Evidence-based comparison of the disorder-specific welfare burdens of major canine conditions could better inform targeting of stakeholder resources, to maximise improvement of health-related welfare in UK dogs. Population-level disease related welfare impact offers a quantitative, welfare-centred framework for objective disorder prioritisation, but practical applications have been limited to date due to sparse reliable evidence on disorder-specific prevalence, severity and duration across the canine disease spectrum. The VetCompass™ Programme collects de-identified electronic health record data from dogs attending primary-care clinics UK-wide, and is well placed to fill these information gaps.The eight common, breed-related conditions assessed were anal sac disorder, conjunctivitis, dental disease, dermatitis, overweight/obese, lipoma, osteoarthritis and otitis externa. Annual period prevalence estimates  were highest for dental disorder (9.6%), overweight/obese (5.7%) and anal sac disorder (4.5%). Dental disorder (76% of study year), osteoarthritis (82%), and overweight/obese (70%) had highest annual duration scores. Osteoarthritis (scoring 13/21), otitis externa (11/21) and dermatitis demonstrated (10/21) highest overall severity scores. Dental disorder (2.47/3.00 summative score), osteoarthritis (2.24/3.00) and overweight/obese (1.67/3.00) had highest VetCompass Welfare Impact scores overall.Of the eight common, breed-related disorders assessed, dental disorder, osteoarthritis and overweight/obese demonstrated particular welfare impact, based on combinations of high prevalence, duration and severity. Future work could extend this methodology to cover a wider range of disorders.Dental disorders, osteoarthritis and overweight/obese have emerged as priority areas for health-related welfare improvement in the UK dog population. This study demonstrated applicability of a standardised methodology to assess the relative health-related welfare impact across a range of canine disorders using VetCompass clinical data. Breed-related disorders in dogs have been documented, discussed and investigated for well over a century –6 and coCanine welfare is a dynamic discipline, with standards and definitions constantly evolving alongside ongoing animal welfare research –23. TherEmerging concepts/tools in animal welfare epidemiology now offer an ethically and scientifically sound conceptual framework for disorder comparison according to relative health-related welfare impact at the population level , 36. HisDevelop a data-driven strategy for disorder prioritisation, using novel welfare metrics to evaluate the data available in routinely collected canine EHR.Apply this strategy to a defined set of relevant disorders, providing transparent evidence to support particular disorders as priorities for reform.This project aimed to provide an evidence-led basis for prioritising action on certain canine breed-related disorders over others, according to comparative health-related welfare impact. Broadly, the objectives were to:The eight disorders selected for assessment of relative health-related welfare impact were anal sac disorder, conjunctivitis, dental disorder, dermatitis, lipoma, osteoarthritis, otitis externa and overweight/obese. Disorder groupings were based on previously published VetCompass analyses estimating prevalence of disorders recorded in dogs attending primary-care veterinary practices in England . The denAll-breed annual period prevalence estimates with 95% confidence intervals for the eight disorders assessed are presented in Table Otitis externa, anal sac disorder, conjunctivitis and dermatitis all showed a median of one episode per case over the year, with maximum annual episode counts varying from three (conjunctivitis) to seven  Table . NeitherFor osteoarthritis, lipoma, dental disorder, overweight/obese and conjunctivitis the proportion of cases affected for the entire study period ranged from 39.9%  to 1.2%   and osteoarthritis  provided reference values for calculation of relative prevalence, duration and severity index scores across conditions. Dental disorder ranked highest for the VetCompass Welfare Impact score (VWI score 2.47) followed by osteoarthritis (2.24), overweight/obese (1.67), lipoma (1.41), otitis externa (1.30), dermatitis (1.19), anal sac disorder (1.05) and conjunctivitis (0.96) , overweight/obese (5.7%) and anal sac disorders (4.5%) had the highest prevalence and showed results that were broadly in line with previous reports , 52. TheDuration of individual health problems is increasingly emphasised by animal welfare scientists as a key contributor to the overall welfare impact, as recognised in human medicine . DisordeBased on the median age at earliest disorder-associated record, dermatitis, anal sac disorders and conjunctivitis appeared to affect a relatively younger demographic than osteoarthritis and lipoma. Although late-onset (geriatric) health issues are often cited as of particular concern , 56, it Osteoarthritis, dental disorder, lipoma and overweight/obese had median annual duration estimates that exceeded 50, 60, 70 and 80% respectively with over a quarter of cases affected for the entire annual period. In contrast, otitis externa, dermatitis, anal sac disorder, conjunctivitis had much lower median durations (< 4%). In the latter group of conditions, the number of episodes per case could generally be more reliably determined than individual episode durations. Ambiguous or absent revisit data introduced degrees of uncertainty and subjective interpretation when time delimiting distinct episodes, with median duration often based on only small numbers of episodes. Even where feasible episode ‘start’ and ‘end’ dates were available, these likely underestimated true duration as disorders were likely clinically present before the diagnostic visit. In addition, the median duration metric itself potentially underestimated the longer term, cumulative importance of seasonally recurring episodic disorders. Annual recurrence patterns seen in seasonally triggered allergic skin disorders can manifest as a single, annual ‘flare up’ of dermatitis, otitis externa, conjunctivitis and/or anal sac irritation , 57. ThiObjective and quantitative methods are required to compare duration across the spectrum of canine disease. The current study estimated the annual duration for each disorder but given that this was across a sample of 250 cases, it also reflected the varying age structures of these cases. Annual disorder duration could also be useful to estimate the lifetime duration for each disorder although this remains challenging until large-scale birth-to-death health data on dogs become available. In the meantime, the novel median duration metric developed in the current study offers a reproducible, comparable alternative to true lifetime duration measures (or WALYs), by restricting retrospective review of EHR data to a single study year and using information typically recorded in these datasets across a wide spectrum of disorders.Overall severity scores were highest for osteoarthritis (score 13), otitis externa  and dermOverweight/obese and dental disorders had comparatively low overall severity scores (3 and 7), which appears contradictory to established veterinary consensus on the critical importance of these issues to health-related canine welfare , 49–51. Owner uptake (not just veterinary recommendation) of interventions, re-examination or referral were key when assigning disorder-specific sub-scores for individual severity metrics. This helps reflect owner perceptions of disorder importance but could mask veterinary-perceived severity in disorders where owners frequently declined recommended interventions or failed to return for follow-up care, perhaps due to lack of concern or limited resources. As applied here, the severity scoring system did not consider the welfare consequences of common secondary conditions or exacerbation of co-morbidities when assigning severity scores for assessed disorders. For example, no overweight/obese cases received directly therapeutic referral, analgesia/anti-inflammatories or procedures under GA/sedation, but potentially important detrimental consequences of sequelae/co-morbidities such as osteoarthritis –64 also Dental disorder, osteoarthritis, overweight/obese and lipoma ranked highest among the 8 studied disorders based on VetCompass Welfare Impact scores. Top-ranking duration and severity scores, interpreted alongside individual severity metric sub-scores characterised osteoarthritis as a relatively frequent primary reason for veterinary presentation, with multiple related visits per year, frequent association with chronic pain management and a notable level of associated referral. This evidence supports osteoarthritis as a priority disorder for targeted reform .Recent surveys by the British Veterinary Association (BVA) and Peoples Dispensary for Sick Animals (PDSA) reported that UK veterinary surgeons consider obesity/excessive bodyweight , 49, 51 A number of breeds were numerically over-represented in every disorder group. Assessing breed over-representation within a disorder by comparing breed-specific annual period prevalences with overall period prevalence was intended to avoid highlighting spurious potential disorder-associations in generally popular breeds . The authors acknowledge that these comparisons were only undertaken for the most commonly represented breeds in each disorder , thus less likely to flag-up potential disorder associations in less common breeds. However it is important to stress that these studies were based on relatively small samples and were likely underpowered to identify all true breed associations regardless of this approach. Exploration of individual breed-disorder predispositions was not a primary objective of this study but rather to establish evidence for breed associations in general, in support of disorders that may have an inherited element. It remains interesting to note that the majority of breed-disorder over-representations seen were consistent with previously reported breed-disorder associations, including otitis externa in Cocker spaniels , 68, ostThis study aimed to develop and apply a standardised methodology for disorder assessment to maximise comparability of prevalence, duration and severity across disorders, but individual disorder assessments and comparisons remain sensitive to certain ‘critical control points’. As previously discussed, these include selection criteria for disorders to be assessed, specificity of case definitions, inclusion criteria, subjective interpretation of available clinical free text, availability of all required data for parameter estimation, criteria for assigning severity metric sub-scores and methodology for annual duration estimation. Nonetheless, the primary comparisons aimed to establish relative measures and scores rather than absolute numerical indices of health-related welfare. Defining SEHB level 2 in at least one breed as sufficient evidence to consider a disorder ‘breed-related’ did not constitute strong evidence for genetic inheritance/hereditability. However, in this study the interest was in comparing disorders with a tendency for association with certain breeds for various, not necessarily solely genetic, reasons. For example, environmental, epigenetic and socioeconomic factors may also be relevant if certain breeds tend to be owned by less experienced or affluent dog owners, those whose lifestyle/housing offers fewer opportunities for exercise, or are inclined to feed their pets in particular ways. Hence, it was not critical to seek the higher levels of evidence for genetic inheritance which remain relatively scarce within the available literature on canine genetics , 18, 19.Broad diagnostic specificities applied within some disorder groupings may fall short of the fine-grained level achievable in referral or smaller-scale prospective studies. Some case definitions reflected common approaches in primary care practice, rather than confirmed diagnoses, e.g. lipoma diagnosis without pathological supporting evidence or osteoarthritis diagnosis without radiographic