Imaging extrachromosomal DNA (ecDNA) in cancer.

Extrachromosomal DNA (ecDNA) are circular regions of DNA that are found in many cancers. They are an important means of oncogene amplification, and correlate with treatment resistance and poor prognosis. Consequently, there is great interest in exploring and targeting ecDNA vulnerabilities as potential new therapeutic targets for cancer treatment. However, the biological significance of ecDNA and their associated regulatory control remains unclear. Light microscopy has been a central tool in the identification and characterisation of ecDNA. In this review we describe the different cellular models available to study ecDNA, and the imaging tools used to characterise ecDNA and their regulation. The insights gained from quantitative imaging are discussed in comparison with genome sequencing and computational approaches. We suggest that there is a crucial need for ongoing innovation using imaging if we are to achieve a full understanding of the dynamic regulation and organisation of ecDNA and their role in tumourigenesis.

Pollinator shifts despite hybridisation in the Cape's hyperdiverse heathers (Erica, Ericaceae).

Interrogating the ecological and geographic factors that influence population divergence dynamics can reveal why some groups of organisms diversify more prolifically than others. One such group is the heathers (Erica, Ericaceae), the largest plant genus in the Cape Floristic Region. We study Erica abietina, a highly variable species complex with four subspecies differing in geographic range, habitat and pollination syndrome. We test for population differentiation, hybridisation, introgression and pollinator-driven divergence using genotyping-by-sequencing on samples across the entire distribution. We find five variably distinct genetic groups, with one subspecies comprising two independent lineages that are geographically isolated and occur on different soil types. Phylogenetic analysis suggests two independent shifts between bird and insect pollination, with accompanying genetic divergence. However, for one pair of populations with different pollinators, we uncover several individuals of hybrid origin at a site of sympatry. These results suggest that floral differentiation driven by divergent selection acts in concert with geographic isolation to maintain reproductive isolation and promote speciation. Our investigations reveal a highly dynamic system whose diversity has been shaped by a variety of interacting forces. We suggest that such a system could be a model for much of the diversification of the Cape flora.

Genetic differentiation across a steep and narrow environmental gradient: Quantitative genetic and genomic insights into Lake Superior populations of Quercus rubra.

Adaptive differentiation of traits and underlying loci can occur at a small geographical scale if natural selection is stronger than countervailing gene flow and drift. We investigated this hypothesis using coupled quantitative genetic and genomic approaches for a wind-pollinated tree species, Quercus rubra, along the steep, narrow gradient of the Lake Superior coast that encompasses four USDA Hardiness Zones within 100 km. For the quantitative genetic component of this study, we examined phenotypic differentiation among eight populations in a common garden, measuring seed mass, germination, height, stem diameter, leaf number, specific leaf area and survival. For the genomic component, we quantified genetic differentiation for 26 populations from the same region using RAD-seq. Because hybridisation with Quercus ellipsoidalis occurs in other parts of the species' range, we included two populations of this congener for comparison. In the common garden study, we found a strong signal of population differentiation that was significantly associated with at least one climate factor for nine of 10 measured traits. In contrast, we found no evidence of genomic differentiation among populations based on FST or any other measures. However, both distance-based and genotype-environment association analyses identified loci showing the signature of selection, with one locus in common across five analyses. This locus was associated with the minimum temperature of the coldest month, a factor that defines the climate zones and was also significant in the common garden analyses. In addition, we documented introgression from Q. ellipsoidalis into Q. rubra, with rates of introgression correlated with the climate gradient. In sum, this study reveals signatures of selection at the quantitative trait and genomic level consistent with climate adaptation, a pattern that is more often documented at a much broader geographical scale, especially in long-lived wind-pollinated species.

Genomic insights into hybrid zone formation: The role of climate, landscape, and demography in the emergence of a novel hybrid lineage.

