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Peters, S.

Publications and source records attributed to Peters, S..

At least 19 recordsLinked to original sources

Consensus native-like hepatitis C virus E1E2 engages broadly neutralizing antibody precursors

A major goal for hepatitis C virus (HCV) vaccine development is to elicit broadly neutralizing antibodies (bNAbs) against the E1E2 glycoprotein complex located on the viral surface. Inducing HCV bNAbs requires engagement of their germline B cell precursors. HCV glycoproteins usually do not bind and activate inferred germline precursors of bNAbs (igl-bNAbs), possibly because most circulating strains contain non-conserved isolate-specific residues, even in bNAb epitopes. Here, we generated stabilized native-like soluble E1E2 (sE1E2) antigens based on a consensus sequence of HCV (HepCon) to limit the exposure of antigenically rare residues. The antigenicity and glycosylation profiles show that HepCon sE1E2 resembles a native-like E1E2 heterodimer. HepCon sE1E2 induced cross-reactive neutralizing antibody responses as a soluble protein immunogen and as membrane-anchored mRNA-delivered immunogen in animals. Importantly, HepCon sE1E2 engages multiple igl-bNAbs against two major epitopes: antigenic region 3 (AR3), which is targeted by igl-bNAbs derived from the widely expressed human VH1-69 B cell gene, and antigenic region 4 (AR4), which is only present on native-like E1E2. Nanoparticles with HepCon sE1E2 efficiently activated B cell lines expressing AR3 and AR4 igl-bNAb B cell receptors in vitro. Finally, using HepCon sE1E2 we elucidated the atomic contacts of an AR3 igl-bNAb by cryo-electron microscopy. Thus, HepCon sE1E2 is a promising candidate for germline-targeting vaccination strategies.

immunology↗

Cellular senescence is associated with age-related loss of liver zonation and hepatocyte function

The liver is organized into tightly regulated zones with distinct metabolic functions but zonation erodes with age. Cellular senescence contributes to aging and liver diseases, however, its impact on aging biology is ill-defined. As part of The Cellular Senescence Network Consortium, we used multiple spatial transcriptomics approaches (GeoMx, Visium, CosMx) with snRNA-seq to profile senescence signatures, zonation markers, and metabolic pathways in livers from wild-type (WT) mice of multiple ages. We observed a loss of canonical zone signatures in aged mouse livers characterized by "expansion" of midlobular (zone 2) marker gene expression, accompanied by diminished expression of zone 3 marker genes by middle-age (18 months), indicative of loss of cell identity. Multiple analytic approaches identified distinct age-, zone- and sex-specific senescence signatures, which were significantly associated with zonation markers changes. This was recapitulated in Ercc1 mutant models of accelerated senescence, supporting a causal role of senescent cells in liver aging. A "no-zone" hepatocyte-like cluster expanded with age and with the strongest Senescence-Associated Secretory Phenotype (SASP) profile. Gene expression profiles from senescent hepatocytes implicate decreased WNT signaling and increased BMP as contributing to age-related loss of zonation. Together, these data elucidate the role of senescent cells in driving aging biology in non-diseased liver through disruption of cell:cell signaling and the loss of metabolic and cell identity gene expression necessary for hepatocyte function.