imaging. However, these wider case definitions better reflect the typically varied level of diagnostic certainty available to clinicians in the primary care setting and allowed researchers to harness the unique benefits of analytical scale offered by the VetCompass database.Using data available within VetCompass in 2013 introduced certain potential limitations to the generalisability of study findings to all UK primary care practices. The study included a mix of private and corporate but with a predominance of corporate practices. This may have skewed the results towards the standards and policies of clinical management and care that are typical of these corporate practices. Additionally, the study represents the demographic breed structure in dogs in 2013. This structure of the UK veterinary dog population may change over time with consequent changes in the frequency and nature of the most common disorders. The age of the dataset could also mean that certain topical/relevant breed-disorder associations change, e.g. brachycephalic breeds are becoming increasingly popular in the UK. These breed types have strong, well-documented disorder associations that would therefore be expected to elevate the frequency of these disorders in the overall dog population over time. Future studies analysing more recent VetCompass datasets can provide valuable scope to reflect and compare current caseloads within both private and corporate veterinary settings.All welfare metric scoring in this study was designed for disorder  level assessment, thus could not be used to reflect ranges in severity between individual cases of the same disorder. The authors recognise that the range of individual case severity within-disorder is an important area of research, and could provide opportunities to examine relative severity of a disorder between defined sub-groups of affected dogs . Appropriate re-framing of the proposed severity scoring system, or modification of published severity scoring systems such as the GISID  for pracGenerating an overall welfare impact score by summing selected indicators of prevalence, severity and duration may seem to contradict the published concept of population welfare impact as the product of these three key concepts , 36. TakOn balance, this large-scale epidemiological analysis of primary-care EHRs provides valuable evidence, complementary to that from more detailed, smaller scale studies undertaken prospectively or within referral/specialist caseloads. Specific controversies over disorder definitions or researcher judgements should not detract from the applicability of the disorder assessment approach across a range of conditions. Transparency of case definitions, data availability and criteria for individual metric calculation are key to the valid interpretation of findings from future applications of these methods.The practical implications of this study are many and varied. While not aimed directly at pet owners, findings reported can indirectly raise owner awareness through veterinary healthcare professionals, providing the latter with evidence to support care recommendations in priority areas of welfare concern which are routinely underestimated or dismissed by owners as unimportant. We also present a transferable framework for evidence-based disorder prioritisation, potentially useful for canine health and welfare stakeholders with funding to allocate to particular areas of canine health-related welfare concern. Applied to new (or expanded) lists of potential priority disorders, this tool could help canine charities, organisations or research institutions to target available resources at disorders with greatest population-level welfare impact. Strategic funding of public awareness campaigns, further research or lobbying for improvements to regulations governing the key areas of concern highlighted could ultimately raise standards of welfare in pet dogs across the UK. Results from VetCompass studies on breed-related disorders are directly contributing to the ongoing development of breed health strategies such as those of the Kennel Club’s Breed Health and Conservation Plans project  and the Eight of the most common breed-related conditions seen in dogs attending primary care veterinary practices were identified for assessment of disorder-specific health-related welfare impact. Novel assessment tools were developed and applied to evaluate EHR information from affected dogs to score and compare health-related welfare impact at a population level. Dental disorders, osteoarthritis and overweight/obese had highest overall VetCompass Welfare Impact scores. Severity metric sub-scores appear to support mismatches between veterinary and owner perceptions of obesity/excessive bodyweight and dental disease as significant canine health issues , suggestThis study provides proof-of-concept for the applicability of standardised methods for population-level, health-related welfare impact assessment across a spectrum of canine conditions using EHR data. The VetCompass Programme offers substantial practical opportunities for evidence-based disorder prioritisation according to sound epidemiological principles , 36. TheThe project received ethical approval from the RVC Ethics and Welfare committee (RVC URN 2015 1369).The VetCompass Programme collates de-identified EHR data from UK primary-care veterinary practices for epidemiological research. Collaborating practices can record summary diagnosis terms during episodes of care from an embedded VeNom code list . Data fiRelatively high disorder frequency in UK dogs. Disorders with highest estimated prevalence in UK veterinary-attending pet dogs were identified by review of previously published VetCompass analyses .Acceptable evidence in the literature to suggest a disorder may show breed-relatedness, i.e. particular association with at least one canine breed, rather than broadly affecting all breeds/types within the canine population at a similar frequency. The authors determined that an acceptable level of evidence to define a disorder as breed-associated was attainment of a minimum level 2 on the Strength of Evidence for Hereditary Basis (SEHB) scale for at least one canine breed/type. SEHB level 2 requires ‘prior evidence of an apparent breed predisposition based on numerical over-representation/risk compared to a suitable baseline or reference population’ upon review of existing literature of relevance to that disorder .Disorders were considered for assessment based on fulfilment of two criteria:Within the time restraints of this project it was possible to complete welfare impact assessment studies in eight of the most prevalent disorders meeting both these criteria.and after 2013 were defined as ‘under veterinary care during 2013’. An animal was considered to have an EHR if a VeNom diagnosis term, free-text clinical note, treatment or bodyweight was recorded.The study population included all dogs under veterinary care at VetCompass participating clinics between January 1st and December 31st 2013 (inclusive). Dogs with a) at least one EHR recorded during 2013 or b) at least one EHR recorded both before Assessment of the welfare impact of each selected disorder on the UK dog population was undertaken via disorder-specific studies of standardised design, using VetCompass EHR data from 250 randomly selected cases recorded during 2013. Power calculations to report the prevalence of individual conditions with a precision of +/− 2–3% indicated each disorder study required approximately 200–250 cases per condition .A multi-stage approach was used for case selection. First, dogs in the study population likely to be affected by the disorder during 2013 were identified as ‘candidate cases’ (CCs), using keyword searches designed to achieve good sensitivity and specificity for case capture. Keyword searches were based on disorder-associated terms  likely to be recorded in clinical notes, recorded as VeNom terms or within parameter fields of relevance to individual disorders (e.g. body condition score). Initial keyword selection was based around disorder-specific literature reviews, relevant VeNom diagnostic terms and expert opinion . Search terms were combined into keyword search strategies which were then trialed, refined and expanded iteratively using additional terms suggested by natural language processing (NLP) searches . NLP seaEHR data must have provided a ‘clear, recorded diagnosis’ of the relevant disorder; i.e. the diagnosis must have been recorded in at least one clinical note from the year 2013 using accepted terms or via recognisable synonyms, abbreviations, misspellings or typing errors.Where relevant health parameters were embedded within the clinical records, values acceptable as directly indicative of disorder were stated .Notable additional inclusion criteria were specified; e.g. dogs where 2013 notes indicated a permanent disorder-associated abnormality originating before 01.01.2013, resulted in the animal being classified as disorder positive during 2013 according to the relevant case definition.Examples of acceptable disorder-specific recording terminology and common conventions in note-taking were specified .Next, EHR data from a random sample of all CCs identified was manually reviewed against specified case definition criteria, to determine a disorder-specific study groups of 250 verified cases. A sub-group of all CCs identified by the final keyword search strategy was randomly sampled for potential inclusion. Randomisation was achieved using the RAND function in Microsoft Excel (2013) to assign each CC a random identification (ID) number. The CCs were then sorted by descending magnitude of ID. Maintaining this order, EHR data from individual CCs were manually reviewed in detail against the case definition to verify or reject disorder-affected status in 2013. For consistency, manual review was undertaken by an individual researcher, experienced in reviewing primary care veterinary clinical notes, according to protocols developed in consultation with other VetCompass research team members over a preliminary study design period. Case definitions for the selected disorders were finalised with reference to published literature. The main elements consistent across all disorder case definitions were as follows:250/total CCs reviewed to verify 250 cases).Manual reviewing continued down the list until 250 cases were verified for inclusion in each disorder group. The case verification rate within the CC sample was calculated based on the number of CCs reviewed to achieve 250 cases  recognised breed or type, or as generic ‘Crossbreed’) and dates of birth and death. All additional clinical data required for welfare metric generation were extracted via structured manual case review using a bespoke software application . Relevant data were extracted from cases using standardised study questions relating to severity and duration during EHR review (see measures described below), then data for each disorder group were pooled for generation of disorder-level welfare metrics, as described below. Case-finding and data interrogation were limited to data from 2013 in order to derive annual prevalence estimates and evaluate disorder severity and duration based on a consistent time period, for optimal comparability.Annual period prevalences (i.e. the proportion of the population affected by each disorder at any point during the study year) were estimated via a scaling method, as previously described . The proA number of metrics were defined to reflect different aspects of disorder duration across the spectrum of diseases. Duration metrics included: median individual episode duration (days), median number of episodes per year, median total annual duration (median % of year affected), median age at first disorder presentation or diagnosis in 2013 and the proportion of each disorder group affected by the disorder for the full year (%).Metrics were generated as appropriate for individual disorders using clinical knowledge of their nature and patterns of occurrence where recorded in the EHR. Disorders with evidence of at least one start and end date available in clinical records, within the study period, were