The Masai giraffe has experienced a population decline from 70,000 to 35,000 in the past three decades and was declared an endangered subspecies by the IUCN in 2019. The remaining number of Masai giraffe are geographically separated by the steep cliffs of the Gregory Rift escarpments (GRE) in Tanzania and Kenya dividing them into two populations, one west and one east of the GRE. The cliffs of the GRE are formidable barriers to east–west dispersal and gene flow and the few remaining natural corridors through the GRE are occupied by human settlements. To assess the impact of the GRE on Masai giraffe gene flow, we examined whole genome sequences of nuclear and mitochondrial DNA (mtDNA) variation in populations located east (Tarangire ecosystem) and west (Serengeti ecosystem) of the GRE in northern Tanzania. Evidence from mtDNA variation, which measures female‐mediated gene flow, suggests that females have not migrated across the GRE between populations in the Serengeti and Tarangire ecosystems in the past ~289,000 years. The analysis of nuclear DNA variation compared to mtDNA DNA variation suggests that male‐mediated gene flow across the GRE has occurred more recently but stopped a few thousand years ago. Our findings show that Masai giraffes are split into two populations and fulfill the criteria for designation as distinct evolutionary significant units (ESUs), which we denote as western Masai giraffe and eastern Masai giraffe. While establishing giraffe dispersal corridors across the GRE is impractical, conservation efforts should be focused on maintaining connectivity among populations within each of these two populations. The importance of these efforts is heightened by our finding that the inbreeding coefficients are high in some of these Masai giraffe populations, which could result in inbreeding depression in the small and fragmented populations. The steep escarpments of the GRV are formidable barriers to migration of Masai giraffe. To assess the impact of the GRV on Masai giraffe gene flow, we examined nuclear and mitochondrial DNA variation in subpopulations east and west of the GRV in Tanzania. Strikingly, evidence from mtDNA variation suggests that females have not migrated across the GRV between major subpopulations in the Serengeti and Tarangire ecosystems since the approximate time that Masai giraffes emerged as a (sub)species 250 kya. However, male‐mediated gene flow across the GVR has occurred over most of the history of Masai giraffes but may have ceased in the recent past as natural migratory routes across the escarpment have been blocked by human activities. These findings suggest that the Masai giraffe is now split into two separate metapopulations and in the absence of gene flow will eventually become separate species with populations of <20,000. Both ecosystems conserve biodiversity and large landscapes, and support two of Africa's few remaining long‐distance migrations of large mammals including the white‐bearded wildebeests (Connochaetes taurinus) and the plains zebras (Equus quagga) and along with their major predators including lions (Panthera leo) and leopards (Panthera pardus)  bisect the Masai giraffe populations in northern Tanzania into two distinct regions: west of the GRE including the Serengeti Ecosystem and east of the GRE including the Tarangire Ecosystem Figure . In tota2 for females and 157 km2 for males  , shot from a Model 196 Pneu‐Dart cartridge‐fired projector, to obtain skin biopsies. The type U Pneu‐darts are designed to collect a 3cc tissue plug and then immediately drop off the animal. The veterinarian aimed the rifle at flat surfaces on thighs or shoulders from a distance between 10 and 30 m. We removed the tissue samples from the needles, placed them in a 2 mL microcentrifuge tube, and secured the tube in a cool box with ice packs and froze them to −20°C within 6 h of collection. The samples were collected from different giraffe groups within a short time to minimize sampling closely related individuals and by choosing animals with dissimilar coat spot patterns and/or separated by a 1‐km distance. Animals with similar coat patterns are more likely to be related  and isolated tissue DNA with the Monarch Nucleic Acid Purification Kits using the manufacturer's protocol, but we increased incubation time to 12 h. to ensure the whole tissue was completely lysed. We extracted DNA from the samples at the Nelson Mandela African Institution of Science and Technology.d) and nucleotide diversity (π) using DnaSP whereas pairwise genetic fixation (FST) was calculated using the Arlequin version 3.5   of the 100 Masai giraffe dart biopsy samples using short read Illumina sequencing. We sequenced 25 samples at a medium coverage level  and 75 samples at low coverage level . Low coverage genomic sequence data are sufficient to accurately estimate most population genetic parameters utilizing genotype likelihood estimations as described below. To assess mitochondrial genetic variation, we extracted and assembled the whole mitochondrial genome  for the 100 dart biopsy samples. Uniquely indexed libraries for each of the samples were prepared at the Pennsylvania State University Huck Institute's genomic core and sequenced at the Pennsylvania State University Hershey genomics core on an Illumina NovaSeq. The samples were prepared with unique index sequence attached to the template. All samples were sequenced at the Sequencing facility in Hershey Medical Center, as 150 bp paired‐end reads on a multiplexed sequencing NovaSeq Illumina instrument. Bcl2fastq software was used to demultiplex the mixed sequences. During the demultiplexing, reads were checked against the adapter sequence (‐‐mask‐short‐adapter‐reads = 10) and the bases run into the adapter were turned to Ns. The 75 samples sequenced at low coverage yielded ~1.66× average peak depth of mapped reads and the 25 samples sequenced at medium coverage yielded ~24.1× average peak depth of mapped reads. In order to avoid the magnitude difference in coverage in the population analyses, we down‐sampled the 25 medium coverage reads by 85% using the random sample process (seed = 11) in seqkit v0.11.0  that was previously reported  and a full implementation of EM ML algorithm. The former produces an estimate of all parameters such