Genomic variation in montane bumblebees in Scandinavia: High levels of intraspecific diversity despite population vulnerability.

Populations of many bumblebee species are declining, with distributions shifting northwards to track suitable climates. Climate change is considered a major contributing factor. Arctic species are particularly vulnerable as they cannot shift further north, making assessment of their population viability important. Analysis of levels of whole-genome variation is a powerful way to analyse population declines and fragmentation. Here, we use genome sequencing to analyse genetic variation in seven species of bumblebee from the Scandinavian mountains, including two classified as vulnerable. We sequenced 333 samples from across the ranges of these species in Sweden. Estimates of effective population size (NE ) vary from ~55,000 for species with restricted high alpine distributions to 220,000 for more widespread species. Population fragmentation is generally very low or undetectable over large distances in the mountains, suggesting an absence of barriers to gene flow. The relatively high NE and low population structure indicate that none of the species are at immediate risk of negative genetic effects caused by high levels of genetic drift. However, reconstruction of historical fluctuations in NE indicates that the arctic specialist species Bombus hyperboreus has experienced population declines since the last ice age and we detected one highly inbred diploid male of this species close to the southern limit of its range, potentially indicating elevated genetic load. Although the levels of genetic variation in montane bumblebee populations are currently relatively high, their ranges are predicted to shrink drastically due to the effects of climate change and monitoring is essential to detect future population declines.

MU variability in CBCT-guided online adaptive radiation therapy.

PURPOSE: CBCT-guided online-adaptive radiotherapy (oART) systems have been made possible by using artificial intelligence and automation to substantially reduce treatment planning time during on-couch adaptive sessions. Evaluating plans generated during an adaptive session presents significant challenges to the clinical team as the planning process gets compressed into a shorter window than offline planning. We identified MU variations up to 30% difference between the adaptive plan and the reference plan in several oART sessions that caused the clinical team to question the accuracy of the oART dose calculation. We investigated the cause of MU variation and the overall accuracy of the dose delivered when MU variations appear unnecessarily large. METHODS: Dosimetric and adaptive plan data from 604 adaptive sessions of 19 patients undergoing CBCT-guided oART were collected. The analysis included total MU per fraction, planning target volume (PTV) and organs at risk (OAR) volumes, changes in PTV-OAR overlap, and DVH curves. Sessions with MU greater than two standard deviations from the mean were reoptimized offline, verified by an independent calculation system, and measured using a detector array. RESULTS: MU variations relative to the reference plan were normally distributed with a mean of -1.0% and a standard deviation of 11.0%. No significant correlation was found between MU variation and anatomic changes. Offline reoptimization did not reliably reproduce either reference or on-couch total MUs, suggesting that stochastic effects within the oART optimizer are likely causing the variations. Independent dose calculation and detector array measurements resulted in acceptable agreement with the planned dose. CONCLUSIONS: MU variations observed between oART plans were not caused by any errors within the oART workflow. Providers should refrain from using MU variability as a way to express their confidence in the treatment planning accuracy. Clinical decisions during on-couch adaptive sessions should rely on validated secondary dose calculations to ensure optimal plan selection.

Screening Incarcerated Women for Opioid Use Disorder.

The overall aim of the present study is to examine the utility of the DSM OUD Checklist and the NM-ASSIST screening tools to identify symptoms consistent with OUD among incarcerated women in county jails. This study contributes to the existing literature because research on screening and assessment approaches for incarcerated women has been limited. The focus of the current study is to describe the screening procedures and study recruitment for a larger parent study focused on increasing treatment linkages. Study findings indicate a positive correlation between indicators of OUD using the two screening tools, as well as a high degree of correlation between street opioid misuse and other high-risk drug indicators (overdose and injection practices). These findings underscore the importance of outreach, screening, and intervention in real-world settings, including jails, in order to increase access to OUD treatment among this vulnerable sample of women.

A quick guide on implementing and quality assuring 3D printing in radiation oncology.

