and raise the possibility that, similar to mammals, receptor function is required for the development and/or maintenance of the highly complex olfactory processing areas in the ant brain. VIDEO ABSTRACT.

Transcriptomics and neuroanatomy of the clonal raider ant implicate an expanded clade of odorant receptors in chemical communication.

A major aim of sociogenomic research is to uncover common principles in the molecular evolution of sociality. This endeavor has been hampered by the small number of specific genes currently known to function in social behavior. Here we provide several lines of evidence suggesting that ants have evolved a large and novel clade of odorant receptor (OR) genes to perceive hydrocarbon-based pheromones, arguably the most important signals in ant communication. This genomic expansion is also mirrored in the ant brain via a corresponding expansion of a specific cluster of glomeruli in the antennal lobe. We show that in the clonal raider ant, hydrocarbon-sensitive basiconic sensilla are found only on the ventral surface of the female antennal club. Correspondingly, nearly all genes in a clade of 180 ORs within the 9-exon subfamily of ORs are expressed exclusively in females and are highly enriched in expression in the ventral half of the antennal club. Furthermore, we found that across species and sexes, the number of 9-exon ORs expressed in antennae is tightly correlated with the number of glomeruli in the antennal lobe region innervated by odorant receptor neurons from basiconic sensilla. Evolutionary analyses show that this clade underwent a striking gene expansion in the ancestors of all ants and slower but continued expansion in extant ant lineages. This evidence suggests that ants have evolved a large clade of genes to support pheromone perception and that gene duplications have played an important role in the molecular evolution of ant communication.

The Vivir Mejor! Consortium: A Rural, US-México Collaborative Model to Prevent and Treat Diabetes.

The Vivir Mejor! (Live Better!) System of Diabetes Prevention and Care Consortium is a multi-sector partnership to establish an integrated diabetes system of care in Santa Cruz County, Arizona on the U.S.-Mexico border. Major outcomes include improved healthy eating and active living knowledge and behaviors and lowered HbA1c.