considered ‘episodic’. Disorders considered to be permanent or continuous, even with therapeutic management/intervention were considered ‘non-episodic’.. Where the upper limit was calculated as greater than 365 days this was reported as 100%. The interquartile range was not calculated for episodic disorders.Duration metric calculation was approached separately for episodic and non-episodic conditions. For episodic conditions, the median episode duration (episodic disorders) was estimated using durations of all episodes of a disorder recorded in the EHRs in 2013. A case could contribute more than one episode to this calculation, or none if diagnosis and resolution dates were not available for any episodes. The median number of episodes per case was calculated directly from the number of episodes seen in cases in 2013. The median percentage of the year affected was calculated from the median number of episodes multiplied by the median episode duration during 2013 (as below). The range for duration was calculated from the minimum and maximum number of episodes and their respective durationsMedian % of year affected (episodic disorders) = [(median number of episodes per case*median episode duration)/365]*100.Range of % study year affected = (minimum number of episodes per case during year*minimum episode duration) to (maximum episodes per case during year*maximum episode duration).For non-episodic conditions the median percentage of 2013 affected, was calculated directly from the affected periods of individual cases. For cases with a start date prior to 1st January 2013 and no evidence of death during the year this was classified as a 100% year duration. For dogs first diagnosed in 2013 the duration was from the start date until the end of 2013 or the date of death if there was evidence the animal died during 2013.Additional duration metrics calculated  included the proportion of cases with > 1 episodes per study year (episodic disorders only), the proportion of cases affected by the disorder for the entire year and the age at first disorder presentation or diagnosis in 2013.Seven individual severity metrics were defined based on both their potential to objectively reflect aspects of veterinarian and/or owner perceived disorder severity and the types of clinical data commonly recorded in EHRs Table . The sevData relevant to each metric were manually extracted from the EHRs of all cases using a structured set of questions, as previously described. Disorder-level sub-scores on a four-point ordinal scale  were assigned at a disorder  level. These were based on the combined findings within each disorder, to reflect an ‘average’ severity level for the disorder. A zero score was assigned only where no cases contributed to a given severity metric . Individual metrics and sub-score criteria are summarised in Table Of the welfare metrics explored annual period prevalence (%), median proportion of year affected (%) and disorder overall severity score (maximum 21) were selected as the representative measures of disorder prevalence, duration and severity, respectively. Disorder-level scores for these three metrics were mathematically standardised and used to generate a novel health-related VetCompass Welfare Impact (VWI) score, directly comparable between disorders. For each disorder, absolute metric values scores were transformed to index values  by setting the highest scoring disorder assessed as the denominator within each metric. For example the prevalence index value for a given disorder was the annual period prevalence for that disorder divided by the annual period prevalence for the disorder with highest prevalence. This allowed direct comparison of disorders within each key metric and ensured equal contribution of each metric to the disorder-level VWI score. Using the highest scoring disorder as the metric baseline provided a baseline relevant to the specific set of disorders being compared, rather than using external benchmarks of frequency, duration and severity which could distort this comparison. Disorder-specific prevalence, duration and severity indices were summed to generate summative VWI scores, reflecting the population-level welfare impact of each disorder relative to the others assessed in this study . VWI scoFor each disorder, breed–specific annual period prevalence  estimates were also calculated for breeds with ≥5 cases using the breed-specific expected case numbers and total relevant breed counts in the VetCompass study population denominator. This provided key within-breed annual period prevalences alongside all-breed annual period prevalence for each disorder. Evidence for potential breed-disorder associations was generated by comparing breed-specific estimates to overall  annual period prevalence within individual disorders. A breed-specific:all-breed annual period prevalence ratio > 1 without overlap of 95% confidence intervals was accepted as sufficient evidence for breed predisposition in accordance with the SEHB scale, Level 2 . ExploraCategorical data used to allocate scores for welfare metrics were summarised using numbers of cases (n) and proportion of study group (%), whilst quantitative data were assessed graphically for normality and summarised with median, interquartile range and range, as appropriate . Confide


AbstractHTS) technologies and thus fast accumulation of HTS data, there has been a growing need and interest for developing tools for HTS data processing and communication. In particular, a number of bioinformatics tools have been designed for analysing metabarcoding data, each with specific features, assumptions and outputs. To evaluate the potential effect of the application of different bioinformatics workflow on the results, we compared the performance of different analysis platforms on two contrasting high-throughput sequencing data sets. Our analysis revealed that the computation time, quality of error filtering and hence output of specific bioinformatics process largely depends on the platform used. Our results show that none of the bioinformatics workflows appears to perfectly filter out the accumulated errors and generate Operational Taxonomic Units, although PipeCraft, LotuS and PIPITS perform better than QIIME2 and Galaxy for the tested fungal amplicon dataset. We conclude that the output of each platform requires manual validation of the OTUs by examining the taxonomy assignment values.Along with recent developments in high-throughput sequencing ( Fungi are major ecological and functional players in terrestrial ecosystems. The full diversity of fungi remains largely uncharted due to their largely unculturable nature, the lack of tangible morphological manifestations and shortcomings of the mycological community to sample beyond traditional habitats and substrates (ITS) region is the best region for delimiting fungal taxa at the species level across a variety of fungal groups (HTS) have made it possible to sequence millions of reads and identify thousands of fungal taxa from a single sample. Handling this enormous amount of data is often complicated and requires extensive bioinformatics expertise.bstrates . As a rel groups . Recent HTS data. However, most of these software suites were developed for the prokaryotic 16S rRNA gene and may therefore perform poorly with other markers and other organisms, in particular ITS sequences due to their length variation and non-alignability across taxonomic expanses. To accommodate this, several tailored platforms have recently been developed to specifically address fungal ITS datasets (OTU) abundance tables. Many of these platforms cover all these analysis steps, whereas others do not.Multiple analysis platforms have been introduced to facilitate the bioinformatics treatment of datasets . These pOTU tables  Sequel machine, amplified from soil samples. PacBio data set is available through PlutoF database (https://plutof.ut.ee/#/datacite/10.15156%2FBIO%2F781236). For bioinformatics analyses, we used multiple platforms that support all steps in the analysis of HTS-based metabarcoding datasets: QIIME2 . Quality filtered sequences were passed through chimeric reads removal algorithms as implemented in USEARCH (ITS extraction using ITSx (ITS region. OTU formation (clustering) was performed using USEARCH or VSEARCH as outlined below (Platform specific options). For each platform, we relied on de-novo single linkage clustering, which is the most popular approach in fungal community studies, knowing that reference-based clustering methods can provide similar results  and quality filtered using DADA2  with defde novo chimera filtering  and USEARCH reference-based chimera filtering using UNITE v7.2 as reference database. Flanking genes of the ITS region were discarded using ITSx . ITS reads were clustered to OTUs with USEARCH/UPARSE algorithm .In LotuS pipline, data was assembled (Illumina data), quality filtered  and demultiplexed with sdm . Chimera filtering was undertaken using USEARCH Using web-based Galaxy pipeline, Illumina data were assembled with Fastq joiner  and quality filtered using VSEARCH . Demultiplexing was undertaken using mothur . In this step, sequences are also re-orientated into the 5’-3’ orientation based on primers .de novo  and reference-based (UNITE v7.2 as reference) chimera filtering algorithms. In the chimera filtering step, the PipeCraft supported option for “primer artefact” removal was also used (sequences where primer strings were found in the middle of the sequence were removed). ITS reads were extracted using ITSx (default options). Clustering was performed using USEARCH/UPARSE algorithm .Chimeric sequences were removed using VSEARCH Using PIPITS, sequences were assembled with VSEARCH and quality-filtering was undertaken with fastx through the PIPITS command pispino_createreadpairslist. The ITSx was executed through the PIPITS command pipits_funits. Chimera filtering and clustering were undertaken using VSEARCH through the PIPITS command pipits_process.OTU table filtering was based on the BLAST similarity scores when run against UNITE (v7.2) reference database. Any OTUs that had no BLAST hit or that were not classified to the kingdom Fungi were discarded from the OTU table. The remaining OTUs were filtered based on BLAST e-value and query coverage. OTUs with higher e-value than 1e-25 and query coverage less than 70% were excluded from the dataset . Additionally, OTUs with low numbers of sequences per sample were removed . The numbers of sequences per sample were included in the analysis as covariates. Rarefaction curves were generated based on OTU abundance matrices for each dataset using the RTK package  data.Bioinformatics platforms differ by specific requirements to the input data, with the options being a raw multiplexed file  and multiple demultiplexed files (reads split into separate files based on indexes). PipeCraft and Galaxy use raw multiplexed data, whereas QIIME2 and PIPITS require demultiplexed files. Only LotuS allows both, multiplexed and demultiplexed files as input. As the raw data files are multiplexed by default, QIIME2 and PIPITS platforms required additional steps of analyses outside these tools to meet the input requirements. Using a Python script, we demultiplexed the raw Illumina data, allowing 2 and 1 mismatches to primer and index strings, respectively. However, PacBio datasets, the final OTU richness (singleton OTUs excluded) differed considerably amongst the tested workflows , we observed a weak but significant effect of pipeline choice on overall OTU composition for the Illumina data set . For the PacBio dataset , differences amongst platforms were slightly stronger .For both the Illumina and ws Table . We foun Figures  and 3. BITS trainset optimistically classified all OTUs to Fungi (100% confidence). Numerous papers have evaluated the performance of different methods on the accuracy of taxonomic assignment and performance inevitably hinges on the completeness of the reference database used . In spite of its relatively rapid performance, the RDP Fungal ITS trainset does not include any non-fungal data, which explains its shortcomings in detecting non-fungal OTUs. However, the confidence