As three-dimensional (3D) printing becomes increasingly common in radiation oncology, proper implementation, usage, and ongoing quality assurance (QA) are essential. While there have been many reports on various clinical investigations and several review articles, there is a lack of literature on the general considerations of implementing 3D printing in radiation oncology departments, including comprehensive process establishment and proper ongoing QA. This review aims to guide radiation oncology departments in effectively using 3D printing technology for routine clinical applications and future developments. We attempt to provide recommendations on 3D printing equipment, software, workflow, and QA, based on existing literature and our experience. Specifically, we focus on three main applications: patient-specific bolus, high-dose-rate (HDR) surface brachytherapy applicators, and phantoms. Additionally, cost considerations are briefly discussed. This review focuses on point-of-care (POC) printing in house, and briefly touches on outsourcing printing via mail-order services.

Influence of air mapping errors on the dosimetric accuracy of prostate CBCT-guided online adaptive radiation therapy.

PURPOSE: CBCT-guided online adaptive radiotherapy (oART) plans presently utilize daily synthetic CTs (sCT) that are automatically generated using deformable registration algorithms. These algorithms may have poor performance at reproducing variable volumes of gas present during treatment. Therefore, we have analyzed the air mapping error between the daily CBCTs and the corresponding sCT and explored its dosimetric effect on oART plan calculation. METHODS: Abdominopelvic air volume was contoured on both the daily CBCT images and the corresponding synthetic images for 207 online adaptive pelvic treatments. Air mapping errors were tracked over all fractions. For two case studies representing worst case scenarios, dosimetric effects of air mapping errors were corrected in the sCT images using the daily CBCT air contours, then recalculating dose. Dose volume histogram statistics and 3D gamma passing rates were used to compare the original and air-corrected sCT-based dose calculations. RESULTS: All analyzed patients showed observable air pocket contour differences between the sCT and the CBCT images. The largest air volume difference observed in daily CBCT images for a given patient was 276.3 cc, a difference of more than 386% compared to the sCT. For the two case studies, the largest observed change in DVH metrics was a 2.6% reduction in minimum PTV dose, with all other metrics varying by less than 1.5%. 3D gamma passing rates using 1%/1 mm criteria were above 90% when comparing the uncorrected and corrected dose distributions. CONCLUSION: Current CBCT-based oART workflow can lead to inaccuracies in the mapping of abdominopelvic air pockets from daily CBCT to the sCT images used for the optimization and calculation of the adaptive plan. Despite the large observed mapping errors, the dosimetric effects of such differences on the accuracy of the adapted plan dose calculation are unlikely to cause differences greater than 3% for prostate treatments.

Genetic Barriers to Historical Gene Flow between Cryptic Species of Alpine Bumblebees Revealed by Comparative Population Genomics.

Evidence is accumulating that gene flow commonly occurs between recently diverged species, despite the existence of barriers to gene flow in their genomes. However, we still know little about what regions of the genome become barriers to gene flow and how such barriers form. Here, we compare genetic differentiation across the genomes of bumblebee species living in sympatry and allopatry to reveal the potential impact of gene flow during species divergence and uncover genetic barrier loci. We first compared the genomes of the alpine bumblebee Bombus sylvicola and a previously unidentified sister species living in sympatry in the Rocky Mountains, revealing prominent islands of elevated genetic divergence in the genome that colocalize with centromeres and regions of low recombination. This same pattern is observed between the genomes of another pair of closely related species living in allopatry (B. bifarius and B. vancouverensis). Strikingly however, the genomic islands exhibit significantly elevated absolute divergence (dXY) in the sympatric, but not the allopatric, comparison indicating that they contain loci that have acted as barriers to historical gene flow in sympatry. Our results suggest that intrinsic barriers to gene flow between species may often accumulate in regions of low recombination and near centromeres through processes such as genetic hitchhiking, and that divergence in these regions is accentuated in the presence of gene flow.

A genomic and morphometric analysis of alpine bumblebees: Ongoing reductions in tongue length but no clear genetic component.