cell biology↗

Song sparrows fail to discriminate between current and historical songs

Song evolves rapidly in songbirds, as has been proven for a substantial number of songbird species by demonstrating acoustic differences between current songs and "historical" songs recorded 20 or 30 years previously. In two species of songbirds, white-crowned sparrows and savannah sparrows, it has been further shown that evolutionary changes over such time spans are sufficient to affect the response of receivers, with territorial males responding more aggressively to current songs than to historical ones. These two species, however, have especially low population variability in song, with most males in any population singing the same, single song type; this background of song uniformity makes it especially easy to discern temporal changes. Here we examine response to temporal change in song in a third sparrow species, the song sparrow, in which population variation in song is much higher: males sing 5-13 song types each, with low song-sharing between males, so that hundreds of distinct song types occur in a local population. We find evidence that temporal change has occurred in the songs of our study population, in that 24 song current song types share more introductory phrases with other current song types than do 24 historical song types recorded 27-29 years earlier. Nevertheless, in a song playback experiment, current males showed no difference in response to current and historical songs. The results are in accord with the hypothesis that high levels of population variability in song make temporal changes in song difficult to discern. Significance statementThe song of songbirds is shaped by social learning, making birdsong a model system for the study of cultural evolution. Because cultural evolution is thought to occur more rapidly than genetic evolution, many studies have examined changes in song over time periods thought too short to allow measurable genetic evolution. Here we show that song has evolved in our study population of song sparrows, with the relative abundance of introductory phrases changing over a period of just 27-29 years. We find, however, that the song sparrows themselves fail to discern these changes, responding just as strongly to the old ("historical") song types as to current ones. Measurable cultural evolution thus occurs but without functional consequences.

animal behavior and cognition↗

Response to geographic variation in song is associated with differential gene expression in the blood of a songbird

Birdsong mediates territory acquisition and mate choice. In agonistic interactions, local songs generally elicit stronger responses than songs from more distant populations. However, the molecular mechanisms associated with differential responses to local vs. foreign songs are poorly understood. We addressed this knowledge gap by combining behavioral assays in the field with blood transcriptomic analysis, using a within-subjects design to ask whether male song sparrows (Melospiza melodia) show differential gene expression when exposed to playback of local and foreign songs. Transcriptomic profiles reflected the difference in behavioral response to local vs. foreign songs, with individuals exposed to local songs showing greater expression of genes associated with song perception and production, anti-inflammatory responses and energy metabolism. Our study suggests that changes in expression of key molecular pathways correlate with behavioral responses to geographic song variation, providing insight into the potential mechanisms regulating signal recognition and response to social challenges. HighlightsO_LIGene expression in sparrow blood was measured after simulated territorial intrusion. C_LIO_LIStronger response to local songs was associated with differential gene expression. C_LIO_LISong-associated genes (FOXP2, NRXN1) had higher expression when birds heard local songs. C_LIO_LIGene expression in the blood contains potential biomarkers of song recognition. C_LI

molecular biology↗

Dissecting the coordinated progression of cell states in spatial transcriptomics with CoPro

Spatial transcriptomics enables the study of how cells coordinate their molecular states within tissue, providing insight into both normal function and disease processes. A key challenge is to identify gene expression programs that vary continuously across space and are coordinated between cell types. We present CoPro, a computational framework for detecting the spatially coordinated progression of cellular states. CoPro can operate in both supervised and unsupervised modes to identify gene programs that co-vary within or between cell types, and to disentangle multiple overlapping spatial patterns. CoPro can be applied to single-cell-level spatial transcriptomics datasets, including MERFISH, SeqFISH+, Xenium, and histology-imputed transcriptomic data. We demonstrate the utility of CoPro with data collected from colon, brain, liver, and kidney tissues. In the colon, CoPro separates epithelial differentiation along the crypt axis from spatially localized inflammatory signals. In the aging liver, it identifies multiple aging-associated cellular programs superimposed on anatomical zonation. In the brain, the flexible kernel design enables the decoupling of the gene expression gradient along the dorsal-ventral and medial-lateral axes. In the kidney, CoPro identifies tubule-vasculature coordination that is essential in nephron function. These results demonstrate CoPros utility for analyzing spatial coordination of gene expression in complex tissues and disentangling overlapping biological processes, such as anatomical organization and disease-associated variation.