score of an RDP classifier did not exceed 64% for non-fungal OTUs, mostly overestimating the group of unclassified fungi.Taxonomic annotation tools differed in the ability to classify OTUs. In general, BLAST searches revealed many cases of high-quality matches to non-fungal organisms (in some cases for hundreds of OTUs), while RDP when combined with the Warcup Fungal OTU table filtering was applied to jointly clustered reads from different pipelines, the significant effect of pipeline choice on the community composition diminished .We also observed that the quality-filtered datasets included up to ~10% of obvious erroneous/chimeric OTUs that produced matches with low query coverage and confidence scores. A long tail of satellite OTUs, assigned to a single species hypothesis with 99–100% BLAST identity and RDP classifier confidence level, were also common – especially in the results where a relatively high number of OTUs was observed . After filtering the spurious OTUs manually (see Methods), we found that richness estimates per sample became more homogeneous across pipelines  and technical quality. For PacBio, the tools implemented in PipeCraft were most suitable for the long-read analysis. Conversely, the widely used platform in prokaryote 16S-based studies, our options chosen in Galaxy, performed relatively poorly on the ITS data. While QIIME2 implements an accurate quality filtering algorithm of DADA2, the lack of ITS region extraction lowers the accuracy for mycological studies. Of classification tools, BLAST searches against the UNITE database provided more accurate results on the kingdom and phylum levels compared with the RDP and Warcup ITS trainset combined. We emphasise that none of the tested bioinformatics workflows is able to fully filter out the errors that accumulated during sample preparation and sequencing, even when using the most elaborate error-filtering options. Therefore, manual curation of OTU tables continues to be an important step in obtaining robust datasets, although semi-automatic tools to assist evaluation are becoming available (In conclusion, our results indicate that bioinformatics analysis pipelines greatly differ in their relative performance on vailable . It is a


Solanum lycopersicum Mill. plants to drought stress by analyzing the expression of sly-miR159 and its target SlMYB transcription factor genes in tomato plants of cv. Ailsa Craig grown in deprived water conditions or in response to mechanical damage caused by the Colorado potato beetle, a devastating insect pest of Solanaceae plants. Results showed that sly-miR159 regulatory function in the tomato plants response to distinct stresses might be mediated by differential stress-specific MYB transcription factor targeting. sly-miR159 targeting of SlMYB33 transcription factor transcript correlated with accumulation of the osmoprotective compounds proline and putrescine, which promote drought tolerance. This highlights the potential role of sly-miR159 in tomato plants’ adaptation to water deficit conditions.In a scenario of global climate change, water scarcity is a major threat for agriculture, severely limiting crop yields. Therefore, alternatives are urgently needed for improving plant adaptation to drought stress. Among them, gene expression reprogramming by microRNAs (miRNAs) might offer a biotechnologically sound strategy. Drought-responsive miRNAs have been reported in many plant species, and some of them are known to participate in complex regulatory networks via their regulation of transcription factors involved in water stress signaling. We explored the role of miR159 in the response of  To analyze each condition, ten independent biological replicates per sample were generated and three independent experiments were conducted.Polyamine analysis was conducted according to the method described by Sánchez-López et al. , using a


Drosophila have identified a larger repertoire of IBPs. How insulators function at the molecular level is not fully understood, but it is currently thought that they fold chromosomes into conformations that affect regulatory element-promoter communication. Here, we review the discovery of insulators and describe their properties. We discuss recent genetic studies in flies and mice to address the question: Is gene insulation important for animal development? Comparing and contrasting observations in these two species reveal that they have different requirements for insulation, but that insulation is a conserved and critical gene regulation strategy.Development is orchestrated by regulatory elements that turn genes ON or OFF in precise spatial and temporal patterns. Many safety mechanisms prevent inappropriate action of a regulatory element on the wrong gene promoter. In flies and mammals, dedicated DNA elements (insulators) recruit protein factors  to shield promoters from regulatory elements. In mammals, a single IBP called CCCTC-binding factor (CTCF) is known, whereas genetic and biochemical analyses in  During development and cell differentiation, genes are turned ON and OFF in robust spatial and temporal patterns. Gene expression is regulated at multiple levels, a central one being the control of gene transcription. Regulatory elements, such as enhancers and silencers, recruit transcription factors that respectively activate or silence transcription at promoters. In flies and mammals, a developmental enhancer can activate multiple genes ,2 over lWhereas the importance of enhancers and silencers for controlling developmental gene expression has been demonstrated, the contribution of insulation to this process is less clear. This review discusses the relevance of insulation for animal development. We first review the discovery of insulators and their binding proteins  and descDrosophila melanogaster genome to maintain the independence of neighboring regulatory domains came from studies of the Hox gene, Abdominal-B (Abd-B). A spontaneous chromosomal deletion removed the boundary between two enhancer domains that each drive Abd-B expression in separate body segments. This deletion resulted in Abd-B activation by the wrong enhancer in the wrong body segment, as if the two adjacent enhancer domains had lost their abilities to act independently in different segments  insulators that flank the heat-shock gene locus that becomes “puffed” upon heat-shock induction [5’HS4 insulator located near the boundary between the decondensed beta-globin locus expressed in erythroid cells and a chromatin domain that remains condensed in those cells [Using a newly developed reporter assay, the first genomic regions were tested for insulator activity in flies in 1991. Test loci were selected based on their locations between transcriptionally active and inactive genomic regions. The first loci tested were the specialized chromatin structure . The first IBP to be discovered was CCCTC-binding factor (CTCF). CTCF was purified from chicken cell extracts through its interaction with the beta-globin insulator  Figure . To dateDrosophila have identified a dozen or more proteins with insulator activity  er assay ,22, and er assay ,24,25. Ier assay ,27. FlieIBPs bind to many sites in the genome. For example, fly and mammalian CTCF binds to thousands of sites that are often intergenic and invariantly bound in different cell types ,29. A miAs previously mentioned, insulators are defined by their ability to block the communication between enhancers or silencers and gene promoters when interposed ,10,30,31In the few studies to date in which insulators have been analyzed in situ, deletion of endogenous insulators in mammals and flies often resulted in ectopic activation of a gene by a formerly insulated enhancer. This is referred to as enhancer-adoption or enhancer-hijacking . There aBypass is the property of fly insulators by which two tandem copies of insulators cancel their activities and no longer block the communication between regulatory elements and promoters when interposed ,35 FiguB. In somHox genes in mouse cells [Drosophila cells, knock-downs of IBPs in cultured cells have led to conflicting results regarding changes in histone mark distributions, but IBPs are also unlikely to globally regulate, for example, H3K27me3 domain boundaries [Some insulators lie at borders between different chromatin landscapes and may block the spreading of histone modifications C. This hse cells  and fly se cells ,39,40. Yse cells  and lessse cells . In Drosundaries ,43.cut locus) [Though many enhancers act locally, developmental enhancers can act over large distances in mammals (up to over 1 Mb) and flies (up to 80 kb at the t locus) ,44,45. Et locus) , so how t locus) ). This it locus) ,49,50,51Though it may seem contradictory given their characteristic ability to block the communication between regulatory elements and promoters, insulators may also foster long-range regulation. In mice, CTCF may bridge certain developmental enhancers and promoters to enable their communication see .. Hox genes (see eve) locus [eve, two insulators have been characterized that are capable of highly specific pairing with themselves or with each other when they are placed in specific orientations on the same chromosome or on homologous chromosomes [eve insulators placed more than 140 kb apart was visualized in live fly embryos and preceded transcriptional activation of a reporter gene linked to one insulator by distal enhancers linked to the other insulator, indicating that insulator pairing mediated long-distance enhancer-promoter communication [Hox gene boundaries to support long-distance regulation has been shown to be separable from their insulator activity and independent of IBP binding  locus . At eve,omosomes . Positionication . Importading see .. On theDrosophila after early embryogenesis [cis and in trans and increased the stability, though not the frequency, of pairing of these loci in very early embryos when homolog pairing is still inefficient [Transvection is a phenomenon described in flies in which regulatory elements on one chromosome regulate a gene on the homologous chromosome . Transveogenesis . Insulatogenesis ,61. Whenfficient .trans-homolog regulation has not been so clearly demonstrated, likely because homologous chromosome pairing is not as pervasive as in flies.In mammals, Chromosomes are partitioned into contiguous and often cell-type invariant Topologically Associated Domains (TADs) in many organisms including flies and mammals. TADs are visualized in Chromosome Conformation Capture (3C) techniques like Hi-C that measure chemical crosslinking frequencies between distant genomic loci as a proxy for their proximity in 3D space ,64,65.In mammals, CTCF plays a central role in forming the boundaries of TADs and other chromosomal loops. Chromosomal loops of various sizes are widespread in mammalian Hi-C maps, and TADs are in fact the larger of these loops. Loops are believed to be formed by extrusion of chromatin by cohesin until cohesin reaches a pair of convergently oriented CTCF-bound sites ,67,68. TDrosophila genome appear different from mammals. It is not clear whether TADs arise from loop extrusion and whether CTCF or other IBPs participate in this process [Drosophila TADs do not have focal peaks at their corners like many mammalian TADs do [Drosophila Hi-C maps [The folding principles of the  process . Earlier process ,84, yet  process  to >90%  process ). Most D TADs do , chromosi-C maps ,88,89 coi-C maps ), and nei-C maps ,87,89. Ti-C maps ,90,91. Ri-C maps ,83,91,92i-C maps ,94 or ini-C maps ,82,95 hai-C maps . Future CTCF is essential for the viability of mammalian cells, including mouse embryonic stem cells (mESCs) ,97 and mCTCF binds pervasively in the genome ,103 and Igf2/H19 genes [Hox genes [The manipulation of specific CTCF binding sites or broader regions have revealed that CTCF insulates critical developmental genes including the imprinted 19 genes , Hox genox genes ,105, pluox genes ,107 and ox genes . Below, HoxD gene cluster which lies at the boundary between two TADs that each drive expression of specific HoxD genes in the proximal part of limbs or in digits, respectively [HoxD, clustered CTCF-bound sites point from