An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

The dominance and growth of shallow groundwater resources in continuous permafrost environments.

Water is a limited resource in Arctic watersheds with continuous permafrost because freezing conditions in winter and the impermeability of permafrost limit storage and connectivity between surface water and deep groundwater. However, groundwater can still be an important source of surface water in such settings, feeding springs and large aufeis fields that are abundant in cold regions and generating runoff when precipitation is rare. Whether groundwater is sourced from suprapermafrost taliks or deeper regional aquifers will impact water availability as the Arctic continues to warm and thaw. Previous research is ambiguous about the role of deep groundwater, leading to uncertainty regarding Arctic water availability and changing water resources. We analyzed chemistry and residence times of spring, stream, and river waters in the continuous permafrost zone of Alaska, spanning the mountains to the coastal plain. Water chemistry and age tracers show that surface waters are predominately sourced from recent precipitation and have short (<50 y) subsurface residence times. Remote sensing indicates trends in the areal extent of aufeis over the last 37 y, and correlations between aufeis extent and previous year summer temperature. Together, these data indicate that surface waters in continuous permafrost regions may be impacted by short flow paths and shallow suprapermafrost aquifers that are highly sensitive to climatic and hydrologic change over annual timescales. Despite the lack of connection to regional aquifers, continued warming and permafrost thaw may promote deepening of the shallow subsurface aquifers and creation of shallow taliks, providing some resilience to Arctic freshwater ecosystems.

Modulation of tooth regeneration through opposing responses to Wnt and BMP signals in teleosts.

Most vertebrate species undergo tooth replacement throughout adult life. This process is marked by the shedding of existing teeth and the regeneration of tooth organs. However, little is known about the genetic circuitry regulating tooth replacement. Here, we tested whether fish orthologs of genes known to regulate mammalian hair regeneration have effects on tooth replacement. Using two fish species that demonstrate distinct modes of tooth regeneration, threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio), we found that transgenic overexpression of four different genes changed tooth replacement rates in the direction predicted by a hair regeneration model: Wnt10a and Grem2a increased tooth replacement rate, whereas Bmp6 and Dkk2 strongly inhibited tooth formation. Thus, similar to known roles in hair regeneration, Wnt and BMP signals promote and inhibit regeneration, respectively. Regulation of total tooth number was separable from regulation of replacement rates. RNA sequencing of stickleback dental tissue showed that Bmp6 overexpression resulted in an upregulation of Wnt inhibitors. Together, these data support a model in which different epithelial organs, such as teeth and hair, share genetic circuitry driving organ regeneration.

A new approach to RNA synthesis: immobilization of stably and functionally co-tethered promoter DNA and T7 RNA polymerase.

Current approaches to RNA synthesis/manufacturing require substantial (and incomplete) purification post-synthesis. We have previously demonstrated the synthesis of RNA from a complex in which T7 RNA polymerase is tethered to promoter DNA. In the current work, we extend this approach to demonstrate an extremely stable system of functional co-tethered complex to a solid support. Using the system attached to magnetic beads, we carry out more than 20 rounds of synthesis using the initial polymerase-DNA construct. We further demonstrate the wide utility of this system in the synthesis of short RNA, a CRISPR guide RNA, and a protein-coding mRNA. In all cases, the generation of self-templated double stranded RNA (dsRNA) impurities are greatly reduced, by both the tethering itself and by the salt-tolerance that local co-tethering provides. Transfection of the mRNA into HEK293T cells shows a correlation between added salt in the transcription reaction (which inhibits RNA rebinding that generates RNA-templated extensions) and significantly increased expression and reduced innate immune stimulation by the mRNA reaction product. These results point in the direction of streamlined processes for synthesis/manufacturing of high-quality RNA of any length, and at greatly reduced costs.

High-salt transcription from enzymatically gapped promoters nets higher yields and purity of transcribed RNAs.