Population demographic changes, alongside landscape, geographic and climate heterogeneity, can influence the timing, stability and extent of introgression where species hybridise. Thus, quantifying interactions across diverged lineages, and the relative contributions of interspecific genetic exchange and selection to divergence at the genome-wide level is needed to better understand the drivers of hybrid zone formation and maintenance. We used seven latitudinally arrayed transects to quantify the contributions of climate, geography and landscape features to broad patterns of genetic structure across the hybrid zone of Populus trichocarpa and P. balsamifera and evaluated the demographic context of hybridisation over time. We found genetic structure differed among the seven transects. While ancestry was structured by climate, landscape features influenced gene flow dynamics. Demographic models indicated a secondary contact event may have influenced contemporary hybrid zone formation with the origin of a putative hybrid lineage that inhabits regions with higher aridity than either of the ancestral groups. Phylogenetic relationships based on chloroplast genomes support the origin of this hybrid lineage inferred from demographic models based on the nuclear data. Our results point towards the importance of climate and landscape patterns in structuring the contact zones between P. trichocarpa and P. balsamifera and emphasise the value whole genome sequencing can have to advancing our understanding of how neutral processes influence divergence across space and time.

Genomic, morphological and physiological data support fast ecotypic differentiation and incipient speciation in an alpine diving beetle.

An intricate interplay between evolutionary and demographic processes has frequently resulted in complex patterns of genetic and phenotypic diversity in alpine lineages, posing serious challenges to species delimitation and biodiversity conservation planning. Here we integrate genomic data, geometric morphometric analyses and thermal tolerance experiments to explore the role of Pleistocene climatic changes and adaptation to alpine environments on patterns of genomic and phenotypic variation in diving beetles from the taxonomically complex Agabus bipustulatus species group. Genetic structure and phylogenomic analyses revealed the presence of three geographically cohesive lineages, two representing trans-Palearctic and Iberian populations of the elevation-generalist A. bipustulatus and another corresponding to the strictly-alpine A. nevadensis, a narrow-range endemic taxon from the Sierra Nevada mountain range in southeastern Iberia. The best-supported model of lineage divergence, along with the existence of pervasive genetic introgression and admixture in secondary contact zones, is consistent with a scenario of population isolation and connectivity linked to Quaternary climatic oscillations. Our results suggest that A. nevadensis is an alpine ecotype of A. bipustulatus, whose genotypic, morphological and physiological differentiation likely resulted from an interplay between population isolation and local altitudinal adaptation. Remarkably, within the Iberian Peninsula, such ecotypic differentiation is unique to Sierra Nevada populations and has not been replicated in other alpine populations of A. bipustulatus. Collectively, our study supports fast ecotypic differentiation and incipient speciation processes within the study complex and points to Pleistocene glaciations and local adaptation along elevational gradients as key drivers of biodiversity generation in alpine environments.

Gastric cancer molecular classification based on immunohistochemistry and in-situ hybridisation and mortality.

BACKGROUND AND AIMS: Gastric cancers (GC) are divided into subtypes based on molecular profile: Epstein-Barr virus (EBV)-positive, microsatellite instability (MSI), chromosomal instability (CIN) and genomically stable (GS) tumours. The prognostic impact of this classification is unclear. The aim was to evaluate whether the molecular subtypes determined using in-situ hybridisation (ISH) and immunohistochemistry (IHC) are associated with clinicopathological parameters and prognosis. METHODS AND RESULTS: The study included 503 GC patients. Based on ISH (EBV) and IHC (MSI and TP53), tumours were divided into EBV-positive, MSI, CIN (EBVneg/MSS/TP53aberrant) and GS (EBVneg/MSS/TP53wild-type) subgroups. Survival analyses with intestinal- and diffuse-type tumours were examined separately. EBV-positive tumours associated with male sex. Both EBV-positive and MSI tumours associated with intestinal type. CIN tumours associated with intestinal-type and positive lymph node status. GS tumours associated with diffuse-type and negative lymph node status. In the total cohort, no significant differences in the 5-year survival were observed. In intestinal tumours, the 5-year survival was better in EBV-positive tumours compared with GS tumours [hazard ratio (HR) = 0.57, 95% confidence interval (CI) = 0.33-0.99]. In diffuse tumours, the 5-year survival was worse in CIN tumours compared with GS tumours (HR = 1.57, 95% CI = 1.14-2.18). In radically resected diffuse tumours, the 5-year survival was worse in MSI tumours compared with GS tumours (HR = 3.26, 95% CI = 1.20-8.82). CONCLUSIONS: The molecular classification is associated with histological type but not prognosis in GC. As the prognostic effects of molecular subtypes in intestinal- and diffuse-type cancers may differ, combining histological and molecular information is recommended for future studies.