the gene cluster towards convergently oriented CTCF sites located near enhancers within the flanking TADs, as well as at the flanking TADs’ boundaries [Compared to flies, mammals have a ~15-fold larger genome for only ~1.5-times more genes. Many mammalian developmental genes lie in large gene deserts comprising enhancers hundreds of kb away. Analyses of hundreds of enhancer trap insertions in mouse embryos revealed that enhancers are active in broad domains, typically a few hundred kb long, that correlate strikingly well with TADs . Anotherectively ,110. Intundaries . Do TADsShh) gene locus encoding a morphogen important for limb and brain development. Shh and its enhancers are comprised within a TAD [Shh expression in the developing limb bud [Shh and the ZRS within the TAD did not affect Shh expression, but others that placed Shh and the ZRS in separate TADs disrupted Shh expression in limbs, resulting in malformations [One example supporting this notion is the Sonic hedgehog  in many mouse tissues [Shh? Precise deletions of individual CTCF binding sites near Shh and/or the ZRS caused Shh and ZRS to move further apart, but Shh transcription was still activated in limbs [Shh and ZRS on opposite sides of a TAD boundary (discussed previously). Pre-formed CTCF-anchored topologies are therefore not strictly required for enhancer-promoter communication, but they may make this communication more robust. A similar observation was made in the Sox9 TAD. Sox9 enhancers could still activate Sox9 transcription in limb buds of mice in which all CTCF sites, both inside the Sox9 TAD and at its border with the neighboring Kcnj2 TAD were deleted resulting in TAD fusion. Sox9 levels were only reduced by ~10% without any phenotypic consequence [Within TADs, CTCF-anchored chromosomal loops can bridge a gene to its enhancers. Some of these loops are constitutive (present in cells in which the gene is ON and in cells in which it is OFF) and others are tissue-specific ,110,114. tissues . This coin limbs  albeit iin limbs . These min limbs . This cosequence .Epha4 gene locus containing a potent enhancer cluster and adjacent CTCF binding sites resulted in formation of “architectural stripes” visualized by Hi-C that emanated from the clustered CTCF sites at the base of the enhancer cluster [In another example, a CTCF-mediated topology supported gene activation by a long-distance enhancer in an ectopic context. Chromosomal inversions engineered in mice near the  cluster . Ectopic cluster . The extNkx2-2 and Pax1 developmental regulator genes that are expressed in different patterns in mouse embryos. A survey of the regulatory landscape between these genes using enhancer trap insertions did not provide evidence for the presence of an insulator between them. Instead, the natural decay of each gene’s enhancer activities with increasing distance was sufficient to prevent cross-regulation [Large genomic distances between adjacent developmental genes are in many cases sufficient to prevent interactions between genes and non-cognate enhancers without the need of insulators . This isgulation . How impTADs boundaries appear to generally delimit enhancer function since enhancer activity is detected at many locations within a TAD but not beyond its boundaries ,112. LarHox gene clusters  that harbor CTCF binding sites. On the one hand, deletions of specific CTCF binding sites between independently regulated Hox genes in HoxA or HoxC led to homeotic transformations of mice skeletons [HoxD had mild effects [HoxD form a boundary between the proximal limb and digit regulatory TADs mentioned before. Deletions of even large portions of HoxD encompassing several CTCF sites in many cases did not result in ectopic HoxD gene activation by enhancers from the wrong TAD, even though increasingly larger deletions did end up leading to noticeable ectopic activation [Yet, more precise manipulations of individual CTCF-binding sites have led to a spectrum of effects, from strong to undetectable. This is, for example, illustrated at keletons . On the  effects . CTCF bitivation .Kcnj2 and Sox9 genes, which are expressed in different cells in developing mouse limbs, caused weak ectopic activation of Kcnj2 transcription in a Sox9 pattern but no phenotype [Kcnj2/Sox9 locus were genetically engineered in mice to separately test the relevance of insulation at CTCF binding sites on one hand, and of rearranged distances between putative enhancers and the Kcnj2 and Sox9 genes on the other. Only the combination of both CTCF site deletions and intra-TAD rearrangements led to severe gene misexpression and developmental phenotypes [It may seem paradoxical that in several reported cases, the severe pathological effects of chromosomal rearrangements breaking TADs were not recapitulated by smaller rearrangements  or maniphenotype . Why do enotypes   and mechanisms that limit promiscuous enhancer-promoter communication between regulatory elements and genes that are in proximity (points 5–8 below).In brief, TADs and other CTCF-mediated topologies provide robustness to gene expression. In specific, but not all genomic contexts, insulation is critical for developmental gene regulation.Hox gene expression along the anterior-posterior body axis. For Hox genes of the bithorax complex (BX-C) that specify the identities of thoracic and abdominal segments, independent regulatory domains containing enhancers and silencers drive expression of their respective Hox gene in specific body segments [Hox boundaries also coincide with topological boundaries between TADs that span regulatory domains, and boundary deletion results in TAD fusion [One of the best understood developmental roles of fly insulators is to regulate segments   function of different boundaries is generic, but swapped boundaries frequently fail to support long-range regulation of Hox genes by their respective regulatory domains [Abd-B gene swapped into the boundary separating abd-A and Abd-B regulatory domains ectopically directed an abd-A regulatory domain (iab-4) to regulate the Abd-B promoter [Hox boundaries are separable. Whereas the insulator activity of Hox boundaries relies on IBPs and can even be recapitulated by synthetic multimerized IBP binding sites [The regulatory domains located distally from  domains ,133,134 ng sites ,131, theng sites . This isDrosophila IBPs have not yet been fully explored. Several IBPs with described mutants are essential for viability [su(Hw) and Cp190 that may act as a transcriptional repressor [The developmental roles of many iability ,140,141.epressor  or activepressor ,144 respepressor . To dateepressor ,43,136.Hox gene expression patterns. CTCF mutants completely lacking zygotically-expressed and maternally inherited CTCF develop until the late pupal stage [Abd-B is expressed at more homogeneous levels in different parasegments compared to wildtype [Abd-B regulatory domains  [BEAF-32 mutants have been reported to mis-express Hox genes that control anterior body segment identity [The strongest evidence to date that IBPs insulate developmental genes is the fact that several IBP mutants show homeotic phenotypes due to altered al stage . Their mwildtype . This is domains . Similarxpressed . Cp190 meletions ,147 compes. Ibf2  and mod(od(mdg4)  mutants identity .Gene insulation remains enigmatic compared to other paradigms of transcriptional regulation yet work reviewed here highlights its importance for animal development. Two main challenges to study the biological relevance of insulation in mammals are the facts that global perturbation of CTCF results in cell lethality, and that effects of perturbing individual CTCF binding sites can be masked by the robust nature of regulatory and topological interactions in the genome. The small effects on gene expression observed in several experiments have moderated our view of how impactful CTCF may be on gene regulation. Yet, recent genetic studies have demonstrated that loss of CTCF binding in specific genomic contexts leads to transcriptional and developmental defects. Insulators prevent enhancer-promoter interactions between certain elements that are close . Moreover, TADs can help regulatory elements and promoters overcome otherwise prohibitory linear distances between them to enable regulation. Hox genes, which have maintained CTCF binding sites for hundreds of millions of years to help organize animal body plans [The requirement for insulation is thus conserved in flies and mammals, notably at dy plans . In flieThe conspicuous topological role of CTCF in mammals has led to the view that it controls transcription through chromosomal loop formation. This is supported by the fact that TADs correspond to both structural and gene regulatory units and by the TAD perturbation experiments reviewed here. The “loop model” also reconciles many of the activities exerted by insulators discussed in


Solitary, persistent wave packets called solitons hold potential to transfer information and energy across a wide range of spatial and temporal scales in physical, chemical, and biological systems. Mechanical solitons characteristically emerge either as a single wave packet or uncorrelated propagating topological entities through space and/or time, but these are notoriously difficult to control. Here, we report a theoretical framework for programming static periodic topological solitons into a metamaterial, and demonstrate its implementation in real metamaterials computationally and experimentally. The solitons are excited by deformation localizations under quasi-static compression, and arise from buckling-induced kink-antikink bands that provide domain separation barriers. The soliton number and wavelength demonstrate a previously unreported size-dependence, due to intrinsic length scales. We identify that these unanticipated solitons stem from displacive phase transitions with periodic topological excitations captured by the well-known  Mechanical solitons are notoriously difficult to control. Here, the authors report a theoretical framework for programming static periodic topological solitons into a metamaterial, and demonstrate its implementation in real metamaterials computationally and experimentally. Localizations associated with solitons explain many fundamental features of dislocations, ferroelectric domain walls, surface diffusion, conducting polymers, intrinsic localized modes, and nonlinear excitations in biological molecules12. Although they have been observed in solitary excitations of mechanical metamaterials14 and domain-wall formations of van der Waals layered materials17, they have previously been stochastic and scale-independent discrete and/or single-soliton systems. A longstanding goal has been development of programmable, localized topological structures to control solitons18. This technology would hold prospects not only in tunable photonic and phononic rectification19, but also in designing smart soft robots and high sensitivity devices20.The localized deformation underlying kink-excitations drives numerous nonlinear physical and mechanical phenomena including fault zones in earthquake22, but also on designing desired patterns and interfaces in architected metamaterials24. The central challenge is the widely-recognized problem that localized deformations are sensitive to inherent or externally induced defects, rendering the location and dimension of localization zones hard to predict26. Successful previous efforts include ordered configurations achieved by introducing long-range interactions27, geometric frustration28, periodic dopants or macro-defects29. However, generation of programmable, ordered and robust static solitons and ensuing localized instabilities in nominally defect-free mechanical systems still remains largely unexplored.Extensive work has been focused not only on engineering localizations into civil structures 30, here we develop a general framework for analyzing this entire class of materials through the formalism of static periodic solitons. Many have previously addressed the bi-stability of metamaterials through conventional snap-though equilibrium techniques32. The general framework presented here is based on an approach to the