bioinformatics↗

Three novel genomes broaden the wild side of the Capsicum pangenome

This study presents three genome assemblies within the Capsicum genus, enabling comprehensive comparative analyses for the Annuum and Baccatum complexes within the genus. We produced highly continuous assemblies of the nuclear genomes and complete chloroplast assemblies. Subsequent genome annotation identified 34,580 genes in non-pungent C. annuum cv. ECW, and 32,704 and 33,994 genes in pungent C. chacoense and C. galapagoense, respectively. These assemblies, including the first complete genomes for C. chacoense and C. galapagoense, provide additional genomic resolution within the Capsicum genus. The novel genomes were analyzed within a pangenomic framework, integrating 16 Capsicum genomes across the Annuum, Baccatum, and Pubescens complexes. Homology grouping was used to identify core, accessory and unique genes and showed a wide spectrum of genetic diversity, particularly in homology groups exclusive to C. chacoense and C. galapagoense. Out of 79,267 homology groups identified, 13% were core groups, present in all accessions, corresponding to approximately 30% of core genes per genome. Comparative analyses revealed distinct species and genus specific genomic characteristics. Additionally, we used the graph pangenome to illustrate locus-level exploration by examining the Pun1 locus associated with capsaicinoid biosynthesis, identifying multiple Pun1-like genes including their genomic position and homology information. The integration of these new resources into a dynamic Capsicum pangenome framework provides a versatile platform for extracting genetic information relevant to both fundamental research and breeding applications.

genomics↗

Did Iron Suppress Eukaryote Emergence and Early Radiation?

The last eukaryotic common ancestor (LECA) is widely thought to have been an oxygen-respiring organism, arising through endosymbiosis when a free-living bacterium became the mitochondrion. Owing to the mitochondrions central metabolic task of oxidative phosphorylation, oxygen availability has long been a hypothesized driver of eukaryogenesis. However, this hypothesis is challenged by a temporal disconnect, spanning several hundred million years, between the earliest geochemical evidence for oxygen in the environment ([~]3.2-2.5 Ga) and the oldest widely accepted eukaryotic fossils ([~]1.7 Ga). Notably, the earliest candidate eukaryotes appear contemporaneous with the cessation of major iron deposits and rise of sulfide- and sulfate-rich marine sediments in coastal environments. Here, we integrate Proterozoic surface geochemical records with the microbial biochemistry of iron to examine potential environmental constraints on early eukaryotic evolution. Iron bioavailability exerts complex and often antagonistic effects on both aerobic and anaerobic microbial lineages that contributed to LECA. Elevated iron levels likely disrupted cellular homeostasis, particularly by destabilizing labile iron pools and promoting oxidative damage to bacterial lipids. The programmed cell death pathways known as ferroptosis, which is widespread among eukaryotic lineages, may trace its origins to iron-rich conditions in Archaean and Paleoproterozoic seawater and LECA. Our findings challenge oxygen-centered paradigms of eukaryogenesis and reframes the long-recognized temporal gap as a consequence of iron-mediated physiological constraints.

evolutionary biology↗

From Transcripts to Cells: Dissecting Sensitivity, Signal Contamination, and Specificity in Xenium Spatial Transcriptomics

Spatial transcriptomics has transformed our ability to map gene expression within intact tissues at cellular and subcellular resolution. Among current platforms, Xenium is widely adopted for its reliability, accessibility, and high data quality. Yet, the properties and limitations of Xenium-derived data remain poorly characterized. Here, we present one of the most comprehensive Xenium datasets to date, encompassing over 40 breast and lung tumor sections profiled using a diverse set of gene panels. Leveraging this resource, we systematically dissect technical noise--including transcript diffusion--alongside assay specificity, panel performance, and segmentation strategies. Our comparison of targeted panels with the newer 5K panel reveals that although the latter captures more transcripts overall, it suffers from reduced per-gene sensitivity and persistent diffusion, even with enhanced chemistry. We demonstrate that single-nucleus RNA-seq (snRNA-seq) markedly improves cell type annotation and enables more precise quantification of diffusion. Building on this, we introduce SPLIT (Spatial Purification of Layered Intracellular Transcripts), a novel method that integrates snRNA-seq with RCTD deconvolution to enhance signal purity. SPLIT effectively resolves mixed transcriptomic signals, improving background correction and cell-type resolution. Together, our findings provide a critical benchmark for Xenium performance and introduce a scalable strategy for signal refinement--advancing the accuracy and utility of spatial transcriptomics.

cancer biology↗

High-resolution genome and genetic map of tetraploid Allium porrum expose pericentromeric recombination