HER2-positive grade 1 invasive carcinomas of the breast.

AIMS: The American Society of Clinical Oncology and College of American Pathologists HER2-guidelines recommend repeat testing for most grade 1 mammary carcinomas that are HER2-positive in the core biopsy. This study aimed to assess the value of repeat HER2-testing and the histological features of HER2-positive grade 1 carcinomas. METHODS AND RESULTS: A case-series of HER2-results of grade 1 carcinomas was conducted of patients with no pre-operative systemic treatment over a 5-year period. HER2-positive carcinomas had histological review. Twelve HER2-positive carcinomas were initially reported as grade 1. On review, two were reclassified as grade 2. The remaining 10 carcinomas represented 2% of the 508 grade 1 carcinomas. Eight HER2-positive grade 1 carcinomas from other years were also studied. HER2-positive carcinomas more often had marked nuclear pleomorphism (50 versus 6%) and were more often oestrogen receptor-negative (17 versus 0.8%) and progesterone receptor-negative (28 versus 8%) compared with HER2-negative grade 1 carcinomas. Six carcinomas that were HER2 3+ in the core biopsy were also 3+ on repeat assessment. Five of seven carcinomas that were 2+ amplified in the core biopsy were also HER2-positive in the excision. CONCLUSIONS: HER2-positive grade 1 carcinomas are uncommon, and more often have marked nuclear pleomorphism and lack oestrogen receptor and progesterone receptor expression compared with HER2-negative grade 1 carcinomas. A HER2-poitive result in the core biopsy was confirmed in 11 of 13 tumours that had repeat testing.

Best practices for achieving consensus in HER2-low expression in breast cancer: current perspectives from practising pathologists.

AIMS: Human epidermal growth factor receptor 2 (HER2) expression is an important biomarker in breast cancer (BC). Most BC cases categorised as HER2-negative (HER2-) express low levels of HER2 [immunohistochemistry (IHC) 1+ or IHC 2+/in-situ hybridisation not amplified (ISH-)] and represent a clinically relevant therapeutic category that is amenable to targeted therapy using a recently approved HER2-directed antibody-drug conjugate. A group of practising pathologists, with expertise in breast pathology and BC biomarker testing, outline best practices and guidance for achieving consensus in HER2 IHC scoring for BC. METHODS AND RESULTS: The authors describe current knowledge and challenges of IHC testing and scoring of HER2-low expressing BC and provide best practices and guidance for accurate identification of BCs expressing low levels of HER2. These expert pathologists propose an algorithm for assessing HER2 expression with validated IHC assays and incorporate the 2023 American Society of Clinical Oncology and College of American Pathologist guideline update. The authors also provide guidance on when to seek consensus for HER2 IHC scoring, how to incorporate HER2-low into IHC reporting and present examples of HER2 IHC staining, including challenging cases. CONCLUSIONS: Awareness of BC cases that are negative for HER protein overexpression/gene amplification and the related clinical relevance for targeted therapy highlight the importance of accurate HER2 IHC scoring for optimal treatment selection.

CDKN2A/B deletion in IDH-mutant astrocytomas: An evaluation by Fluorescence in-situ hybridization.