underlying physics whereby interactions between double-well on-site potentials of the unit cell and the strong coupling energies of adjacent unit cells determine how metamaterial architecture and loading produce buckling-induced kink and antikink deformation bands that provide domain separation barriers.Inspired by recent numerical work indicating that excitations of ordered localizations can be achieved via carefully designed architectures34. This unanticipated behavior originates from a structural phase transition analogous to mechanical soliton-lattices of magnetic systems35. We theoretically illustrate that the period of the soliton-lattice in fact introduces an intrinsic length scale perpendicular to the compression direction, which leads to novel size-dependence of the soliton number and wavelength in the metamaterial.We therefore explored the instability of cellular metamaterials subjected to quasi-static compression. We found that, beyond a critical loading, deformation symmetry breaks down, leading to two distinct macroscopic buckling modes separated by localized topological interfaces that are static periodic solitons (kink-antikink pairs) governed by the well-known 37. Between these two states, highly localized topological domain walls emerged to accommodate the incompatible deformation . To verify the structural origin of this order, we performed 2D plane strain finite element (FE) simulations that reproduced experimental observations  are excited in the metamaterial, inducing an orderly localized configuration. A discrete size-dependence of the wavelength of the periodic solitons caused by an intrinsic length scale is also unveiled. We emphasize that the double-well potential and the strong coupling energy are pivotal factors that ensure the displacive phase transition and, evidently, can be programmed into the unit cell geometries to prescribe soliton-lattice configurations.43, atomic-scale frictions16, and domain switching44. This holds potential to be leveraged for a range of new metamaterials with programmable localized deformations for fields such as smart molecular robotics. Besides, the alternative kinks and antikinks count the reversals of the topological mechanical polarizations along the quasi-1D metamaterial chain. By encoding the inward and outward domain-states as mechanical bits which can be identified by probing the local order parameters, we envisage such robust reversal features are promising for enabling information storage and retrieval on static soliton-lattices45. More broadly, the results enable design of unit cell topologies with multistable states that can pave novel pathways to prescribing ordered localization excitations and controllable geometric phase transitions across a range of physical contexts.The striking size-dependent property of kink-antikink pairs, denoted by a ceiling function in our metamaterial systems, applies across multistable systems including molecular transport in restricted spacesWe fabricated our samples via a double-molding process. Firstly, we cast polyurethane solution into a 3D printed mold . Before testing, sample surfaces and loading platens were covered with white Vaseline to reduce friction. Tests were conducted at a constant nominal strain rate of entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaThe commercial finite element software Abaqus/Standard  was employed in our static finite element simulations. Based on the uniaxial testing results, the Mooney-Rivlin hyperelastic constitutive model was adopted to characterize the metamaterial. Material constants were taken as mentclass2pt{minimSimulation of full samples: we conducted nonlinear FE simulations on the full metamaterial, sandwiched between two rigid plates, with frictionless contact between the sample and the plates (Supplementary Fig. entclass1pt{minimaOn-site potential of the unit cell: two static steps were employed to obtain the on-site potential of the unit cell. First, a static compressive displacement, i.e. Hinge stiffness: the hinge stiffness of the simplified model is dependent on the geometry of the elliptical holes and neck thickness46. In the ideal case such as necks of constant cross-section, or circular holes with the slender limit The general framework for predicting solitons in metamaterials was verified by considering a range of classes of metamaterials. For all of these see Fig. –12, sampSupplementary InformationSupplementary Movie 1Supplementary Movie 2Description of Additional Supplementary Files


The growing availability of virtual cranial endocasts of extinct and extant vertebrates has fueled the quest for endocranial characters that discriminate between phylogenetic groups and resolve their neural significances. We used geometric morphometrics to compare a phylogenetically and ecologically comprehensive data set of archosaurian endocasts along the deep evolutionary history of modern birds and found that this lineage experienced progressive elevation of encephalisation through several chapters of increased endocranial doming that we demonstrate to result from progenetic developments. Elevated encephalisation associated with progressive size reduction within Maniraptoriformes was secondarily exapted for flight by stem avialans. Within Mesozoic Avialae, endocranial doming increased in at least some Ornithurae, yet remained relatively modest in early Neornithes. During the Paleogene, volant non-neoavian birds retained ancestral levels of endocast doming where a broad neoavian niche diversification experienced heterochronic brain shape radiation, as did non-volant Palaeognathae. We infer comparable developments underlying the establishment of pterosaurian brain shapes. Certain cognitive requirements of vertebrate flight, particularly those demanded in processing sensory input and controlling the intricate flight apparatus5, have been used to explain important changes in relative volume and organisation of the brain along the evolutionary pathway towards a “flight-adapted” brain6. Associated evolutionary patterns, including cranial shape shifts9 and overall body size reduction along the avian stem10, propose paedomorphosis as an important developmental mechanism underlying the establishment of characteristic properties of avian anatomy, including the inflated brain. Such overarching trends in the evolutionary lineage leading up to modern birds are consistent with the hypothesis that heterochronic developments were crucial towards the establishment of archosaurian volancy in general and dinosaurian flight in particular.Among Diapsida, the avian brain is uniquely enlarged relative to body size, which renders birds the only animal group that rivals the encephalisation of mammalsArchaeopteryx, considered exemplary for the transition between non-volant and volant theropods12, did not present an anatomy profoundly contrasting those of non-volant Maniraptoriformes1. Only a single cerebral structure, the wulst, until then only recognised in modern birds (Neornithes) but conclusively absent in non-volant Maniraptoriformes and Mesozoic (non-avian) Ornithurae14, was found to also be potentially present in Archaeopteryx1. The apparent lack of unambiguous flight-related cerebral adaptations in the oldest volant avialan identified to date has confused our understanding of the relation between inferred dinosaurian volancy and the development of a correspondingly flight-capable brain15. Furthermore, because the variation in relative endocranial and cerebral volumes of modern birds and their non-avian ancestors was found to not primarily reflect the presence or absence of powered flight16, other geometrical parameters should be explored to resolve the potential influence of volancy on the evolution of the archosaurian brain.Detailed comparative investigations into brain shape and size at the very onset of dinosaurian flight have been incapable of identifying specific endocranial conditions unequivocally linked with dinosaurian volancy. It has been concluded that the brain of the early volant avialan 17. Although conventional CT setups typically allow for reliable endocranial visualisation of modern material, fossils encased in and filled with a sedimentary matrix may require the application of more elaborate tomographical techniques, especially when the fossil is preserved in a plate-like slab composed of a lithic substrate with a density comparable to that of the sample of interest. More advanced CT approaches do not only enable the visualisation of samples that cannot be confidently resolved otherwise, but will generally also offer improved contrast and spatial resolution to allow for more reliable reconstruction and comparison of multiple samples, and a better appreciation of deformation incurred during taphonomy. Synchrotron propagation phase-contrast X-ray microtomography (PPC-SRµCT) in particular offers unparalleled contrast and detail in palaeontological data and has emerged as the pre-eminent approach to imaging challenging fossils today23.Where early palaeoneurological investigations on lithified cranial fossils typically involved perilous physical casting and occasionally demanded sacrifice of complete osseous braincases, the advent and development of X-ray computed tomography (CT) has enabled reliable and non-destructive reconstruction of the size and shape of an endocranial cavity25, palaeoneurology resorts to studying the cranial endocast that reflects the surface geometry of the endocranial cavity in which the brain was housed during life17. Non-avian dinosaurs generally exhibit an overall brain shape resembling the crocodilian brain more strongly than the modern avian brain27. We aimed to exploit the conservative preservation of the osseous braincase in both extinct and extant taxa while avoiding uncertainty regarding whole-brain volume or exact delimitation of cerebral components. Comparative analysis of endocast geometries covering a wide phylogenetic, size, and shape range confidently permits identification of broadly supported trends relevant to identifying the evolutionary processes that shaped the modern avian brain.Because brain tissue itself rarely fossilises28. PPC-SRµCT with optimised imaging protocols at beamlines BM05, ID17, and ID19 of the European Synchrotron Radiation Facility (ESRF) ensured reliable yet non-destructive visualisation of the endocasts in two specimens of Archaeopteryx and other fossil as well as extant archosaurs30. The data set was supplemented with conventional CT data representing a sufficiently wide selection of modern avian endocasts to resolve endocranial variation across the ecological and body size range occupied by present-day birds. Following qualitative characterisation, shape variation was quantified through two-dimensional landmarking in lateral view and shape changes were subsequently analysed with Principal Component Analysis (PCA). PCA identified dorsal endocast curvature, originating from brain axis flexure and relative telencephalic and cerebellar size, as the dominant shape variable in the data set. The C/D parameter, defined as the ratio of dorsal endocast length following the convex hull between the anteriormost tip of the cerebrum and the opisthion (C) over the linear distance between those two locations (D), was recovered as a straightforward geometric ratio conservatively capturing and quantifying endocast doming. The biological significance of C/D was tested by investigating the relationship between this ratio and physiological properties, including alert head posture31 and encephalisation quotient32. We compared embryonic C/D development in Crocodylus niloticus, Gallus gallus, and Ficedula albicollis to characterise and compare developmental timing during their in ovo trajectories towards revealing heterochronic influences on embryonic ontogeny. We furthermore plotted endocast doming (C/D) against log D for all adult archosaurs subjected to PCA, but also included a broad selection of lepidosaurs, as an outgroup to archosaurs, and an ontogenetic growth series of Crocodylus niloticus. This resolved a partially overlapping distribution of archosaurian endocranial shapes and sizes that can be directly compared against