We present the first reference genome of highly heterozygous autotetraploid Allium porrum (leek). Combining long-read sequencing with SNP-array screening of two experimental F1 populations, we generated a genetic map with 11,429 SNP markers across 8 linkage groups and a chromosome-scale assembly of Allium porrum (leek) totaling 15.2 Gbp in size. High quality of the reference genome is substantiated by 97.2% BUSCO completeness and a mapping rate of 96% for full-length transcripts. The linkage map exposes the recombination landscape of leek and confirms that crossovers are predominantly proximal located to the centromeres, contrasting with distal recombination landscapes observed in other Allium species. Comparative genomics revealing structural rearrangements between A. porrum and its relatives (A. fistulosum, A. sativum, A. cepa), suggests a closer genomic relationship to A. sativum. Our annotated high-quality reference genome delivers crucial insights into the leek genome structure, recombination landscape, and evolutionary relationships within the Allium genus, offering significant implications for breeding programs, facilitating marker-assisted selection and genetic improvement in leek.

plant biology↗

Associative coding of conditioned fear in the thalamic nucleus reuniens in rodents and humans

The nucleus reuniens (RE) is a midline thalamic structure interconnecting the medial prefrontal cortex (mPFC) and the hippocampus (HPC). Recent work in both rodents and humans implicates the RE in the adaptive regulation of emotional memories, including the suppression of learned fear. However, the neural correlates of aversive learning in the RE of rodents and humans remains unclear. To address this, we recorded RE activity in humans (BOLD fMRI) and rats (fiber photometry) during Pavlovian fear conditioning and extinction. In both rats and humans, we found that conditioned stimulus (CS)-evoked activity in RE reflects the associative value of the CS. In rats, we additionally found that spontaneous neural activity in RE tracks defensive freezing and shows anticipatory increases in calcium activity that precede the termination of freezing behavior. Single-unit recordings in rats confirmed that individual RE neurons index both the associative value of the CS and defensive behavior transitions. Moreover, distinct neuronal ensembles in the RE encode fear versus extinction memories. These findings suggest a conserved role of the RE across species in modulating defensive states and emotional memory processes, providing a foundation for future translational research on fear-related disorders.

neuroscience↗

Efficient creation and characterization of multi-kilobase knockins by CRISPR/Cas9 and multiple rAAVs

Multi-kilobase knock-ins (KIs) are a necessary, yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9/gRNA ribonucleoprotein complex (RNP) enables highly efficient KI, but the insert size is limited by the viral packaging capacity. Here, we report the creation of up to 6.6 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs, we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets), where relevant genomic regions are enriched via hybridization, achieving over 100-fold greater coverage compared to other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of the KI allele, genotypes non-KI alleles and more importantly, uniquely identifies donor concatenation in the KI allele, and localizes random integration of the full or partial donor. Additionally, the multi-rAAV donor approach is successfully applied to cell lines, including lines intolerant of plasmid DNA, whereas LOCK-seq reliably and efficiently screens for KI clones. Together, the two approaches significantly improve the creation and precision of knock-in models. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/625457v3_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1619b77org.highwire.dtl.DTLVardef@125b0b2org.highwire.dtl.DTLVardef@173b12dorg.highwire.dtl.DTLVardef@7519b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

Transcriptome Analysis of Archived Tumor Tissues by Visium, GeoMx DSP, and Chromium Methods Reveals Inter- and Intra-Patient Heterogeneity

Recent advancements in probe-based, full-transcriptome, high-resolution technologies for Formalin-Fixed Paraffin-Embedded (FFPE) tissues, such as Visium CytAssist, Chromium Flex (10X Genomics), and GeoMx DSP (Nanostring), have opened new opportunities for studying decades-old archival samples in biobanks, facilitating the generation of data from extensive cohorts. However, the experimental protocols can be labor-intensive and costly; therefore, it is thus essential for researchers to carefully evaluate the strengths and limitations of each technology in relation to their specific research objectives. Here, we report the results of a comparative analysis of the three methods mentioned above on FFPE archival tumor samples from four non-small cell lung cancer, four breast cancer and six diffuse large B-cell lymphoma. We highlight some relative advantages and disadvantages of each method in the context of operational challenges, bioinformatic analysis and biological discovery. Our results show that: 1) all three methods yielded good-quality, highly reproducible transcriptomic data from serial sections of the same FFPE block; 2) GeoMx data contained mixtures of cell types, even when pre-selecting areas with cell type-specific markers; 3) high-throughput spot-level (Visium) or cell-level (Chromium) data enabled the identification of tumor heterogeneity within and between patients, which could be used to identify targeted therapies. Our data support the use of Visium and Chromium for high-throughput and discovery-driven projects, while the GeoMx platform could be suited for addressing specialized questions on targeted regions. All data generated from this study, including GeoMx, Visium, Chromium, H&E, and expert annotations are publicly available.