T7 RNA polymerase is commonly used to synthesize large quantities of RNA for a wide variety of applications, from basic science to mRNA therapeutics. This in vitro system, while showing high fidelity in many ways, is also well known for producing longer than encoded RNA products, particularly under high-yield reaction conditions. Specifically, the resulting product pool is contaminated by an often disperse collection of longer cis-primed extension products. In addition to reducing yield via the conversion of correctly encoded RNA to longer products, self-primed extension generates partially double-stranded RNAs that can trigger the innate immune response. Extensive and low-yield purifications are then required to produce therapeutic RNA. Under high-yield conditions, accumulating concentrations of RNA effectively compete with promoter DNA for polymerase binding, driving self-primed extension at the expense of correct initiation. In the current work, we introduce a simple and novel modification in the DNA to strengthen promoter binding, shifting the balance back toward promoter-driven synthesis and so dramatically reducing self-primed extension. The result is higher yield of the encoded RNA at the outset and reduced need for extensive purifications. The approach can readily be applied to the synthesis of mRNA-length products under high-yield conditions.

Evolution of Spatial and Temporal cis-Regulatory Divergence in Sticklebacks.

Cis-regulatory changes are thought to play a major role in adaptation. Threespine sticklebacks have repeatedly colonized freshwater habitats in the Northern Hemisphere, where they have evolved a suite of phenotypes that distinguish them from marine populations, including changes in physiology, behavior, and morphology. To understand the role of gene regulatory evolution in adaptive divergence, here we investigate cis-regulatory changes in gene expression between marine and freshwater ecotypes through allele-specific expression (ASE) in F1 hybrids. Surveying seven ecologically relevant tissues, including three sampled across two developmental stages, we identified cis-regulatory divergence affecting a third of genes, nearly half of which were tissue-specific. Next, we compared allele-specific expression in dental tissues at two timepoints to characterize cis-regulatory changes during development between marine and freshwater fish. Applying a genome-wide test for selection on cis-regulatory changes, we find evidence for lineage-specific selection on several processes between ecotypes, including the Wnt signaling pathway in dental tissues. Finally, we show that genes with ASE, particularly those that are tissue-specific, are strongly enriched in genomic regions of repeated marine-freshwater divergence, supporting an important role for these cis-regulatory differences in parallel adaptive evolution of sticklebacks to freshwater habitats. Altogether, our results provide insight into the cis-regulatory landscape of divergence between stickleback ecotypes across tissues and during development, and support a fundamental role for tissue-specific cis-regulatory changes in rapid adaptation to new environments.

Parthenolide As a Therapeutic for Disseminated Canine Neoplasms.

This study provides a unique translational research opportunity to help both humans and dogs diagnosed with diseases that carry dismal prognoses in both species: histiocytic sarcoma (HS), hemangiosarcoma (HSA), and disseminated mastocytosis/mast cell tumor (MCT). Although exceedingly rare in humans, these so called "orphan diseases" are relatively more common in dogs. For these and other more commonplace cancers like lymphoma (Lym), dogs are an excellent translational model for human disease due to remarkably similar disease biology. In this study, assays were performed to assess the therapeutic potential of parthenolide (PTL), a known canonical nuclear factor kappa B (NF-κB) signaling inhibitor with additional mechanisms of antineoplastic activity, including alteration of cellular reduction-oxidation balance. Canine cell lines and primary cells are sensitive to PTL and undergo dose-dependent apoptosis after exposure to drug. PTL exposure also leads to glutathione depletion, reactive oxygen species generation, and NF-κB inhibition in canine cells. Standard-of-care therapeutics broadly synergize with PTL. In two canine HS cell lines, expression of NF-κB pathway signaling partners is downregulated with PTL therapy. Preliminary data suggest that PTL inhibits NF-κB activity of cells and extends survival time in a mouse model of disseminated canine HS. These data support further investigation of compounds that can antagonize canonical NF-κB pathway signaling in these cancers and pave the way for clinical trials of PTL in affected dogs. As dogs are an excellent natural disease model for these cancers, these data will ultimately improve our understanding of their human disease counterparts and hopefully improve care for both species. SIGNIFICANCE STATEMENT: Disseminated neoplasms in human and canine cancers are challenging to treat, and novel therapeutic approaches are needed to improve outcomes. Parthenolide is a promising treatment for histiocytic sarcoma, hemangiosarcoma, and mast cell neoplasia.