the complete ontogenetic variability of C. niloticus.To test the hypothesis that the endocranial cavities of extinct bird-line archosaurs conservatively documented the evolutionary pathway of the brain through time, we compiled a representative database of endocranial shapes encompassing the largest possible diversity of extant and extinct archosaurs, and included selected lepidosaurs as an outgroup. In order to evaluate the influence of developmental heterochrony on the evolutionary trajectory towards modern birds, we furthermore investigated ontogenetic series of extant crocodilians and birds to assess associated brain shape developments while bracketing non-avian dinosaurs phylogenetically1  and galliform Phasianus colchicus (n = 3) appear to follow the same pattern  adult archosaurs are corroborated by Principal Component Analysis. PCA morphospaces for two-dimensional Type I landmarks and semilandmarks assigned to archosaurian endocasts in lateral view (see Methods) exhibit broadly comparable shape distributions Fig.  that areThe dominant shape shift in the semilandmark set, reflected in PC1, can be reliably expressed as a ratio dividing the total distance of the dorsal margin of the endocast between the anteriormost point of the telencephalon and the dorsalmost point of the foramen magnum (C) over the shortest distance between these two points (D). The correlation between PC1 in semilandmark PCA and the novel geometrical ratio C/D was found to be substantial , share a discrete domain in endocast shape and size morphospace with Galliformes  and Anseriformes  that partially overlaps with the hulls of non-avian maniraptoriforms and pterosaurs. Sparsely available metrical and endocast data on a cranium of the Eocene anseriform Presbyornis sp. (USNM 299846)34 indicates a C/D ratio between 1.24 and 1.37 for this taxon, which is well within the range occupied by present-day anseriforms. Non-volant palaeognaths (“ratites”) share their average degree of endocast doming with volant palaeognaths but encompass a relatively broad C/D range . The neoavian radiation spans a particularly broad endocast shape variability  that partially overlaps with all other groups but also includes the highest C/D values recorded. Notably, the Late Cretaceous ornithurine Cerebavis cenomanica exhibits a C/D ratio in the range of modern volant neoavians (C/D = 1.54), which is higher than in any volant palaeognath, galliform, or anseriform.Across the entire size range, adult lepidosaurs (C/D range 1.00–1.16) and crocodilians (C/D range 1.01–1.10) exhibit strongly elongated endocast shapes (average C/D = 1.04) that broadly overlap in morphospace Fig. . Non-manHalszkaraptor and the early volant avialan Archaeopteryx present comparable endocranial geometries that are intermediate between those of non-maniraptoriform dinosaurs and those found in modern birds6  1.11 on day 93  and explain the relative brain enlargement in Eumaniraptora as a paedomorphic feature45. Progenesis has been argued to represent the most important heterochronic pathway culminating in evolutionary novelties47. Progressive progenetic expression recognised in the evolutionary history of bird-line dinosaurs can be mirrored on corresponding (and increasingly earlier) developmental stages in the crocodilian ontogenetic series , this indicates that hatchlings of both taxa represent equivalent developmental stages upon hatchling, which is corroborated by the observation that near-adult C/D values are observed in the hatchlings of each species. In ovo embryonic reduction of C/D in G. gallus proceeds more rapidly than in C. niloticus in absolute time  adult C/D ratios are achieved in G. gallus whereas embryonic C/D decrease in C. niloticus proceeds below adult ratios of G. gallus towards values approaching those of the adult crocodilian condition upon hatching. Consistent with the progenetic signature recognised in overall cranial shape7, the divergence between C. niloticus and G. gallus in magnitude and timing of endocranial shape shifts during embryonic development is best explained by progenesis in bird-line archosaurs. This interpretation is reinforced by the reduced olfactory tracts and bulbs, a relatively smaller cerebellum, and posteroventral rotation of the adult avian brain relative to the crocodilian condition, as these characters are invariably shared with embryonic crocodilians but not with later crocodilian ontogenetic stages.In crocodiles, ontogenetic progression has been demonstrated to involve an “unrollment” of the brain through a reduction of cephalic and pontine flexures and relative increase in cerebellar volume, olfactory tract length, and olfactory bulb sizeovo Fig. . Comparilus Fig.  illustraime Fig.  but occuage Fig. . In effeHalszkaraptor exhibits a high level of endocast coiling among non-avian maniraptoriform dinosaurs . Despite the clearly non-volant anatomy of Halszkaraptorinae and its ancestors29, Halszkaraptor does combine a body size within the range of flying birds with a degree of endocast coiling shared with some modern birds. Archaeopteryx also shows endocast coiling within the range of Maniraptoriformes. The endocranial geometry of Archaeopteryx is broadly shared with small yet non-volant maniraptorans, particularly Halszkaraptor, indicating that its endocranial shape resulted mainly from heterochronic developments at the root of Paraves and did not arise in the context of its incipient dinosaurian volancy. Progenetic reduction in the relative size of the adult olfactory bulbs, as observed in modern birds, was not yet achieved in Archaeopteryx11, which has been used to infer an olfactory acuity ranging from “typical to high” in Archaeopteryx relative to Neornithes56. This again illustrates that particular properties of the characteristic avian endocranial anatomy arose within already volant Avialae, implying they did not represent absolute functional prerequisites for flight but rather adaptations towards improved flight or other ecological demands.urs Fig. , as doesCerebavis57 is only known from an incomplete and abraded skull preserving a sufficiently complete braincase to reliably reveal a brain shape involving strong flexure and a “stacked” geometry, similar to those of some extant Neornithes14. The light cranium itself was furthermore reported to exhibit a degree of fusion indicating advanced flight adaptation, such as also seen in Neoaves14.The Mesozoic ornithurine 58, coupled with the recent recognition of a basal anseriform clade that originated before the end-Cretaceous extinction event59, indicates that the divergences between Palaeognathae-Neognathae, Galloanserae-Neoaves, and Galliformes-Anseriformes all occurred during the Cretaceous60. This implies that at least four extant avian clades  crossed the K-Pg boundary and subsequently negotiated an obligatory non-arboreal phase during the earliest Paleogene61. All living birds, including flightless palaeognaths, descended from volant stock62, although likely not from dexterous flyers comparable to aerial specialists within modern Neoaves. Furthermore, the present-day palaeognathous diversity originated exclusively during the Paleogene58. This group stemmed from a volant tinamou-like ancestor akin to the earliest Paleogene (and possible latest Mesozoic)63 members of the clade Lithornithidae65, despite the true (but presently obscured) origin of Palaeognathae possibly having occurred as far back as the Early Cretaceous65.The current avian phylogenyArchaeopteryx, which indicates that these groups exhibit generally similar endocast doming levels at comparable endocast lengths. All referred taxa share essentially ground-dwelling ecologies66, which, for the referred avians, likely aided their survival of the K-Pg event (possibly in broadly “shorebird-like” ecologies)67 and were retained after the earliest Paleogene recovery of arboreal habitats61. The overall C/D range shared by modern-day members of basal avian sister taxa that have occupied analogous habitats since the K-Pg event suggests their level of endocast doming preserved the ancestral, Mesozoic condition for Neornithes. This conclusion is supported by the pivotal positions of Lithornis and Presbyornis in C/D versus log D morphospace , the Enantiornithes, and the Pterosauria, may have exhibited endocast coiling levels exceeding those of non-neoavian birds. Nevertheless, their physiological and ecological demands proved incompatible with the global short- and long-term effects of the Chicxulub impact event61.Within Aves, the galliforms, anseriforms, and volant palaeognaths occupy discrete, restricted, yet broadly overlapping hulls in C/D vs log D morphospace Fig. . This pa69.As such, the proportionally large endocast size and C/D range of secondarily non-volant extant Palaeognathae reflects the Paleogene radiative colonisation of ecological niches by volant ancestors followed by independent evolutionary histories after these taxa became flightless70, no Mesozoic neoavians are presently known. The highly diverse and speciose distribution of present-day Neoaves arose during and following their explosive early Paleogene ecological radiation67. In a comparative context, we conclude that the degree of endocast doming in Cerebavis exceeding those of volant non-neoavians was achieved independently from the  neoavian radiation. This observation is consistent with an expression of cognitive challenges surpassing those of the largely ground-dwelling ecologies retained by non-neoavian birds included in our study, and may indicate progressive adaptation to more demanding aerial requirements in Cerebavis. Similar progenetic developments appear to have coincided with the origins of other volant archosaurian clades, such as earlier at the root of Pygostylia, but also in Pterosauria  uncoupled from the generalised non-neoavian archosaurian C/D curve and may have originated to meet the cognitive requirements required for elaborate flight in ecological niches adopted by (passeriform) Neoaves. Nevertheless, Mesozoic chapters of basal avialan and, later, ornithurine brain inflation did not involve the development of a wulst of wulst-like homologue. Until a wulst is positively identified in a (Mesozoic) ornithurine closer to Neornithes than Cerebavis, the wulst is again considered a uniquely neornithine synapomorphy17.Most striking is the large diversity in C/D across a broad range of endocast sizes seen in Neoaves that dominates the C/D vs log D plot Fig. . Althougria Fig. . Progeneent Fig.  suggest Halzskaraptor, and their early volant relatives including Archaeopteryx. This larger phylogenetic range thus includes the endocast size and degree of endocast doming corresponding to the first dinosaurian “flight-adapted” brain. Endocast doming was found to resolve the developmental strategies associated with the evolution from non-maniraptoriform dinosaurs to Avialae and the subsequent evolutionary pathway towards and into neoavian birds. Nevertheless, strong group overlap and limited phylogenetic resolution at the origin and during the early evolutionary history of Avialae prevents identification of discrete conditions resolvable in lateral whole-endocast shape that either enabled volancy or unambiguously indicate volancy. Volancy originated in a dinosaurian group characterised by reduced body sizes relative to their ancestral condition that achieved sufficient cognitive performance to permit aerial locomotion. However, this locomotory innovation did not preserve a characteristic morphological hallmark on the endocranium, rendering the recognition of (early) dinosaurian volancy through whole-endocast volume1 and geometry (this contribution) impossible on presently known material.Among extant birds, volant palaeognaths, galliforms, and anseriforms share an ancestral level of endocast doming that remained close to those of some non-volant Maniraptoriformes, such as Archaeopteryx. Agreement with the endocast shapes of the Paleocene volant palaeognath Lithornis and the Eocene anseriform Presbyornis furthermore indicate that these endocranial shapes are conservative relicts that do not represent (relatively) recent convergences. Among modern birds (Aves), volant