as the individual inbreeding and site frequency, which is required as the initial values to start the iterations in the latter. To avoid the algorithm converging to local maxima, the approximated EM was run 20 times with a convergent criteria (‐‐min_epsilon) of 1e−5 and random generated initial values  in each run. We chose the parameters file of the run that exhibited the largest global log likelihood as the initial values in the full EM ML run (without ‐‐approx_EM flag) where a convergent criterion of 1e−9 was used.We estimated individual inbreeding coefficients 2.10K from 1 to 5 for five subpopulations where K is the number of ancestry states. The log likelihood of each K was extracted from the output log file and plotted against the K value. From the plot, the value of K was determined as the point at which the slope of line segments shifts downward. The result from NGSadmix (.qopt file) contains the inferred proportions for each individual and was plotted using the R package “xadmix”   haplotypes as they are to each other  as the calibration time and 289 kya using Giraffa .To assess mtDNA variation we sequenced a 1140 bp fragment for 320 individuals Table  and the G. c. thornicrofti), which mtDNA whole genome sequence data are available, exhibited the WMG1 haplotype  also revealed a large degree of differentiation on the PCoA1 axis between populations lying east and west of the GRE  haplotype, which may have resulted from an introgression event  sites. The Tarangire NP (TNP) and Manyara Ranch (MRC) giraffes exhibited the smallest FST (0.0140) whereas the highest FST values (0.0773–0.0832) were between the four western and eastern Masai giraffe pairwise populations that lie across the Manyara‐Natron escarpment from each other . We evaluated three potential dispersal routes  on geographic distance , while significant correlations for nuDNA FST were found for the Engaresero‐Salei WEP (R2 = 0.880**) and the Manyara‐Eyasi WEP (R2 = 0.874**) using the Rousset method , we estimated the correlation between pairwise population es Table  of mtDNAs Figure  as well Rousset, , and theRousset, . A highld Figure . No sign4The Masai giraffe population has plummeted in the past 30 years as the result of human activities including illegal hunting and land use changes creating fragmented populations with reduced opportunities for dispersal among them  the founders of the western and eastern sides of the GRE were independent, arising from ancestral Masai giraffe populations that already possessed different mtDNA haplotypes and (2) the western side of the GRE was founded first and subsequently the eastern side was founded by dispersal from the west. The independent origin hypothesis would require circumnavigating the steep GRE either from the north between the gap separating the western and eastern branches of the Great Rift in Uganda and Kenya or possibly from the south through gaps in the Tanzanian Craton in southern Tanzania and Zambia  is positive for all eastern giraffes and negative for all western giraffes. While the  Wright,  and furt Wright, . The ManFST values between eastern and western Masai giraffe populations range between 0.0773 and 0.0832, which are comparable to the difference between plains zebra populations in Etosha NP and Luangwa Valley NP separated by 1800 km  populations in British Columbia and Alaska separated by 2000 km of mountainous terrain and between wolf populations in Russia separated by 2100 km  for Masai giraffe in our study were high, comparable to the F of cheetah (Acinonyx jubatus jubatus)  utilizing the same method for estimating F , common elands (Taurotragus oryx), and giraffes  with no more than 20,000 in each. The proposed designation of western Masai giraffe and eastern Masai giraffe as ESUs is based on meeting the specific criteria that the two populations in question are reproductively isolated Waples,  and that2 Serengeti Ecosystem, with no natural barriers that should impede dispersal and gene flow between them, but dispersal and movement rates among WMG populations are unknown. WMG populations are subject to predation pressure and illegal hunting . LMNP is comprised of a narrow strip of land sandwiched between the Manyara‐Natron Escarpment to the west and Lake Manyara to the east. Wildlife dispersal routes around the southwest and northeast ends of Lake Manyara linking to other areas of the Tarangire Ecosystem  ; data curation (lead); formal analysis ; investigation ; methodology ; project administration (supporting); resources ; supervision (supporting); visualization ; writing – original draft ; writing – review and editing (supporting). Derek E. Lee: Investigation (supporting); writing – original draft (supporting); writing – review and editing (supporting). Lan Wu‐Cavener: Formal analysis (supporting); resources (supporting); writing – review and editing (supporting). David L. Pearce: Formal analysis (supporting); methodology (supporting); software (supporting); writing – review and editing (supporting). Monica L. Bond: Writing – original draft (supporting); writing – review and editing (supporting). Xiaoyi Hou: Resources (supporting); writing – review and editing (supporting). Douglas R. Cavener: Conceptualization (lead); data curation ; formal analysis ; funding acquisition (lead); investigation ; methodology ; project administration (lead); resources ; supervision (lead); validation (lead); visualization ; writing – original draft ; writing – review and editing .Benefits Generated: A research collaboration was developed with scientists from the Tanzania providing genetic samples, all collaborators are included as co‐authors and/or acknowledged for their contributions, and the research addresses a priority concern, in this case the conservation of Masai giraffe in Tanzania and more broadly giraffe East Africa. Our group is committed to international scientific partnerships, as well as institutional capacity building.Permission to conduct this research was provided by the Commission of Science and Technology #2020‐185‐NA‐1990‐172, Tanzania Wildlife Research Institute, Tanzania National Park Authority, Ngorongoro Conservation Authority, Manyara Ranch Conservancy, and IACUC# PROTO201901219 by Penn State University.Appendix S1.Click here for additional data file.