Global mesozooplankton communities show lower connectivity in deep oceanic layers.

Mesozooplankton is a key component of the ocean, regulating global processes such as the carbon pump, and ensuring energy transfer from lower to higher trophic levels. Yet, knowledge on mesozooplankton diversity, distribution and connectivity at global scale is still fragmented. To fill this gap, we applied DNA metabarcoding to mesozooplankton samples collected during the Malaspina-2010 circumnavigation expedition across the Atlantic, Indian and Pacific oceans from the surface to bathypelagic depths. We highlight the still scarce knowledge on global mesozooplankton diversity and identify the Indian Ocean and the deep sea as the oceanic regions with the highest proportion of hidden diversity. We report no consistent alpha-diversity patterns for mesozooplankton at a global scale, neither across vertical nor horizontal gradients. However, beta-diversity analysis suggests horizontal and vertical structuring of mesozooplankton communities mostly attributed to turnover and reveals an increase in mesozooplankton beta-diversity with depth, indicating reduced connectivity at deeper layers. Additionally, we identify a water mass type-mediated structuring of mesozooplankton bathypelagic communities instead of an oceanic basin-mediated as observed at upper layers. This suggests limited dispersal at deep ocean layers, most likely due to weaker currents and lower mixing of water mass types, thus reinforcing the importance of oceanic currents and barriers to dispersal in shaping global plankton communities.

The STAT3-MYC axis promotes survival of leukemia stem cells by regulating SLC1A5 and oxidative phosphorylation.

Acute myeloid leukemia (AML) is characterized by the presence of leukemia stem cells (LSCs), and failure to fully eradicate this population contributes to disease persistence/relapse. Prior studies have characterized metabolic vulnerabilities of LSCs, which demonstrate preferential reliance on oxidative phosphorylation (OXPHOS) for energy metabolism and survival. In the present study, using both genetic and pharmacologic strategies in primary human AML specimens, we show that signal transducer and activator of transcription 3 (STAT3) mediates OXPHOS in LSCs. STAT3 regulates AML-specific expression of MYC, which in turn controls transcription of the neutral amino acid transporter gene SLC1A5. We show that genetic inhibition of MYC or SLC1A5 acts to phenocopy the impairment of OXPHOS observed with STAT3 inhibition, thereby establishing this axis as a regulatory mechanism linking STAT3 to energy metabolism. Inhibition of SLC1A5 reduces intracellular levels of glutamine, glutathione, and multiple tricarboxylic acid (TCA) cycle metabolites, leading to reduced TCA cycle activity and inhibition of OXPHOS. Based on these findings, we used a novel small molecule STAT3 inhibitor, which binds STAT3 and disrupts STAT3-DNA, to evaluate the biological role of STAT3. We show that STAT3 inhibition selectively leads to cell death in AML stem and progenitor cells derived from newly diagnosed patients and patients who have experienced relapse while sparing normal hematopoietic cells. Together, these findings establish a STAT3-mediated mechanism that controls energy metabolism and survival in primitive AML cells.

Ceramide-induced integrated stress response overcomes Bcl-2 inhibitor resistance in acute myeloid leukemia.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

Multi-gene measurable residual disease assessed by digital polymerase chain reaction has clinical and biological utility in acute myeloid leukemia patients receiving venetoclax/azacitidine.