palaeognaths, galliforms, and anseriforms are therefore concluded to preserve brain geometries most closely resembling that of the avian ancestor, which renders them most appropriate for reconstructing early avian cerebral developments.The whole-endocast geometries of volant palaeognaths, galliforms, and anseriforms quantified in this study have deep evolutionary histories, as corroborated by their general proportions shared with some Maniraptoriformes, including 61 and set the stage for the exploitation of a broad ecological range occupied by present-day birds. This finding renders the explosive Paleogene avian niche expansion predominantly a neoavian affair, and opens up opportunities for studying endocranial shape variation in direct relation to avian ecological strategies. Nevertheless, it also challenges the employment of neoavian brain shape variation in reconstructing the early “flight-adapted” dinosaurian brain shape. Neoavian brain shape diversity reflects the influence of several successive developments resulting in a highly apomorphic brain shape range that has advanced beyond and thereby likely obscured informative morphological properties associated with the onset of dinosaurian volancy.The large brain shape diversity of Neoaves is mostly the product of complex and progressive niche partitioning that started during the early Paleogene neoavian diversification phaseFollowing convergent progenetic developments associated with size reduction in the ancestral and early evolutionary histories of both birds and pterosaurs, we infer that certain evolutionary thresholds were negotiated in small-bodied ornithodirans that enabled the exploitation of volant ecologies, partially  through exaptation of already available brain capacity Fig. . After sArchaeopteryx may already have been capable of flight30, its brain was probably not yet capable of controlling the complex volancy seen in most modern birds. Enantiornithes were probably proficient flyers73, and the basal ornithurine Cerebavis (circa 122 Ma) already featured clear flight-related adaptations in the cranium14. However, the fossil record surrounding the origin of Neornithes74, as well as generally limited aerial performance across the basalmost extant neornithine clades, suggests that the last Mesozoic ancestors of modern birds were likely much less capable flyers. None of the volant Palaeognathae or Galloanseriformes are capable of particularly dexterous aerial locomotion, and we found that they universally share low cerebral doming levels. However, neoavian taxa that rely on aerobatics for survival, including aerial foraging and predation in falcons and bee-eaters, hunting in and evading predation by negotiating the three-dimensional landscapes of woodlands and the undergrowth by respectively owls and small passerines, and optimising the aerial approach before plunge diving by Sulidae, tend to share substantial cerebral doming. We propose that demands for increased aerial dexterity, as those associated with behavioural optimisation in, for example, aerial foraging, or those imposed by habitat constraints, such as when traversing densily populated floral landscapes, partially drove the convergent cerebral doming that occurred in several neornithine groups. Although the modern avian diversity renders a categorical test of these relations particularly challenging, this hypothesis is consistent with earlier findings that visual cues stimulated both avian cerebral changes and volant capacities.A secondary radiation within neoavian birds was recorded in an explosive diversification of endocranial shapes, which reflected the establishment of a broad ecological range with variable volant requirements. Although recent results propose that even 16, nor does phylogeny76. However, cerebral organisation does respond to certain ecological changes77 and behavioural modifications79, such as those related to migration80. The evolution of avian flight closely ties in with changes in eye size and position81, and visual acuity82. Changing foraging modes are reflected in diverging brain shapes83, which has been related to the influence of increasing visual requirements81. Although many non-neornithine Mesozoic Avialae may have been proficient flyers relative to early Neornithes, this close relation between the wulst and visual acuity in extant birds84 nevertheless suggests that they were not necessarily employing flight as their dominant foraging mode.Avian flight mode itself does not directly influence brain volumeCrocodylus niloticus, 72 additional adult crocodilian individuals, two adult pterosaurs, four non-avian dinosaurs, four specimens of Archaeopteryx, 190 adult specimens of Neornithes, and ontogenetic series of Gallus gallus, Phasianus colchicus, Ficedula albicollis and Passer domesticus, was created using synchrotron X-ray microtomography at beamlines BM05, ID17 and ID19 of the European Synchrotron Radiation Facility in Grenoble, France. Synchrotron X-ray microtomography was conducted using an optimised polychromatic beam with sufficient coherence to allow for the use of PPC-SRμCT. Three-dimensional volume reconstruction was conducted through filtered back-projection following a single distance phase retrieval protocol that relies on an assumption of homogeneity86. The synchrotron data set was supplemented with an additional Crocodylus niloticus cranium visualised with a medical tomograph at the Centre Hospitalier Universitaire in Grenoble, France, and crania of one specimen of Paleosuchus palpebrosus and 146 adult Neornithes visualised using laboratory tomographical techniques at the Institute of Physiology and the Institute for Clinical and Experimental Medicine in Prague, Czech Republic. Additional cranial scanning data of 45 lepidosaurs, two crocodilians, and five Neornithes were accessed through DigiMorph (www.digimorph.com) and processed in tandem with the data obtained in the scope of this study. Material and data provenance as well as repositories curating the digital data are listed in Extended Data Table A digital library of high-resolution tomographical data on crania of 15 adult lepidosaurs, an ontogenetic series including 42 specimens of Halszkaraptor escuilliei presents valuable three-dimensional insight into morphological conditions of a basal Dromaeosauridae29, including those of its cranial endocast. Although its skeletal integrity has been extensively demonstrated29, we care to offer additional demonstration of the non-avialan paravian identity of the Halszkaraptor cranium. To that effect, we here briefly recall the cranial features in the H. escuilliei holotype that ally the cranium with Paraves but exclude it from Avialae. The holotype of H.escuillei presents typical paravian characteristics, including a frontoparietal suture located at the level of the postorbital process of the frontal87, a short lacrimal, and a tooth row reaching posteriorly to the base of the preorbital bar29. The specimen also exhibits a suite of three dromaeosaurid cranial synapomorphies that unequivocally render the skull of Halszkaraptor an appropriate representative for the non-avialan eumaniraptoran condition of the cranial endocast. These are the enlarged paraquadrate fenestra, the shallow and elongate preorbital maxilla, and the subparallel trending ventral and alveolar margins of the dentary88. The Halszkaraptor cranium does recall some avialan conditions, such as the ratio between orbital and anteorbital length exceeding 1.289 and the dorsal border of the anteorbital fossa being formed by the nasal87. Nevertheless, because the lacrimal does not contribute to delimitation of the anteorbital fossa87, the lateral border of the quadrate is not a straight shaft87, and the nasal is longer than the frontal89, the cranium of H. escuillei is not avialan, as was corroborated by phylogenetic analysis29.The holotype of the newly described paravian Bambiraptor feinbergi was first presented as an adult specimen55 but has been suggested to represent a sub-adult at 70% body length by Paul90. In light of our finding that post-hatchling C/D in archosaurs approaches the adult values quite closely, we have adopted its C/D as representative for the adult condition. In order to avoid bias in the C/D ratios resulting from retrodeformation, only braincases that conservatively preserve the braincase geometry informing the C/D ratio were employed in C/D analyses. Similarly, only endocasts obtained from well-preserved and representative specimens were sourced from literature for inclusion in the presented analyses.For specimens sourced from literature, developmental stages used in this study 11 is a nearly complete specimen of which most of the braincase was freed from the limestone slab. The left part of the natural cranial endocast remains largely delimited by cranial bones but the right side is directly observable because the corresponding part of the osseous braincase remains in the counterslab. Of the braincase in the Munich Specimen (BSP 1999 I 50)11, only a right parietal can be readily recognised, but more material may be present ex situ on and within the limestone plates.The endocast of the London specimen of ryx Fig. , includiOn all adult archosaurian endocasts, seven Type I landmarks were conservatively identified in lateral view at discrete morphological locations along the dorsal endocast contour between the anteriormost tip of the telencephalon and the dorsalmost point of the foramen magnum. Two-dimensional landmark digitisation was realised in lateral views. Three-dimensional landmark visualisation was considered tenuous, as dinosaurian endocasts typically do not accurately preserve three-dimensional shape throughout. The definition of Type I landmarks relies on recognition of homologuous borders between cerebral structures, which in turns depend on the cerebral representativity of the endocast. This agreement is generally much lower in crocodilian and dinosaurian specimens than in modern avian material, which renders direct comparison challenging. Landmarks were placed on the borders between or extreme limits of cerebral structures  in MorphoJ to explore shape variation among endocrania of adult archosaurs.Semilandmarks were placed at equidistant intervals along the dorsal endocast contour between the anteriormost tip of the telencephalon and the dorsalmost point of the foramen magnum in all studied adult archosaurian endocasts  to visualise the associated endocast size variation. Relations and underlying patterns were assessed, interpreted, and discussed in relation to the evolutionary establishment of discrete archosaurian groups in the context of dinosaurian volancy lus Fig.  was statStatistical tests for the relations between doming ratio and biological indices were conducted in the R environment (Rstudio 1.2.1355). Normality of body mass and alert cranial angle (i.e. head posture) was assessed using Shapiro-Wilk testing. As at least one of the variables was found to disagree with the assumption of normality, Kendall rank correlation tests were performed. Towards comparing doming ratio and encephalisation quotient, the dataset was first subjected to a Bartlett test to assess homoscedasticity of the dataset. The correlation between doming ratio and encephalisation quotient was subsequently tested independently for the different groups with the Kendall rank correlation test. The Kendall rank correlation test was selected over the Spearman correlation test because of its lower error sensitivity and the higher accuracy of its p-value. Results with p-value > 0.05 were considered as non-significant.94 in recognising the synonymy of Neornithes and Aves. In this definition, only the last common ancestor of modern birds and all of its descendants account for the complete avian diversity. All dinosaurs with feathered wings used for flapping flight are placed within Avialae94, which renders Avialae virtually synonymous with the younger but now abandoned definition of Aves that encompasses the last of common ancestor of Archaeopteryx and Passer domesticus, and all of its descendants95.Towards defining birds, we follow GauthierSupplementary Dataset 2Supplementary Dataset 1Supplementary Dataset 3