bioinformatics↗

Prostate-specific membrane receptor PPAP facilitates E. coli invasion of luminal prostate cells via FimH binding

Bacterial prostatitis caused by uropathogenic Escherichia coli (UPEC) strains is a highly prevalent and recurrent infection responsible for significant morbidity in men. However, the molecular pathogenesis of prostatitis remains poorly understood, partly due to the lack of comprehensive in-vitro models. In this study, we introduce a murine prostate organoid model that replicates the cellular heterogeneity of the prostate epithelium with a cell composition and transcriptional signature comparable to the native prostate tissue. Using this model, we uncovered that UPEC preferentially attaches to, invades, and replicates within luminal prostate cells. This selective interaction is mediated by the binding of the bacterial adhesin FimH to the prostate- specific membrane protein PAPP, which is exclusively expressed on luminal prostate cells. Altogether, we identified a new mechanism by which UPEC infects the prostate epithelium, highlighting FimHs adaptability in engaging host receptors and its potential for targeted therapeutic strategies.

molecular biology↗

An essential and highly selective protein import pathway encoded by nucleus-forming phage

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts. Significance StatementThe phage nucleus is an enclosed replication compartment built by Chimalliviridae phages that, similar to the eukaryotic nucleus, separates transcription from translation and selectively imports certain proteins. This allows the phage to concentrate proteins required for DNA replication and transcription while excluding DNA-targeting host defense proteins. However, the mechanism of selective trafficking into the phage nucleus is currently unknown. Here we determine the region of a phage nuclear protein that targets it for nuclear import and identify a conserved, essential nuclear shell-associated protein that plays a key role in this process. This work provides the first mechanistic model of selective import into the phage nucleus.

microbiology↗

Unveiling the Role of Endoplasmic Reticulum Stress Pathways in Canine Demodicosis

Canine demodicosis is a prevalent skin disease caused by overpopulation of a commensal species of Demodex mite, yet its precise cause remains unknown. Research suggests that T cell exhaustion, increased immunosuppressive cytokines, induction of regulatory T cells, and increased expression of immune checkpoint inhibitors may contribute to its pathogenesis. This study aimed to gain a deeper understanding of the molecular changes occurring in canine demodicosis using mass spectrometry and pathway enrichment analysis. The results indicate that endoplasmic reticulum stress is promoting canine demodicosis through regulation of three linked signalling pathways: eIF2, mTOR, and eIF4 and p70S6K. These pathways are involved in the modulation of Toll-like receptors, most notably TLR2, and have been shown to play a role in the pathogenesis of skin diseases in both dogs and humans. Moreover, these pathways are also implicated in the promotion of immunosuppressive M2 phenotype macrophages. Immunohistochemical analysis, utilizing common markers of dendritic cells and macrophages, verified the presence of M2 macrophages in canine demodicosis. The proteomic analysis also identified immunological disease, organismal injury and abnormalities, and inflammatory response as the most significant underlying diseases and disorders associated with canine demodicosis. This study demonstrates that Demodex mites, through ER stress, unfolded protein response and M2 macrophages contribute to an immunosuppressive microenvironment thereby assisting in their proliferation.

immunology↗

Genome assembly and analysis of Lactuca virosa: implications for lettuce breeding