Neurodevelopmental disorders (NDDs) are complex, heterogeneous, conditions that show considerable clinical overlap. The concept of NDDs were introduced as an overarching category about 10 years ago in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM‐5)  associates with other mental health problems remains important given that most research this far has focused on the most common types of NDDs . Thus, the two papers above add an important focus on understudied subtypes of NDDs.In recent times, there has been a growing recognition, in particular in general medicine and primary care, of the need to take multimorbidity into account. Multimorbidity is typically defined as the co‐occurrence of two or more chronic disorders in the same person. According to some definitions of multimorbidity, these should affect multiple systems of the body (Skou et al., For individuals with NDDs, there is still a lack of research focusing on multimorbidity. A first important task for multimorbidity research in NDDs is to advance the understanding of co‐occurring physical health problems. Emerging evidence indicate that individuals with NDDs are at an increased risk for physical health conditions at various stages of the lifespan, including obesity, asthma, epilepsy, and gastrointestinal diseases (Arrondo et al., Another important task for future research is to identify potentially modifiable factors that influence the risk of multimorbidity in NDDs. The underlying mechanisms are most likely complex, but modifiable lifestyle and social factors most probably play a role. Although an increasing number of studies have examined multimorbidity interventions, the evidence is still limited (Skou et al., Longitudinal research using observational designs will be critical to address the knowledge gaps surrounding multimorbidity in NDDs, but the reporting and methodological standards of such studies needs to remain high or increase. Several publications in JCPP Advances have made contributions that aims to improve the standards of observational research. For example, an editorial from Larsson  highlighUntil recently, there was a general view that NDDs was limited to childhood, but it is now increasingly recognized that NDDs can be lifelong conditions. Not only that, a lifespan perspective on NDDs has been implemented to an increasing extent both in research and clinic settings. Longitudinal studies of individuals with NDDs have played a major role in changing this perspective. Hopefully, longitudinal studies will play an equally important role in transforming the health care of multimorbidity in NDDs.Henrik Larsson: Conceptualization; Writing – original draft; Writing – review and editing.Advances.Henrik Larsson reports receiving grants from Shire Pharmaceuticals; personal fees from and serving as a speaker for Shire Pharmaceuticals and Evolan Pharma AB outside the submitted work; and sponsorship for a conference on attention‐deficit/hyperactivity disorder from Shire Pharmaceuticals outside the submitted work. He is Editor‐in‐Chief of JCPP


The development of DNA‐based methods in recent decades has opened the door to numerous new lines of research in the biological sciences. While the speed and accuracy of DNA methodologies are clearly beneficial, the sensitivity of these methods has the adverse effect of increased susceptibility to false positives resulting from contamination in field or lab. Here, we present findings from a metabarcoding study on the diet of and food availability for five insectivorous birds, in which multiple lepidopteran species not known to occur locally were discovered. After describing the pattern of occurrences of these non‐local species in the samples, we discuss various potential origins of these sequences. First, we assessed that the taxonomic assignments appeared reliable, and local occurrences of many of the species could be plausibly ruled out. Then, we looked into the possibilities of natural environmental contamination, judging it to be unlikely, albeit impossible to fully falsify. Finally, while dissimilar combinations of non‐local species' occurrences across the samples did not initially suggest lab contamination, we found overlap with taxa and sequences handled in the same lab, which was undoubtedly not coincidental. Even so, not all exact sequences were accounted for in these locally conducted studies, nor was it clear if these and other sequences could remain detectable years later. Although the full explanation for the observations of non‐local species remains inconclusive, these findings highlight the importance of critical examination of metabarcoding results, and showcase how species‐level taxonomic assignments utilizing comprehensive reference libraries may be a tool in detecting potential contamination events, and false positives in general. The sensitivity of DNA‐based methodologies provides great benefits while also carrying inherent risks of contamination, and subsequently false positives in the final datasets. Here, we present the observations of sequences assigned to presumed false positive non‐local species, investigate their likely origins, and discuss how best to resolve them. In addition, local fauna and particularly lepidopterans have been mapped and monitored extensively. These factors made metabarcoding a promising method to study the diets of several local insectivorous birds: the crested tit , willow tit , blue tit , great tit  and pied flycatcher . Numerous aspects of the biology of several of these species have a long tradition of research both locally  collected in the same area. We look into the various pathways via which this DNA could have ended up in our samples, namely unknown local occurrences of the species, airborne transfer and subsequent contamination in the field, errors in amplification/sequencing or lab contamination. Finally, we discuss the ramifications of these findings for metabarcoding studies. With metabarcoding and other DNA‐based techniques becoming increasingly widely used and accessible, awareness of the potential for such observations is vital.2Pinus sylvestris, L.), Norway spruce  H.Karst) and birch , interspaced with swamps and clear‐cut areas . These areas consist of a patchwork of younger and older stands of predominantly Scots pine , first using the Species Level Barcode Records database, then the All Barcode Records data if no match was available in the former . As BOLD lacks explicit specified query coverage/overlap, at least one sequence from a public record of the best matching taxon was compared to the relevant query sequence. Due to the short target region (145 bp), we discarded matches based on <96% coverage (<140 bp overlap). Identities discarded this way often stood out as they typically had only a single reference record as a match and were phylogenetically distant from all other matches. The Full Length Record database was used when poor overlap between query sequence and suggested matches was suspected, and overlap could not be verified because of non‐public reference sequences. If a single species presented the best match, this would be the assigned identity; if multiple species were equally likely , the lowest shared taxonomic level would be used instead. If locally occurring species were listed at the same match % as species not known to occur locally, the former were favoured. A threshold of 97% match was used for normal identifications, but for the