INTRODUCTION: CDKN2A/B homozygous deletion is one of the defining features of grade 4 in IDH-mutant astrocytic tumours. AIM: To evaluate CDKN2A/B-deletion in IDH-mutant astrocytic tumours and its clinicopathological impact. MATERIALS AND METHODS: CDKN2A/B-deletion was evaluated by Fluorescence in-situ hybridisation (FISH) and interpreted by two recently accepted methods. RESULTS: Eighty-three out of 94 cases (histologically-grade 2: 3, grade 3: 46, grade 4: 34) were interpretable on FISH. Concordant CDKN2A/B-deletion was observed in 71% (27/38) of lower-grade tumours (n = 49) and 90% (27/30) of histological grade 4 tumours (n = 34). Both the interpretation methods showed good agreement (Kappa = 0.75). CDKN2A/B-deletion showed an inverse correlation for < 10% MIB-1 labeling index (p = 0.01) while that by method-2 showed a significant correlation for grade 4 (p = 0.02). No significant correlation was observed for any other clinicopathological parameters. Twenty-four patients showed progression/recurrence (including deaths), and no significant difference in frequency of CDKN2A/B deletion was observed among cases with disease progression across different histological grades. CONCLUSIONS: CDKN2A/B-deletion was observed across all the histological grades of IDH-mutant astrocytic tumours, expectedly more in the higher grade. FISH, as a method, can be used for the detection of CDKN2A/B homozygous deletion, when there is concordant interpretation.

Aquincola agrisoli sp. nov., isolated from rhizospheric soil of eggplant and in silico genome mining for the prediction of biosynthetic gene clusters.

A Gram-stain negative, aerobic, rod-shaped, motile and flagellated novel bacterial strain, designated MAHUQ-54T, was isolated from the rhizospheric soil of eggplant. The colonies were observed to be light pink coloured, smooth, spherical and 0.2-0.6 mm in diameter when grown on R2A agar medium for 2 days. MAHUQ-54T was able to grow at 15-40 °C, at pH 5.5-9.0 and in the presence of 0-0.5 % NaCl (w/v). The strain gave positive results for both catalase and oxidase tests. The strain was positive for hydrolysis of l-tyrosine, urea, Tween 20 and Tween 80. On the basis of the results of 16S rRNA gene sequence comparisons, the isolate was identified as a member of the genus Aquincola and is closely related to Aquincola tertiaricarbonis L10T (98.8 % sequence similarity) and Leptothrix mobilis Feox-1T (98.2 %). MAHUQ-54T has a draft genome size of 5 994 516 bp (60 contigs), annotated with 5348 protein-coding genes, 45 tRNA and 5 rRNA genes. The average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values between MAHUQ-54T and its closest phylogenetic neighbours were 75.8-83.3 and 20.8-25.3 %, respectively. In silico genome mining revealed that MAHUQ-54T has a significant potential for the production of novel natural products in the future. The genomic DNA G+C content was determined to be 70.4 %. The predominant isoprenoid quinone was ubiquinone-8. The major fatty acids were identified as C16  :  0, summed feature 3 (comprising C16  :  1ω7c and/or C16  :  1ω6c) and summed feature 8 (comprising C18  :  1ω7c and/or C18  :  1ω6c). On the basis of dDDH, ANI value, genotypic analysis, chemotaxonomic and physiological data, strain MAHUQ-54T represents a novel species within the genus Aquincola, for which the name Aquincola agrisoli sp. nov. is proposed, with MAHUQ-54T (=KACC 22001T = CGMCC 1.18515T) as the type strain.

Synthesis of new trypanocidal agents from the hybridisation of metronidazole and eugenol analogues.

Nitroimidazole compounds are well-known bioactive substances, and the structural activity relationship has been reported whereby the position of the nitro group within the imidazole ring has a large influence on the activity. This study focuses on synthesising new trypanocidal agents from the hybridisation of metronidazole with different natural phenols (eugenol, dihydroeugenol and guaiacol). Two different coupling methodologies have been explored in order to analyse the influence of the connector on bioactivity: i) classic direct esterification (AD compounds) and ii) "click" chemistry using a triazole connector (AC compounds). The in vitro trypanocidal tests show good results for both AC and AD hybrid compounds against both epimastigote and trypomastigote forms of T. cruzi. In silico studies showed positive data for most of the synthesised compounds and, in general present low toxicological risks. The AC compounds present lower ClogP (lipophilicity) values than those found for the AD series and higher TPSA (topological polar surface area) values, suggesting lower lipophilicity may be related to the presence of the triazole connector. The AD series compounds have higher Drug Score values than the AC series derivatives, suggesting better general properties for a pharmacological action.