Over the last six decades, populations of the bumblebees Bombus sylvicola and Bombus balteatus in Colorado have experienced decreases in tongue length, a trait important for plant-pollinator mutualisms. It has been hypothesized that this observation reflects selection resulting from shifts in floral composition under climate change. Here we used morphometrics and population genomics to determine whether morphological change is ongoing, investigate the genetic basis of morphological variation, and analyse population structure in these populations. We generated a genome assembly of B. balteatus. We then analysed whole-genome sequencing data and morphometric measurements of 580 samples of both species from seven high-altitude localities. Out of 281 samples originally identified as B. sylvicola, 67 formed a separate genetic cluster comprising a newly-discovered cryptic species ("incognitus"). However, an absence of genetic structure within species suggests that gene flow is common between mountains. We found a significant decrease in tongue length between bees collected between 2012-2014 and in 2017, indicating that morphological shifts are ongoing. We did not discover any genetic associations with tongue length, but a SNP related to production of a proteolytic digestive enzyme was implicated in body size variation. We identified evidence of covariance between kinship and both tongue length and body size, which is suggestive of a genetic component of these traits, although it is possible that shared environmental effects between colonies are responsible. Our results provide evidence for ongoing modification of a morphological trait important for pollination and indicate that this trait probably has a complex genetic and environmental basis.

Phylogenomics and evolutionary history of Oreobates (Anura: Craugastoridae) Neotropical frogs along elevational gradients.

Mountain ranges offer opportunities for understanding how species evolved and diversified across different environmental conditions. Neotropical frogs of the genus Oreobates (Anura: Craugastoridae) are adapted to highland and lowland habitats along the Andes, but many aspects of their evolution remain unknown. We studied their evolutionary history using ~18,000 exons enriched by targeted sequence-capture. Since capture success was very variable across samples, we evaluated to what degree differing data filtering produced robust inferences. The inferred evolutionary framework evidenced phylogenetic discordances among lowland species that can be explained by taxonomic misidentification or admixture of ancestral lineages. Highland species showed smaller effective populations than lowland frogs, probably due to greater habitat fragmentation in montane environments. Stronger genetic drift likely decreased the power of purifying selection and led to an increased proportion of nonsynonymous mutations in highland populations that could play an important role in their adaptation. Overall, our work sheds light on the evolutionary history and diversification of this group of Neotropical frogs along elevational gradients in the Andes as well as on their patterns of intraspecific diversity.

Informed breeding dispersal following stochastic changes to patch quality in a pond-breeding amphibian.

The unidirectional movement of animals between breeding patches (i.e. breeding dispersal) has profound implications for the ecological and evolutionary dynamics of spatially structured populations. In spatiotemporally variable environments, individuals are expected to adjust their dispersal decisions according to information gathered on the environmental and/or social cues that reflect the fitness prospects in a given breeding patch (i.e. informed dispersal). A paucity of empirical work limited our understanding of the ability of animals to depart from low-quality breeding patches and settle in high-quality breeding patches. We examined the capacity of individuals to respond to stochastic changes in habitat quality via informed breeding dispersal in a pond-breeding amphibian. We conducted a 5-year (2015-2019) capture-recapture study of boreal toads Anaxyrus boreas boreas (n = 1,100) that breed in beaver ponds in western Wyoming, USA. During early spring of 2017, an extreme flooding event destroyed several beaver dams and resulted in the loss of breeding habitat. We used multi-state models to investigate how temporal changes in pond characteristics influenced breeding dispersal, and determine whether movement decisions were in accordance with prospects for reproductive fitness. Boreal toads more often departed from low-quality breeding ponds (without successful metamorphosis) and settled in high-quality breeding ponds (with successful metamorphosis). Movement decisions were context-dependent and associated with pond characteristics altered by beaver dam destruction. Individuals were more likely to depart from shallow ponds with high vegetation cover and settle in deep ponds with low vegetation cover. The probability of metamorphosis was related to the same environmental cues, suggesting that boreal toads assess the fitness prospects of a breeding patch and adjust movement decisions accordingly (i.e. informed breeding dispersal). We demonstrated that stochastic variability in environmental conditions and habitat quality can underpin dispersal behaviour in amphibians. Our study highlighted the mechanistic linkages between habitat change, movement behaviour and prospects for reproductive performance, which is critical for understanding how wild animals respond to rapid environmental change.

Evolution of Darwin's finches and their beaks revealed by genome sequencing.