non‐local species presented here, we accepted only those with a stricter >99% match (a maximum of 1 bp difference between queried and reference sequences). If no matches were available above this threshold, ASVs received the ‘No match’ identity instead. ‘No match’ ASVs are included in the full data (Appendix gbif.org). ‘Non‐local species’ are defined as species with known distribution ranges >200 km from the study area. Given typically limited mobility in the lepidopterans studied here and their widespread monitoring, this was considered sufficient to rule out local occurrences. Many species found outside this range would furthermore be constrained by the lack of host plants and climatic conditions, preventing local settlement in the focal boreal area. For the purposes of this paper, we only consider the not locally occurring Lepidoptera encountered, as other arthropod taxa are typically too limited in both known distribution ranges and abundance of reference barcodes for similar assessments.ASVs (Amplicon Sequence Variants) were assigned taxonomic identities primarily using the Identification Engine in BOLD  and the creation of Neighbor‐Joining trees to investigate similarities were performed using MEGA‐X, version 10.1.8 .To investigate specific potential sources of contamination, we compared our findings to sequencing results from two unrelated studies for which samples were handled in the same lab around the same time as the original 2017 batch. The first were faecal samples for a dietary study  collected near Barcelona, Spain, from blue tits and great tits, which would have been processed using the same protocol described above. We performed DNA extractions and PCR of these samples in December–January 2018. The second concerned a phylogenetic study on Cochylina  for Rileyiana fovea; Satyrium spini (Denis & Schiffermüller 1775) for Satyrium esculi; Aethes baloghi (Sabourin & Metzler 2002) for Aethes seriatana; cases 18, 19 and 22, respectively in Appendix Eighty four ASVs were identified as 25 different non‐local species  and C. sponsa (Linnaeus 1767) were presented as slightly less likely matches. Overall, results from BOLD were supported for many non‐local but still Finnish taxa , while no probable alternative identities were provided for any of the residual ASVs.Some species had such numerous reference material in BOLD that the full Top 100 only displayed identical matches to that species, possibly hiding alternative identities from view. The Protax taxonomic assignments, used to investigate the validity of these identities and explore similarities to local species, are displayed alongside their BOLD counterparts in Appendix 3.3Read counts for individual ASVs matched to non‐local species ranged from 19 to 6036 per sample (after 0.1% RRA filter). The abundances of individual ASVs matching non‐local species ranged from 0.10% to 22.75% per sample, and the total contribution of non‐local species in a sample ranged from 0.10% to 55.35% .3.4The non‐local species were present in material from all four bird species sampled in 2017 as well as the frass (sheet) samples, as displayed in Figure 3.5A. marginaria, B. bicolorana, M. lunaris, L. dispar, M. neustria; Peridea anceps, Xestia agathina) and only three species more frequently seen further inland . Eleven of the remaining species have European distributions, varying from widely ranging across the continent to more limited distributions typically concentrated in the west or south. Examples of non‐local Finnish and southwestern European distributions are illustrated by L. monacha and S. esculi in Figure A. seriatana, Henricus cognatus, H. umbrabasanus, Platphalonidia felix) are North American; all ASVs assigned to North American species were rare (occurring in one to two samples) and had low abundances (0.11%–0.26% per sample). Maps illustrating the approximate occurrences of all 25 species are provided in Appendix Of the non‐local species described here, 10 have occurrences or at least observations from southern Finland (approx. 200–500 km south of the study area), with most restricted to the southern coastal or archipelago region (3.6L. dispar) present in 35 samples. The various species and specific ASVs occurred in numerous different combinations and quantities across the samples, lacking any clear pattern. We placed samples in order of field collection date to look into the onset, peak and end of specific species' appearances  was placed among the C. nymphagoga ASVs.A phylogenetic tree constructed for the full set of 952 different ASVs  did reveal other sequences matched to P. anceps and X. areola in the Barcelona data. Of the four North American species from our data similarly compared to the locally handled Cochylina, one of the H. cognatus ASVs (with a 100% match to reference data) and the P. felix ASV matched completely to sequences from that study. The second H. cognatus (with a 99.31% match) and the A. seriatana sequence both deviated by 1 bp for the nearest equivalents, while the H. umbrabasanus ASV differed for three separate and distant base pairs. The closest matches for all ASVs were identical to the taxonomic assignments given by BOLD.Of the 84 ASVs assigned non‐local species identities, 22 fully matched those in the Barcelona data. We also specifically compared only those ASVs from our study that were identified based on 100% matches to the BOLD reference library, to prevent potential sequencing errors from inflating the apparent differences. Of the 22 ASVs found in both studies, 21 were those with 100% matches to reference data, leaving 13 of our ASVs with 100% matches with no equivalents in the Barcelona data. Among the ASVs with overlap in both studies were representatives of 19 of the 25 non‐local species, with only 3.9L. dispar) with an abundance of 0.45%. Three new and three previously seen non‐local species were found in 6 samples in the next batch (#4), with a new sequence for C. nymphagoga. The only new non‐local species without a European distribution  was found here, although it had a wide range of alternative matches in BOLD at slightly lower percentages, none of which represented Finnish taxa. Abundances of non‐local species were predominantly low at <1% for five samples and 11.44% in the last. The latter was one of a pair of replicates for which subsets had been taken from the same original sample, but neither of the two non‐local species found in this sample were recorded for the replicate—limited similarities were found for the taxa in these two subsamples in general. Another pair of replicates for which only one contained a non‐local species (a new L. monacha haplotype) was found in the last large sequencing batch (#5). Here, both replicates were otherwise largely in agreement, but differences existed for ASVs with abundances <1%, and the L. monacha ASV had an abundance of 0.46% in the one sample. The same ASV was found at 0.61% in a different sample in the same batch. Of the in total 15 new ASVs for non‐local species found in later years, none fully matched material from Barcelona, although eight were identified as species that were previously found from there. For the new non‐local species, only Stauropus fagi (Linnaeus 1758), observed in the second batch, was detected in these data, although the exact sequences differed. None of the aforementioned non‐local species, nor any others, were found in any of the negative controls that were sequenced.No ASVs