Genomic diversity and signals of selection processes in wild and farm-reared red-legged partridges (Alectoris rufa).

The genetic dynamics of wild populations with releases of farm-reared reinforcements are very complex. These releases can endanger wild populations through genetic swamping or by displacing them. We assessed the genomic differences between wild and farm-reared red-legged partridges (Alectoris rufa) and described differential selection signals between both populations. We sequenced the whole genome of 30 wild and 30 farm-reared partridges. Both partridges had similar nucleotide diversity (π). Farm-reared partridges had a more negative Tajima's D and more and longer regions of extended haplotype homozygosity than wild partridges. We observed higher inbreeding coefficients (FIS and FROH) in wild partridges. Selective sweeps (Rsb) were enriched with genes that contribute to the reproductive, skin and feather colouring, and behavioural differences between wild and farm-reared partridges. The analysis of genomic diversity should inform future decisions for the preservation of wild populations.

A Review of Probe-Based Enrichment Methods to Inform Plant Virus Diagnostics.

Modern diagnostic techniques based on DNA sequence similarity are currently the gold standard for the detection of existing and emerging pathogens. Whilst individual assays are inexpensive to use, assay development is costly and carries risks of not being sensitive or specific enough to capture an increasingly diverse range of targets. Sequencing can provide the entire nucleic acid content of a sample and may be used to identify all pathogens present in the sample when the depth of coverage is sufficient. Targeted enrichment techniques have been used to increase sequence coverage and improve the sensitivity of detection within virus samples, specifically, to capture sequences for a range of different viruses or increase the number of reads from low-titre virus infections. Vertebrate viruses have been well characterised using in-solution hybridisation capture to target diverse virus families. The use of probes for genotyping and strain identification has been limited in plants, and uncertainty around sensitivity is an impediment to the development of a large-scale virus panel to use within regulatory settings and diagnostic pipelines. This review aims to compare significant studies that have used targeted enrichment of viruses to identify approaches to probe design and potential for use in plant virus detection and characterisation.

Unravelling the limb regeneration mechanisms of Polypedates maculatus, a sub-tropical frog, by transcriptomics.

BACKGROUND: Regeneration studies help to understand the strategies that replace a lost or damaged organ and provide insights into approaches followed in regenerative medicine and engineering. Amphibians regenerate their limbs effortlessly and are indispensable models to study limb regeneration. Xenopus and axolotl are the key models for studying limb regeneration but recent studies on non-model amphibians have revealed species specific differences in regeneration mechanisms. RESULTS: The present study describes the de novo transcriptome of intact limbs and three-day post-amputation blastemas of tadpoles and froglets of the Asian tree frog Polypedates maculatus, a non-model amphibian species commonly found in India. Differential gene expression analysis between early tadpole and froglet limb blastemas discovered species-specific novel regulators of limb regeneration. The present study reports upregulation of proteoglycans, such as epiphycan, chondroadherin, hyaluronan and proteoglycan link protein 1, collagens 2,5,6, 9 and 11, several tumour suppressors and methyltransferases in the P. maculatus tadpole blastemas. Differential gene expression analysis between tadpole and froglet limbs revealed that in addition to the expression of larval-specific haemoglobin and glycoproteins, an upregulation of cysteine and serine protease inhibitors and downregulation of serine proteases, antioxidants, collagenases and inflammatory genes in the tadpole limbs were essential for creating an environment that would support regeneration. Dermal myeloid cells were GAG+, EPYC+, INMT+, LEF1+ and SALL4+ and seemed to migrate from the unamputated regions of the tadpole limb to the blastema. On the other hand, the myeloid cells of the froglet limb blastemas were few and probably contributed to sustained inflammation resulting in healing. CONCLUSIONS: Studies on non-model amphibians give insights into alternate tactics for limb regeneration which can help devise a plethora of methods in regenerative medicine and engineering.

A simplified bacterial community found within the epidermis than at the epidermal surface of atopic dermatitis patients and healthy controls.