Venetoclax with azacitidine (ven/aza) is a lower-intensity therapeutic regimen that has been shown to improve outcomes in elderly patients with acute myeloid leukemia (AML). Measurable residual disease (MRD) using flow cytometry is a valuable tool for the prediction of relapse in AML using conventional therapies and ven/aza; however, the prognostic value for broadscale molecular MRD after ven/aza treatment is less clear. We aimed to determine the utility of retrospective assessment using multi-gene molecular MRD by droplet digital polymerase chain reaction (ddPCR). We found this approach correlates with outcomes in a cohort of patients receiving frontline ven/aza for AML. The predictive value of ddPCR MRD persisted when NPM1 mutations were removed from analysis, as well as after adjustment for the impact of stem cell transplant on outcomes. Late achievement of MRD negativity, including after SCT, was still associated with superior outcomes compared to persistently detectable MRD. We further explored the impact of ven/aza on the burden of different classes of mutations, and identified the persistence of splicing factor mutations, commonly associated with MDS, as a consistent finding after ven/aza treatment. These data add to our understanding of the effects of ven/aza on AML disease biology and provide details on molecular depth of remission that can guide prospective trials in the future.

Lysosomal acid lipase A modulates leukemia stem cell response to venetoclax/tyrosine kinase inhibitor combination therapy in blast phase chronic myeloid leukemia.

The treatment of blast phase chronic myeloid leukemia (bpCML) remains a challenge due at least in part to drug resistance of leukemia stem cells (LSCs). Recent clinical evidence suggests that the BCL-2 inhibitor venetoclax in combination with ABL-targeting tyrosine kinase inhibitors (TKIs) can eradicate bpCML LSCs. In this report, we employed preclinical models of bpCML to investigate the efficacy and underlying mechanism of LSC-targeting with venetoclax/TKI combinations. Transcriptional analysis of LSCs exposed to venetoclax and dasatinib revealed upregulation of genes involved in lysosomal biology, in particular lysosomal acid lipase A (LIPA), a regulator of free fatty acids. Metabolomic analysis confirmed increased levels of free fatty acids in response to venetoclax/dasatinib. Pre-treatment of leukemia cells with bafilomycin, a specific lysosome inhibitor, or genetic perturbation of LIPA, resulted in increased sensitivity of leukemia cells toward venetoclax/dasatinib, implicating LIPA in treatment resistance. Importantly, venetoclax/dasatinib treatment does not affect normal stem cell function, suggestive of a leukemia-specific response. These results demonstrate that venetoclax/dasatinib is an LSCselective regimen in bpCML and that disrupting LIPA and fatty acid transport enhances venetoclax/dasatinib response in targeting LSCs, providing a rationale for exploring lysosomal disruption as an adjunct therapeutic strategy to prolong disease remission.

Genomic population structure associated with repeated escape of Salmonella enterica ATCC14028s from the laboratory into nature.

Salmonella enterica serovar Typhimurium strain ATCC14028s is commercially available from multiple national type culture collections, and has been widely used since 1960 for quality control of growth media and experiments on fitness ("laboratory evolution"). ATCC14028s has been implicated in multiple cross-contaminations in the laboratory, and has also caused multiple laboratory infections and one known attempt at bioterrorism. According to hierarchical clustering of 3002 core gene sequences, ATCC14028s belongs to HierCC cluster HC20_373 in which most internal branch lengths are only one to three SNPs long. Many natural Typhimurium isolates from humans, domesticated animals and the environment also belong to HC20_373, and their core genomes are almost indistinguishable from those of laboratory strains. These natural isolates have infected humans in Ireland and Taiwan for decades, and are common in the British Isles as well as the Americas. The isolation history of some of the natural isolates confirms the conclusion that they do not represent recent contamination by the laboratory strain, and 10% carry plasmids or bacteriophages which have been acquired in nature by HGT from unrelated bacteria. We propose that ATCC14028s has repeatedly escaped from the laboratory environment into nature via laboratory accidents or infections, but the escaped micro-lineages have only a limited life span. As a result, there is a genetic gap separating HC20_373 from its closest natural relatives due to a divergence between them in the late 19th century followed by repeated extinction events of escaped HC20_373.

Aberrant cognitive empathy in individuals with elevated social anxiety and regulation with emotional working memory training.