assigned to the same 25 or any other non‐local species were found in the 50 additional samples from 2017 that were evaluated in subsequent years. Across 474 samples analysed from later years, 22 samples contained a total of 25 different ASVs identified as non‐local species . Without prior knowledge on the distribution ranges of the relevant species, the observations of these species in our data would have appeared perfectly reliable. Assuming the taxonomic assignments of the specimens used for the reference barcodes were correct—highly likely, since many of our identifications were based on multiple independent reference sequences—the detection of DNA belonging to these non‐local species in our samples does appear to be genuine. This then leads to the next question: how did it get here?The first question our findings raised was whether the identities had been correctly assigned. The vast majority of ASVs presented here were assigned species based on multiple matches with high similarities (99%–100%) to reference sequences in BOLD and an absence of probable matches to more locally occurring species. Since complete coverage of all potential haplotypes of a species in the reference library would not be expected, matches lower than 100% are often considered acceptable, with a threshold >98% commonly used in metabarcoding studies , when they might be more suitable prey for the flycatchers. . Finally, local settlement of the non‐local species would likely have resulted in continued observations in later years, which was not the case for many.The most straightforward explanation would be that the Lepidoptera in question did in fact occur locally after all. Although this is impossible to fully disprove, it is unlikely for a number of reasons. Firstly, the field area has a fairly isolated location, near the western coast in northern Finland. Southern species would have had to cross the Gulf of Bothnia from Sweden, or traverse much of Finland, to reach this area, and have done so without observation along the route. For some species, the study area is half the European continent removed from their nearest known occurrences, while the North American species are naturally found even more distantly. That said, many lepidopterans are shifting northwards with ongoing climate change  and pied flycatchers, which arrive to our field areas in late April–early May . While the flycatchers are not known to consume the hairy caterpillars, cuckoos do consume these in large quantities  does hint at a lab‐based origin for the sequences. On the other hand, we have the absence of many specific sequences from the Barcelona data, the continued presence of some species over many years, and the emergence of wholly new variant sequences in later years. To the best of our knowledge, no other studies using these or similar species were conducted in this lab during this time besides those mentioned above.With few technical replicates and no non‐local species encountered in negative controls, we attempted to trace back potential sources of the non‐local species indirectly. The distribution of non‐local occurrences across the samples did not indicate any clear point of contamination, suggesting either another cause or that the contamination was widespread. We cannot rule out that some cross‐contamination occurred when sample tubes were open when drying, but this would not account for the initial introduction of the non‐local species, nor the occurrences at high abundances. The many variable combinations in which non‐local species were present makes it unlikely that, for example, a single reused tool was the cause, even considering methodological stochasticity. Although there were limits to how well we could reconstruct events several years down the line, two other analyses conducted locally around the same time appeared relevant. While the North American Cochylini studied for a phylogenetic study perfect matches to existing species. The non‐local species were distinct from each other and had no particular similarity to local species either, making a shared chimeric origin unlikely. Their frequent semblance to unidentified ASVs (which would include chimeras) is more easily explained by the fact that sequences required a >99% match to be identified as non‐local species, meaning that similar ASVs might have fallen below this threshold. Errors in sequencing could be plausible explanations for some variant ‘haplotypes’ we found, especially since our analyses used ASVs instead of MOTUs. For instance, visual inspection of the aligned sequences showed that a section often having seven repeated T‐bases was shortened to six T‐bases in several sequences, an erroneous call known to occur for homopolymers by the Ion Torrent PGM. While this barely impacted taxonomic assignments , it would have resulted in the apparent existence of a different haplotype. This specific issue was only seen six times, however, and some ASVs with <100% matches are plausibly genuine, simply lacking a reference barcode of the exact same haplotype. Since ASVs with 100% matches are likely to be free of these or other errors, it is puzzling how 100%‐matching ASVs lacking equivalents in the Barcelona data were found in the original 2017 dataset, and that more were encountered in the following years. A further potential origin of apparent haplotypic variation are nuclear mitochondrial DNA transpositions (NUMTs) Leite, . NUMTs o4.7Upon first noticing the non‐local species in the data from 2017, our initial reaction was to effectively run through the steps described above, albeit more superficially: we validated identities, explored the affected samples, attempted to rule out local occurrences and imagined alternative origins of the DNA sequences. In doing so, we strove to keep a balance between narrowing down options by ruling out pathways, while keeping an open mind for unknown factors. Lacking a satisfying answer, we hoped a combination of additional samples from 2017 and further material from later years would eventually resolve matters. The absence of continued high‐abundance occurrences of the non‐local species in later years pointed towards a one‐off (a)biotic transmission of DNA or contamination, while the absence in separately analysed additional samples from 2017 favours the latter explanation. Our methodologies have largely remained unaltered so as to allow fair comparisons over the years—besides the decision to sequence negative controls; a change explicitly made to better detect potential contamination events.L. dispar ; data curation ; formal analysis (lead); funding acquisition ; investigation (lead); methodology (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Marko Suokas: Data curation (lead); investigation ; methodology ; writing – original draft . Tuukka Petäjä: Conceptualization (supporting); investigation (supporting); writing – original draft . Ulla Saarela: Investigation (supporting); methodology (supporting); writing – original draft (supporting). Seppo Vainio: Conceptualization ; investigation (supporting); writing – original draft . Marko Mutanen: Conceptualization (lead); formal analysis ; funding acquisition ; investigation ; methodology ; supervision (lead); writing – original draft .Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.Appendix S4Click here for additional data file.Appendix S5Click here for additional data file.Appendix S6Click here for additional data file.