Darwin's finches, inhabiting the Galápagos archipelago and Cocos Island, constitute an iconic model for studies of speciation and adaptive evolution. Here we report the results of whole-genome re-sequencing of 120 individuals representing all of the Darwin's finch species and two close relatives. Phylogenetic analysis reveals important discrepancies with the phenotype-based taxonomy. We find extensive evidence for interspecific gene flow throughout the radiation. Hybridization has given rise to species of mixed ancestry. A 240 kilobase haplotype encompassing the ALX1 gene that encodes a transcription factor affecting craniofacial development is strongly associated with beak shape diversity across Darwin's finch species as well as within the medium ground finch (Geospiza fortis), a species that has undergone rapid evolution of beak shape in response to environmental changes. The ALX1 haplotype has contributed to diversification of beak shapes among the Darwin's finches and, thereby, to an expanded utilization of food resources.

New dosimetric guidelines for linear Boltzmann transport equations through comparative evaluation of stereotactic body radiation therapy for lung treatment planning.

PURPOSE: To propose guidelines for lung stereotactic body radiation therapy (SBRT) when using Acuros XB (AXB) equivalent to the existing ones developed for convolution algorithms such as analytic anisotropic algorithm (AAA), considering the difference between the algorithms. METHODS: A retrospective analysis was performed on 30 lung patients previously treated with SBRT. The original AAA plans, which were developed using dynamic conformal arcs, were recalculated and then renormalized for planning target volume (PTV) coverage using AXB. The recalculated and renormalized plans were compared to the original plans based on V100% and V90% PTV coverage, as well as V105%, conformality index, D2cm , Rx/Dmax , R50, and Dmin . These metrics were analyzed nominally and on variations according to RTOG and NRG guidelines. Based on the relative difference between each metric in the AAA and AXB plans, new guidelines were developed. The relative differences in our cohort were compared to previously documented AAA to AXB comparisons found in the literature. RESULTS: AAA plans recalculated in AXB had a significant reduction in most dosimetric metrics. The most notable changes were in V100% (4%) and the conformality index (7.5%). To achieve equal PTV coverage, AXB required an average of 1.8% more monitor units (MU). This fits well with previously published data. Applying the new guidelines to the AXB plans significantly increased the number of minor violations with no change in major violations, making them comparable to those of the original AAA plans. CONCLUSION: The relative difference found between AAA and AXB for SBRT lung plans has been shown to be consistent with previous works. Based on these findings, new guidelines for lung SBRT are recommended when planning with AXB.

Social Parasitism in the Honeybee (Apis mellifera) Is Not Controlled by a Single SNP.

The Cape bee (Apis mellifera capensis) is a subspecies of the honeybee, in which workers commonly lay diploid unfertilized eggs via a process known as thelytoky. A recent study aimed to map the genetic basis of this trait in the progeny of a single capensis queen where workers laid either diploid (thelytokous) or haploid (arrhenotokous) eggs. A nonsynonymous single nucleotide polymorphism (SNP) in a gene of unknown function was reported to be strongly associated with thelytoky in this colony. Here, we analyze genome sequences from a global sample of A. mellifera and identify populations where the proposed thelytoky allele at this SNP is common but thelytoky is absent. We also analyze genome sequences of three capensis queens produced by thelytoky and find that, contrary to predictions, they do not carry the proposed thelytoky allele. The proposed SNP is therefore neither sufficient nor required to produce thelytoky in A. mellifera.

Extreme Differences in Recombination Rate between the Genomes of a Solitary and a Social Bee.

Social insect genomes exhibit the highest rates of crossing over observed in plants and animals. The evolutionary causes of these extreme rates are unknown. Insight can be gained by comparing recombination rate variation across the genomes of related social and solitary insects. Here, we compare the genomic recombination landscape of the highly social honey bee, Apis mellifera, with the solitary alfalfa leafcutter bee, Megachile rotundata, by analyzing patterns of linkage disequilibrium in population-scale genome sequencing data. We infer that average recombination rates are extremely elevated in A. mellifera compared with M. rotundata. However, our results indicate that similar factors control the distribution of crossovers in the genomes of both species. Recombination rate is significantly reduced in coding regions in both species, with genes inferred to be germline methylated having particularly low rates. Genes with worker-biased patterns of expression in A. mellifera and their orthologs in M. rotundata have higher than average recombination rates in both species, suggesting that selection for higher diversity in genes involved in worker caste functions in social taxa is not the explanation for these elevated rates. Furthermore, we find no evidence that recombination has modulated the efficacy of selection among genes during bee evolution, which does not support the hypothesis that high recombination rates facilitated positive selection for new functions in social insects. Our results indicate that the evolution of sociality in insects likely entailed selection on modifiers that increased recombination rates genome wide, but that the genomic recombination landscape is determined by the same factors.