Social anxiety may disrupt the empathic process, and well-regulated empathy is critical for navigating the social world. Two studies aimed to further understand empathy in the context of social anxiety. Study 1 compared individuals with elevated or normative social anxiety on a measure assessing cognitive and affective empathy for positive and negative emotions conveyed by other people ("targets"), completed under social threat. Relative to individuals with normative social anxiety, individuals with elevated social anxiety had greater cognitive empathy and no differences in affective empathy, regardless of emotion type. As greater cognitive empathy can be maladaptive, Study 2 tested whether this could be down-regulated. Individuals with elevated social anxiety underwent emotional working memory training (eWMT) for negative emotional information, or control training (CT). Effects on an empathy measure completed under social threat were assessed. Cognitive empathy for negative emotions decreased following eWMT but not CT, and this was only evident for those with higher pre-training working memory capacity. Cognitive empathy for positive emotions and affective empathy were not affected. Overall, social anxiety is associated with aberrant elevated cognitive empathy for negative and positive emotions, and the deviation in cognitive empathy for negative emotions can be regulated with eWMT for certain individuals.Trial registration: Australian New Zealand Clinical Trials Registry identifier: ACTRN12618001196235..

Positive and Negative Gender Role Beliefs and Intimate Partner Violence.

An ethnically diverse sample of 384 male and female undergraduates was assessed for their gender role beliefs based on positive (family responsibility) vs. negative (male dominance and female submissiveness) aspects derived from Hispanic cultural traditions. Negative male and female gender role beliefs were significantly positively correlated with reported victimization by and perpetration of severe intimate partner violence (IPV) for both men and women. Positive male gender role beliefs were negatively correlated with reported victimization by and perpetration of IPV for both men and women, with women also providing some evidence that positive female gender role beliefs were associated with less IPV.

Site pleiotropy of a stickleback Bmp6 enhancer.

Development and regeneration are orchestrated by gene regulatory networks that operate in part through transcriptional enhancers. Although many enhancers are pleiotropic and are active in multiple tissues, little is known about whether enhancer pleiotropy is due to 1) site pleiotropy, in which individual transcription factor binding sites (TFBS) are required for activity in multiple tissues, or 2) multiple distinct sites that regulate expression in different tissues. Here, we investigated the pleiotropy of an intronic enhancer of the stickleback Bone morphogenetic protein 6 (Bmp6) gene. This enhancer was previously shown to regulate evolved changes in tooth number and tooth regeneration, and is highly pleiotropic, with robust activity in both fins and teeth throughout embryonic, larval, and adult life, and in the heart and kidney in adult fish. We tested the hypothesis that the pleiotropy of this enhancer is due to site pleiotropy of an evolutionarily conserved predicted Foxc1 TFBS. Transgenic analysis and site-directed mutagenesis experiments both deleting and scrambling this predicted Foxc1 TFBS revealed that the binding site is required for enhancer activity in both teeth and fins throughout embryonic, larval, and adult development, and in the heart and kidney in adult fish. Collectively these data support a model where the pleiotropy of this Bmp6 enhancer is due to site pleiotropy and this putative binding site is required for enhancer activity in multiple anatomical sites from the embryo to the adult.

Temporal cell fate determination in the spinal cord is mediated by the duration of Notch signalling.

During neural development, progenitor cells generate different types of neurons in specific time windows. Despite the characterisation of many of the transcription factor networks involved in these differentiation events, the mechanism behind their temporal regulation is poorly understood. To address this question, we studied the temporal differentiation of the simple lateral floor plate (LFP) domain in the zebrafish spinal cord. LFP progenitors generate both early-born Kolmer-Agduhr" (KA") interneuron and late-born V3 interneuron populations. Analysis using a Notch signalling reporter demonstrates that these cell populations have distinct Notch signalling profiles. Not only do V3 progenitors receive higher total levels of Notch response, but they collect this response over a longer duration compared to KA" progenitors. To test whether the duration of Notch signalling determines the temporal cell fate specification, we combined a transgene that constitutively activates Notch signalling in the ventral spinal cord with a heat shock inducible Notch signalling terminator to switch off Notch response at any given time. Sustained Notch signalling results in expanded LFP progenitors while KA" and V3 interneurons fail to specify. Early termination of Notch signalling leads to exclusively KA" cell fate, despite the high level of Notch signalling, whereas late attenuation of Notch signalling drives only V3 cell fate. This suggests that the duration of Notch signalling, not simply the level, mediates cell fate specification. Interestingly, knockdown experiments reveal a role for the Notch ligand Jag2b in maintaining LFP progenitors and limiting their differentiation into KA" and V3 interneurons. Our results indicate that Notch signalling is required for neural progenitor maintenance while a specific attenuation timetable defines the fate of the postmitotic progeny.