There has been considerable research into the understanding of the healthy skin microbiome. Similarly, there is also a considerable body of research into whether specific microbes contribute to skin disorders, with atopic dermatitis (AD) routinely linked to increased Staphylococcus aureus (S. aureus) colonisation. In this study, the epidermal surface of participants was sampled using swabs, while serial tape-stripping (35 tapes) was performed to sample through the stratum corneum. Samples were taken from AD patients and healthy controls, and the bacterial communities were profiled by metabarcoding the universal V3-V4 16S rRNA region. Results show that the majority of bacterial richness is located within the outermost layers of the stratum corneum, however there were many taxa that were found almost exclusively at the very outermost layer of the epidermis. We therefore hypothesise that tape-stripping can be performed to investigate the 'core microbiome' of participants by removing environmental contaminants. Interestingly, significant community variation between AD patients and healthy controls was only observable at the epidermal surface, yet a number of individual taxa were found to consistently differ with AD status across the entire epidermis (i.e. both the epidermal surface and within the epidermis). Sampling strategy could therefore be tailored dependent on the hypothesis, with sampling for forensic applications best performed using surface swabs and outer tapes, while profiling sub-surface communities may better reflect host genome and immunological status.

A pathogen effector FOLD diversified in symbiotic fungi.

Pathogenic fungi use secreted effector proteins to suppress immunity and support their infection, but effectors have also been reported from fungi that engage in nutritional symbioses with plants. Sequence-based effector comparisons between pathogens and symbiotic arbuscular mycorrhizal (AM) fungi are hampered by the huge diversity of effector sequences even within closely related microbes. To find sequence-divergent but structurally similar effectors shared between symbiotic and pathogenic fungi, we compared secreted protein structure models of the AM fungus Rhizophagus irregularis to known pathogen effectors. We identified proteins with structural similarity to known Fusarium oxysporum f. sp. lycopersici dual domain (FOLD) effectors, which occur in low numbers in several fungal pathogens. Contrastingly, FOLD genes from AM fungi (MycFOLDs) are found in enlarged and diversified gene families with higher levels of positive selection in their C-terminal domains. Our structure model comparison suggests that MycFOLDs are similar to carbohydrate-binding motifs. Different MycFOLD genes are expressed during colonisation of different hosts and MycFOLD-17 transcripts accumulate in plant intracellular arbuscules. The exclusive presence of MycFOLDs across unrelated plant-colonising fungi, their inducible expression, lineage-specific sequence diversification and transcripts in arbuscules suggest that FOLD proteins act as effectors during plant colonisation of symbiotic and pathogenic fungi.

Widespread introgression of MHC genes in Iberian Podarcis lizards.

Major histocompatibility complex (MHC) genes are crucial for the adaptive immune response of jawed vertebrates. Their variation, reaching extreme levels, is driven mainly by an arms race between hosts and pathogens. One hypothesised mechanism contributing to MHC polymorphism is adaptive introgression, the exchange of genetic variants between hybridising species favoured by selection, yet its effect on MHC variation is poorly understood. Detection of adaptive MHC introgression, though challenging, may be facilitated by the analysis of species complexes forming multiple hybrid zones. Here, we investigated MHC introgression in six hybrid zones formed by seven species of Podarcis lizards inhabiting the Iberian Peninsula. To differentiate adaptive introgression from neutral introgression, we compared the patterns of gene exchange in MHC and genome-wide markers. We found elevated sharing of MHC alleles in the proximity of contact beyond the areas of detectable genome-wide admixture in most hybrid zones and, in half of them, asymmetric MHC exchange. In general, the elevated MHC allele sharing between species pairs with abutting ranges compared to geographically isolated species pairs also supports the prevalence of introgression. Collectively, our results demonstrate widespread MHC introgression in the Iberian Podarcis complex and suggest its adaptiveness. Contrary to previous results from Triturus newts, we did not observe differences in the rate of introgression between MHC classes. Our work adds support to the emerging view of adaptive introgression as a key mechanism shaping MHC diversity. It also raises questions about the effect of elevated MHC variation and factors leading to the asymmetry of adaptive introgression.