Gene flow, ancient polymorphism, and ecological adaptation shape the genomic landscape of divergence among Darwin's finches.

Genomic comparisons of closely related species have identified "islands" of locally elevated sequence divergence. Genomic islands may contain functional variants involved in local adaptation or reproductive isolation and may therefore play an important role in the speciation process. However, genomic islands can also arise through evolutionary processes unrelated to speciation, and examination of their properties can illuminate how new species evolve. Here, we performed scans for regions of high relative divergence (FST) in 12 species pairs of Darwin's finches at different genetic distances. In each pair, we identify genomic islands that are, on average, elevated in both relative divergence (FST) and absolute divergence (dXY). This signal indicates that haplotypes within these genomic regions became isolated from each other earlier than the rest of the genome. Interestingly, similar numbers of genomic islands of elevated dXY are observed in sympatric and allopatric species pairs, suggesting that recent gene flow is not a major factor in their formation. We find that two of the most pronounced genomic islands contain the ALX1 and HMGA2 loci, which are associated with variation in beak shape and size, respectively, suggesting that they are involved in ecological adaptation. A subset of genomic island regions, including these loci, appears to represent anciently diverged haplotypes that evolved early during the radiation of Darwin's finches. Comparative genomics data indicate that these loci, and genomic islands in general, have exceptionally low recombination rates, which may play a role in their establishment.

DNA-Compatible Copper-Catalyzed Oxidative Amidation of Aldehydes with Non-Nucleophilic Arylamines.

We report a DNA-compatible protocol for synthesizing amides from DNA-bound aldehydes and non-nucleophilic arylamines including aza-substituted anilines, 2-aminobenzimidazoles, and 3-aminopyrazoles. The reactions were carried out at room temperature and provided reasonable conversions and wide functional group compatibility. The reactions were also successful when employing aryl and aliphatic aldehydes. In addition, qPCR and NGS data suggested no negative impact on DNA integrity after the copper-mediated oxidative amidation reaction.

Assembly-line synthesis of organic molecules with tailored shapes.

Molecular 'assembly lines', in which organic molecules undergo iterative processes such as chain elongation and functional group manipulation, are found in many natural systems, including polyketide biosynthesis. Here we report the creation of such an assembly line using the iterative, reagent-controlled homologation of a boronic ester. This process relies on the reactivity of α-lithioethyl tri-isopropylbenzoate, which inserts into carbon-boron bonds with exceptionally high fidelity and stereocontrol; each chain-extension step generates a new boronic ester, which is immediately ready for further homologation. We used this method to generate organic molecules that contain ten contiguous, stereochemically defined methyl groups. Several stereoisomers were synthesized and shown to adopt different shapes-helical or linear-depending on the stereochemistry of the methyl groups. This work should facilitate the rational design of molecules with predictable shapes, which could have an impact in areas of molecular sciences in which bespoke molecules are required.

Two extended haplotype blocks are associated with adaptation to high altitude habitats in East African honey bees.

Understanding the genetic basis of adaption is a central task in biology. Populations of the honey bee Apis mellifera that inhabit the mountain forests of East Africa differ in behavior and morphology from those inhabiting the surrounding lowland savannahs, which likely reflects adaptation to these habitats. We performed whole genome sequencing on 39 samples of highland and lowland bees from two pairs of populations to determine their evolutionary affinities and identify the genetic basis of these putative adaptations. We find that in general, levels of genetic differentiation between highland and lowland populations are very low, consistent with them being a single panmictic population. However, we identify two loci on chromosomes 7 and 9, each several hundred kilobases in length, which exhibit near fixation for different haplotypes between highland and lowland populations. The highland haplotypes at these loci are extremely rare in samples from the rest of the world. Patterns of segregation of genetic variants suggest that recombination between haplotypes at each locus is suppressed, indicating that they comprise independent structural variants. The haplotype on chromosome 7 harbors nearly all octopamine receptor genes in the honey bee genome. These have a role in learning and foraging behavior in honey bees and are strong candidates for adaptation to highland habitats. Molecular analysis of a putative breakpoint indicates that it may disrupt the coding sequence of one of these genes. Divergence between the highland and lowland haplotypes at both loci is extremely high suggesting that they are ancient balanced polymorphisms that greatly predate divergence between the extant honey bee subspecies.