To the Editor: We read with interest Foster et al’sWe would like to bring extra attention to one finding in Foster et al’sIt is well established that systemic racism impacts Black patients in the American health care system, limiting access to quality healthcare in many ways. Recruiting and encouraging more Black medical students to enter dermatology could be one straightforward approach to addressing inequities in the field. Notably, a recent study showed limited racial, ethnic, and sexual orientation diversity among students pursuing dermatology and that these students were less likely to pursue care in public health or for marginalized populations.The lack of Black dermatologists, representing only 3% of dermatologists (compared to 12.8% of Americans),None disclosed.


The exponential growth of aquaculture over the past 30 years has been accompanied by a parallel increase in the use of antimicrobials. This widespread use has had negative effects on animal, human and environmental health and affected the biodiversity of the environments where aquaculture takes place. Results showing these harmful effects have been resisted and made light of by the aquaculture industry and their scientific supporters through introduction of misunderstandings and misinterpretations of concepts developed in the evolution, genetics, and molecular epidemiology of antimicrobial resistance. We focus on a few of the most obvious scientific shortcomings and biases of two recent attempts to minimise the negative impacts of excessive antimicrobial use in Chilean salmon aquaculture on human and piscine health and on the environment. Such open debate is critical to timely implementation of effective regulation of antimicrobial usage in salmon aquaculture in Chile, if the negative local and worldwide impacts of this usage are to be avoided. The use of antimicrobials as growth promoters, prophylactics and therapeutics in terrestrial animal husbandry has been repeatedly shown to result in capture of mobile genetic elements and antimicrobial resistance genes derived from agricultural environments by human commensal and pathogen resistomes  than in terrestrial food animal production (140 mg kg−1) or patient care (91.7 mg kg−1)  and 17.3 times its average usage in salmon aquaculture worldwide (27.0 mg kg−1)  where it may be reached by antimicrobials  fish in Chile where it causes no disease and produces no pathology  contained functional genes for resistance to chloramphenicol, tetracyclines, aminoglycosides and sulfonamides  of antimicrobials in the environment. It is also not clear whether Salgado‐Caxito et al.'s risk assessment included any of the many publications documenting the presence of antimicrobial‐resistant commensals and potential pathogens or the presence of antimicrobial residues in fillets from fish raised with antimicrobials  and those of commensals and pathogens of the human intestinal microbiome in people who consumed such fillets ; validation ; writing – original draft ; writing – review and editing . Ana R. Millanao: Conceptualization ; validation ; writing – original draft ; writing – review and editing . Ivonne Lozano‐Muñoz: Conceptualization ; validation ; writing – original draft ; writing – review and editing . Henry P. Godfrey: Conceptualization ; validation ; writing – original draft ; writing – review and editing .The authors declare no conflict of interest.