Speciation without gene-flow in hybridizing deer.

Under the ecological speciation model, divergent selection acts on ecological differences between populations, gradually creating barriers to gene flow and ultimately leading to reproductive isolation. Hybridisation is part of this continuum and can both promote and inhibit the speciation process. Here, we used white-tailed (Odocoileus virginianus) and mule deer (O. hemionus) to investigate patterns of speciation in hybridizing sister species. We quantified genome-wide historical introgression and performed genome scans to look for signatures of four different selection scenarios. Despite ample modern evidence of hybridisation, we found negligible patterns of ancestral introgression and no signatures of divergence with gene flow, rather localized patterns of allopatric and balancing selection were detected across the genome. Genes under balancing selection were related to immunity, MHC and sensory perception of smell, the latter of which is consistent with deer biology. The deficiency of historical gene-flow suggests that white-tailed and mule deer were spatially separated during the glaciation cycles of the Pleistocene and genome wide differentiation accrued via genetic drift. Dobzhansky-Muller incompatibilities and selection against hybrids are hypothesised to be acting, and diversity correlations to recombination rates suggests these sister species are far along the speciation continuum.

Genome-wide variant analyses reveal new patterns of admixture and population structure in Australian dingoes.

Admixture between species is a cause for concern in wildlife management. Canids are particularly vulnerable to interspecific hybridisation, and genetic admixture has shaped their evolutionary history. Microsatellite DNA testing, relying on a small number of genetic markers and geographically restricted reference populations, has identified extensive domestic dog admixture in Australian dingoes and driven conservation management policy. But there exists a concern that geographic variation in dingo genotypes could confound ancestry analyses that use a small number of genetic markers. Here, we apply genome-wide single-nucleotide polymorphism (SNP) genotyping to a set of 402 wild and captive dingoes collected from across Australia and then carry out comparisons to domestic dogs. We then perform ancestry modelling and biogeographic analyses to characterise population structure in dingoes and investigate the extent of admixture between dingoes and dogs in different regions of the continent. We show that there are at least five distinct dingo populations across Australia. We observed limited evidence of dog admixture in wild dingoes. Our work challenges previous reports regarding the occurrence and extent of dog admixture in dingoes, as our ancestry analyses show that previous assessments severely overestimate the degree of domestic dog admixture in dingo populations, particularly in south-eastern Australia. These findings strongly support the use of genome-wide SNP genotyping as a refined method for wildlife managers and policymakers to assess and inform dingo management policy and legislation moving forwards.

Semipermeable species boundaries create opportunities for gene flow and adaptive potential.

Hybridisation and gene flow can have both deleterious and adaptive consequences for natural populations and species. To better understand the extent of hybridisation in nature and the balance between its beneficial and deleterious outcomes in a changing environment, information on naturally hybridising nonmodel organisms is needed. This requires the characterisation of the structure and extent of natural hybrid zones. Here, we study natural populations of five keystone mound-building wood ant species in the Formica rufa group across Finland. No genomic studies across the species group exist, and the extent of hybridisation and genomic differentiation in sympatry is unknown. Combining genome-wide and morphological data, we demonstrate more extensive hybridisation than was previously detected between all five species in Finland. Specifically, we reveal a mosaic hybrid zone between Formica aquilonia, F. rufa and F. polyctena, comprising further generation hybrid populations. Despite this, we find that F. rufa, F. aquilonia, F. lugubris and F. pratensis form distinct gene pools in Finland. We also find that hybrids occupy warmer microhabitats than the nonadmixed populations of cold-adapted F. aquilonia, and suggest that warm winters and springs, in particular, may benefit hybrids over F. aquilonia, the most abundant F. rufa group species in Finland. In summary, our results indicate that extensive hybridisation may create adaptive potential that could promote wood ant persistence in a changing climate. Additionally, they highlight the potentially significant ecological and evolutionary consequences of extensive mosaic hybrid zones, within which independent hybrid populations face an array of ecological and intrinsic selection pressures.