A hybrid de novo genome assembly of the honeybee, Apis mellifera, with chromosome-length scaffolds.

BACKGROUND: The ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map. RESULTS: Each of the assembly steps reduced the number of gaps and incorporated a substantial amount of additional sequence into scaffolds. The new assembly (Amel_HAv3) is significantly more contiguous and complete than the previous one (Amel_4.5), based mainly on Sanger sequencing reads. N50 of contigs is 120-fold higher (5.381 Mbp compared to 0.053 Mbp) and we anchor > 98% of the sequence to chromosomes. All of the 16 chromosomes are represented as single scaffolds with an average of three sequence gaps per chromosome. The improvements are largely due to the inclusion of repetitive sequence that was unplaced in previous assemblies. In particular, our assembly is highly contiguous across centromeres and telomeres and includes hundreds of AvaI and AluI repeats associated with these features. CONCLUSIONS: The improved assembly will be of utility for refining gene models, studying genome function, mapping functional genetic variation, identification of structural variants, and comparative genomics.

Chromosomal inversions associated with environmental adaptation in honeybees.

Chromosomal inversions can facilitate local adaptation in the presence of gene flow by suppressing recombination between well-adapted native haplotypes and poorly adapted migrant haplotypes. East African mountain populations of the honeybee Apis mellifera are highly divergent from neighbouring lowland populations at two extended regions in the genome, despite high similarity in the rest of the genome, suggesting that these genomic regions harbour inversions governing local adaptation. Here, we utilize a new highly contiguous assembly of the honeybee genome to characterize these regions. Using whole-genome sequencing data from 55 highland and lowland bees, we find that the highland haplotypes at both regions are present at high frequencies in three independent highland populations but extremely rare elsewhere. The boundaries of both divergent regions are characterized by regions of high homology with each other positioned in opposite orientations and contain highly repetitive, long inverted repeats with homology to transposable elements. These regions are likely to represent inversion breakpoints that participate in nonallelic homologous recombination. Using long-read data, we confirm that the lowland samples are contiguous across breakpoint regions. We do not find evidence for disruption of functional sequence by these breakpoints, which suggests that the inversions are likely maintained due to their allelic content conferring local adaptation in highland environments. Finally, we identify a third divergent genomic region, which contains highly divergent segregating haplotypes that also may contain inversion variants under selection. The results add to a growing body of evidence indicating the importance of chromosomal inversions in local adaptation.

The genomic basis of adaptation to high-altitude habitats in the eastern honey bee (Apis cerana).

The eastern honey bee (Apis cerana) is of central importance for agriculture in Asia. It has adapted to a wide variety of environmental conditions across its native range in southern and eastern Asia, which includes high-altitude regions. eastern honey bees inhabiting mountains differ morphologically from neighbouring lowland populations and may also exhibit differences in physiology and behaviour. We compared the genomes of 60 eastern honey bees collected from high and low altitudes in Yunnan and Gansu provinces, China, to infer their evolutionary history and to identify candidate genes that may underlie adaptation to high altitude. Using a combination of FST -based statistics, long-range haplotype tests and population branch statistics, we identified several regions of the genome that appear to have been under positive selection. These candidate regions were strongly enriched for coding sequences and had high haplotype homozygosity and increased divergence specifically in highland bee populations, suggesting they have been subjected to recent selection in high-altitude habitats. Candidate loci in these genomic regions included genes related to reproduction and feeding behaviour in honey bees. Functional investigation of these candidate loci is necessary to fully understand the mechanisms of adaptation to high-altitude habitats in the eastern honey bee.