Dispersal is a fundamental process in evolution and ecology. Due to the predominant role of flight in bird movement, their dispersal capabilities can be estimated from their flight morphology. Most predictors of flight efficiency require an estimate of the total wing area, but the existing methods for estimating wing area are multi‐stepped and prone to compounding error. Here, we validated a new method for estimating the total wing area that requires only the measurement of the wingspan plus two measurements from the folded wings of study skin specimens: wing length and wing width. We demonstrate that the new folded‐wing method estimates total wing area with high precision across a variety of avian groups and wing shapes. In addition, the new method performs as well as the old method when used to estimate natal dispersal distances of North American birds. The folded‐wing method will allow for estimates of the total wing to be readily obtained from thousands of specimens in ornithological collections, thus providing critical information for studies of flight and dispersal in birds. Estimating flight capabilities from wing shape is a powerful tool for the comparative analysis of dispersal in ecology and evolution. Here, we describe new methods for estimating the total wing area, which will allow for readily estimating wing aspect ratios and wing loadings for thousands of bird species. We obtained a total of 112 specimens with these characteristics, 93 from the Royal Ontario Museum (ROM) and 19 from the Louisiana State University Museum of Natural Science  to one of the largest swans , and species spanning the full spectrum of wing morphologies, from an albatross  representing extremely elongated wings (aspect ratio 14.1), a tern  representing highly pointed wings (aspect ratio 12.1) to the Little Greenbul  representing short and rounded wings (aspect ratio 4.1). Most species were represented by a single specimen, but we also collected larger samples for three species to analyze how the estimators fare with intraspecific datasets. These three species were selected to represent three different wing shapes: Cyanocitta cristata (n = 16) representing short, rounded wings, Columba livia (n = 18) representing intermediate wings, and Archilochus colubris (n = 17) representing elongated, pointy wings.The specimens represented 60 species from 40 families Appendix . The samWL) and width (S1) on the folded wings of standard skin specimens. Measurements were obtained using digital calipers or rulers depending on the size of the specimen. Wingspans were measured by the authors on fresh specimens or obtained from the specimen labels.We measured wing length . We set the scale using the scale ruler in the image and used the “Threshold” tool to transform the photo into a binary (black and white) image, sometimes followed by the “Fill Holes” option to fill small gaps. Most images were further edited manually using the “Brush” tool to fill small border gaps in broken or disarranged feather vanes, or to fill marginal white spots not distinguished from the background by the thresholding tool. Finally, the “Eraser” tool was used to delete anomalies due to disarranged cover feathers, usually near the shoulder area, and to create a straight proximal border that would represent the root chord  using the wingspan and S1 , analogous to the method used to calculate the root box area in the traditional method , and S1 represents the entire minor axis  in which s Figure . This shs Figure . Howevers Figure  and 4, bAspect ratio. Using the different estimators of the total wing area, we estimated the wings aspect ratio using Equation (Equation .2.310) to eliminate effects of scaling with body size and the resultant heteroscedasticity. Additionally, we assessed how well the folded‐wing method predicted total wing areas produced by the traditional spread‐wing method using phylogenetic generalized least squares models . The increased precision of the new folded‐wing method is likely due to the fewer steps in which error can be introduced. In the traditional spread‐wing method, errors can be introduced during four procedural steps: (1) wing posturing and specimen preparation , (2) photographing , (3) digital processing , and (4) measuring distances. By contrast, the new method only requires measuring three distances: wingspan, wing length, and wing width. The wingspan is known to show relatively low repeatability Winker, , but winNote that although wing areas estimated by the traditional spread‐wing method are a useful reference, they cannot be considered the “ground truth.” Because the avian wing is an articulated limb that can adopt different shapes and surface areas depending on the degree of flexion, there is no such thing as “the true wing area” that can be measured directly and objectively. Even minimizing all human‐related and tool‐related errors, the area estimated by the traditional method will depend on the posture of the wing. In this sense, the traditional method of estimating the surface area on a spread‐wing specimen is also a model, a model that assumes that the posture of the specimen measured is correct and the most relevant for aerodynamic purposes. That assumption does not hold for thousands of spread‐wing specimens that are either underextended or overextended, resulting in underestimation and overestimation of the relevant surface area.The new method also fared well when used to predict empirical data on bird movement. The aspect ratio calculated using wing areas estimated with the folded wing method predicted natal dispersal distances of North American birds as well as the aspect ratio calculated using wing areas estimated with the traditional method. This demonstrates the effectiveness of the new method in studies of bird movement and dispersal.Among the geometric models evaluated for the folded‐wing method, assuming that the shape of the hand‐wing was a half or a quarter of an Ellipse resulted in more accurate and precise estimates as compared to assuming a triangular shape. Because assuming half or a quarter of an ellipse resulted in the same formula for the total wing area, this model has the potential to fit a wide variety of wing shapes. Birds with rounded wings such as jays, wrens and cardinals may be better represented by the half‐ellipse model, whereas species in which the most distal primaries are the longest such as waxwings, swallows, and gulls may be better represented by a quarter of an ellipse Figure . In theoAssuming that estimates from the traditional spread‐wing method are not biased, the simplest Ellipse model evaluated here showed a tendency to overestimate wing areas. This may be due to multiple factors and may depend on the shape of the wing. None of these simple geometrical models take into account the slots or gaps between feather tips, and the wings of many species may be less convex than what the elliptical curvature implies. We experimented with assuming that one of the sides of the wings was straight instead of convex, leading to the Ellipse‐Triangle model Figure , and thiStercorarius . The use of old museum specimens will be particularly beneficial for obtaining estimates of wing area and aspect ratio for rare and poorly known species. In addition, the wing length and secondary length measurements for most bird species are already available in the AVONET database  Fu: Conceptualization ; data curation (lead); formal analysis (lead); writing – original draft (lead). Michelle Su: Data curation (supporting); writing – review and editing (supporting). Jonathan J. Chu: Data curation (supporting); resources (supporting); writing – review and editing (supporting). Alexandra Margaritescu: Data curation (supporting); writing – review and editing (supporting). Santiago Claramunt: Conceptualization ; formal analysis (supporting); funding acquisition (lead); supervision (lead); writing – original draft (supporting).This study received support from the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant RGPIN‐2018‐06747 to S.C.Appendix 1.Click here for additional data file.Appendix 2.Click here for additional data file.