Blood Test Increases Colorectal Cancer Screening in Persons Who Declined Colonoscopy and Fecal Immunochemical Test: A Randomized Controlled Trial.

BACKGROUND & AIMS: The septin 9 blood test is indicated for colorectal cancer screening in individuals who decline first-line tests, but participation in this context is unclear. We conducted a randomized controlled trial to compare reoffering colonoscopy and fecal immunochemical test (FIT) alone versus also offering the blood test among individuals who declined colonoscopy and FIT. METHODS: Screen-eligible Veterans aged 50-75 years who declined colonoscopy and FIT within the previous 6 months were randomized to letter and telephone outreach to reoffer screening with colonoscopy/FIT only (control), or additionally offering the blood test as a second-line option (intervention). The primary outcome was completion of any screening test within 6 months. The secondary outcome was completion of a full screening strategy within 6 months, including colonoscopy for those with a positive noninvasive test. RESULTS: Of 359 participants who completed follow-up, 9.6% in the control group and 17.1% in the intervention group completed any screening (7.5% difference; P = .035). Uptake of colonoscopy and FIT was similar in the 2 groups. The full screening strategy was completed in 9.0% and 14.9% in the control and intervention groups, respectively (5.9% difference; P = .084). CONCLUSIONS: Among individuals who previously declined colonoscopy and FIT, offering a blood test as a secondary option increased screening by 7.5% without decreasing uptake of first-line screening options. However, completion of a full screening strategy did not increase. These findings indicate that a blood test is a promising method to improve colorectal cancer screening, but obtaining a timely colonoscopy after a positive noninvasive test remains a challenge (ClincialTrials.gov number, NCT03598166).

Jaw size variation is associated with a novel craniofacial function for galanin receptor 2 in an adaptive radiation of pupfishes.

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage using in situ hybridization chain reaction (HCR). We then experimentally demonstrated a novel role for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on the reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a circulating Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions.

Genomic evidence for contrasting patterns of host-associated genetic differentiation across shared host-plant species in leaf- and bud-galling sawflies.

Resource specialization and ecological speciation arising through host-associated genetic differentiation (HAD) are frequently invoked as an explanation for the high diversity of plant-feeding insects and other organisms with a parasitic lifestyle. While genetic studies have demonstrated numerous examples of HAD in insect herbivores, the rarity of comparative studies means that we still lack an understanding of how deterministic HAD is, and whether patterns of host shifts can be predicted over evolutionary timescales. We applied genome-wide single nucleotide polymorphism and mitochondrial DNA sequence data obtained through genome resequencing to define species limits and to compare host-plant use in population samples of leaf- and bud-galling sawflies (Hymenoptera: Tenthredinidae: Nematinae) collected from seven shared willow (Salicaceae: Salix) host species. To infer the repeatability of long-term cophylogenetic patterns, we also contrasted the phylogenies of the two galler groups with each other as well as with the phylogeny of their Salix hosts estimated based on RADseq data. We found clear evidence for host specialization and HAD in both of the focal galler groups, but also that leaf gallers are more specialized to single host species compared with most bud gallers. In contrast to bud gallers, leaf gallers also exhibited statistically significant cophylogenetic signal with their Salix hosts. The observed discordant patterns of resource specialization and host shifts in two related galler groups that have radiated in parallel across a shared resource base indicate a lack of evolutionary repeatability in the focal system, and suggest that short- and long-term host use and ecological diversification in plant-feeding insects are dominated by stochasticity and/or lineage-specific effects.