Lettuce (Lactuca sativa L.) is a leafy vegetable crop with ongoing breeding efforts related to quality, resilience, and innovative production systems. Genetic variation of important traits in close relatives is necessary to meet lettuce breeding goals. Lactuca virosa (2x=2n=18), a wild relative assigned to the tertiary lettuce gene pool, has a much larger genome (3.7 Gbp) than Lactuca sativa (2.5 Gbp). It has been used in interspecific crosses and is a donor to modern crisphead lettuce cultivars. Here, we present a de novo reference assembly of L. virosa with high continuity and complete gene space. This assembly facilitated comparisons to the genome of L. sativa and to that of the wild species L. saligna, a representative of the secondary lettuce gene pool. To assess the diversity in gene content, we classified the genes of the three Lactuca species as core, accessory and unique. In addition, we identified three interspecific chromosomal inversions compared to L. sativa, which each may cause recombination suppression and thus hamper future introgression breeding. Using three-way comparisons in both reference-based and reference-free manners, we show that the proliferation of long-terminal repeat elements has driven the genome expansion of L. virosa. Further, we performed a genome-wide comparison of immune genes, nucleotide-binding leucine-rich repeat, and receptor-like kinases among Lactuca spp. and indicate the evolutionary patterns and mechanisms behind their expansions. These genome analyses greatly facilitate the understanding of genetic variation in L. virosa, which is beneficial for the breeding of improved lettuce varieties.

plant biology↗

Thalamic nucleus reuniens coordinates prefrontal-hippocampal synchrony to suppress extinguished fear

Traumatic events result in vivid and enduring fear memories. Suppressing the retrieval of these memories is central to behavioral therapies for pathological fear. The medial prefrontal cortex (mPFC) and hippocampus (HPC) have been implicated in retrieval suppression, but how mPFC-HPC activity is coordinated during extinction retrieval is unclear. Here we show that after extinction training, coherent theta oscillations (6-9 Hz) in the HPC and mPFC are correlated with the suppression of conditioned freezing in male and female rats. Inactivation of the nucleus reuniens (RE), a thalamic hub interconnecting the mPFC and HPC, reduces extinction-related Fos expression in both the mPFC and HPC, dampens mPFC-HPC theta coherence, and impairs extinction retrieval. Conversely, theta-paced optogenetic stimulation of RE augments fear suppression and reduces relapse of extinguished fear. Collectively, these results demonstrate a novel role for RE in coordinating mPFC-HPC interactions to suppress fear memories after extinction.

neuroscience↗

Dieback of stringybark eucalypt forests in the Mount Lofty Ranges

Canopy dieback and concerning rates of tree mortality have been noted in iconic forests of the Mount Lofty Ranges (MLR), South Australia, dominated by the stringybark eucalypt species Eucalyptus baxteri (Brown Stringybark) and E. obliqua (Messmate Stringybark). The extent and causes of stringybark forest decline are not yet fully understood, prohibiting evidence-based management strategies. Here, we explore the distribution of MLR populations of the two species and their position in climate space relative to eastern populations. We also conducted field assessments to investigate stand health and dieback aetiology, and analysed existing tree monitoring data. Stringybarks in the MLR are disjunct from eastern populations and occupy a more summer-arid niche. The species are also susceptible to summer water stress and Phytophthora. Periods of drought during 2006-2009 and 2018-2019 may have contributed to observed dieback. However, field assessments suggest a complex landscape syndrome that includes borer infestations and fire impacts among other factors, rather than solely hydraulic failure. Messmate Stringybark has suffered widespread but patchy stand collapse. There is no obvious common pattern of collapsed sites with respect to topography or local water availability (e.g., swamps and ridges equally affected), although northern range-edge sites are heavily affected. Brown Stringybark is less affected but has notable collapse sites. We hope these studies establish a springboard for future investigations and more widespread sampling of MLR stringybark forests. Further investigations should include regional surveys of stringybark sites to record spatial and temporal patterns of tree mortality combined with multi- or hyperspectral analysis of remotely sensed imagery and visual inspection of dieback from very high resolution aerial images and ground-truthing. Our findings confirm the susceptibility of stringybark forests in the MLR to ecosystem collapse and highlight the urgent need to understand the causes and aetiology of the observed dieback.

ecology↗