Multidisciplinary team meetings (MDTMs), where treatment recommendations are discussed and agreed, are fundamental to effective cancer care. The increasing volume and complexity of caseloads has led to the need to transform MDTM pathways to improve efficiency and allow sufficient time for discussion of complex cases. Understanding of current functioning and inefficiencies is required to inform such transformation.n = 8 MDTs, 96 MDT meetings). Data were collected on meeting attendance and on each discussed case using a validated MDT tool. Semi‐structured interviews were conducted with a range of MDT members and cancer service managers to gain understanding of perceived influences on the efficiency of MDTMs.A mixed‐methods observational study of all lung cancer MDTMs in one UK cancer network over 12 weeks (n = 56) identified addressing clinical and administrative workforce shortages, less reliance on the MDTM for pre‐diagnostic decision‐making and better availability of key clinical information about patients discussed in the MDTM as factors critical to improved MDT function.In total, 1671 case discussions were observed. Models of MDT working, including referral and diagnostic pathway management, varied within the network. Attendance was quorate in only 21% of the observed MDTMs, most often lacking palliative care specialists. Over a third (37%) of observed cases were repeat discussions pre‐diagnosis. Treatment recommendations were agreed in 48% of case discussions but deferred for a quarter (24%) of discussed cases, most commonly due to awaiting results. Information about patients' fitness for treatment and/or performance status score was available for 60% of cases discussed overall (30%–75% by MDT). Interviews (Inefficiencies were prevalent in all MDTMs; improvements would require an individualised approach due to the variation in ways of working. Local, regional and national support is needed for lung MDTs to develop their diagnostic workforce and facilities, and clinical and administrative resource. There is considerable variation in MDTM structures and processes, influenced by organisational contextual factors. Inefficient referral and diagnostic pathways were highlighted, causing repeat discussions. Developing the diagnostic workforce and facilities to support implementation of diagnostic pathways will improve efficiency. This study was reviewed and approved by the Health Research Authority (IRAS 270697) following approval from the NHS London Fulham Research Ethics Committee (ref. 19/LO/1699) and Confidentiality Advisory Group (ref. 19/CAG/0211).2.2Non‐participant observational data was collected in virtual lung cancer MDT meetings in each of the eight lung MDTs over a period of 12 consecutive weeks between April 2021 and June 2021.2.2.1Researchers used a modified version of a validated checklist, the MDT QuIC tool,2.2.2The main lung cancer MDTMs were observed in each hospital. These were defined as meetings where it was intended for all core MDT members to attend, according to the standard set for lung MDT quoracy which specifies the attendance of the following: designated respiratory physician(s); designated thoracic surgeon(s); clinical oncologist; medical oncologist ; imaging specialist; histopathologist; designated cytologist; lung nurse specialist; a core member of the specialist palliative care team; MDT co‐ordinator or secretary.2.3Semi‐structured interviews were conducted between April and August 2021 with MDT members and cancer service managers in the eight hospitals by the researchers collecting observational data in each MDT. Participants were asked about their MDT structure and processes and their views were sought about the efficiency and effectiveness of their lung cancer pathway and MDTMs. Interviews were audio‐recorded and transcribed verbatim. Researchers met weekly to discuss emergent findings, as well as sharing interview notes and transcripts from interviews, so that the topic guide could be adapted iteratively in line with emergent findings.2.3.1Every core MDT member and other regular attenders of each of the eight MDTs participated in the observed MDTMs. Core MDT members and Cancer Services Managers were eligible to be invited for participation in interview(s). Interview participants were purposefully sampled to include members from each of the eight MDTs and represent all core professional groups, as well as each MDT lead clinician , medians and interquartile range (IQR) for continuous data) were calculated overall and by MDT to facilitate reporting of the variation in ways of working between MDTs. All statistical analyses were conducted in Stata 16. To protect the anonymity of the participating MDTs only overall percentages are presented (data available on request) and different sets of pseudonyms are used for sets of data presented.Data relating to MDT ways of working were collated from observation and interviews and cross‐checked for agreement within each MDT. Views relating to the efficiency of MDTMs were analysed thematically, using an inductive approach and constant comparison of data within and between MDTs. Observational and interview data were integrated by using constant comparison between the datasets—cross checking key findings from one dataset with the other, reporting where data were concordant, discordant and/or ‘silent’ on key findings/themes.3A total of 96 MDTMs were observed including 1671 patient case discussions. This ranged from 4 to 33 case discussions per MDTM . Fifty‐six interviews were conducted with MDT members and Cancer Service Managers, lasting between 16 and 51 minutes. Four to eight MDT members were interviewed from each team and at least two members of each core professional group, except palliative care, across the MDTs  and none of the MDTs achieved quoracy for all 12 observed MDTMs, even when incidences of partial attendance were accounted for. In most cases, this was explained by the absence of palliative care members (at 62 MDTMs). In addition, 6 of the MDTs had 2–3 meetings without the required oncology input, five MDTs held between 1 and 3 MDTMs without a surgeon present, and 1 MDT did not have a pathologist present for any of the 12 observed MDTMs.3.2MDTs varied in relation to their MDTM processes including how MDT referrals were triaged for inclusion on the MDTM agenda. Across the eight MDTs, three methods were identified: (1) formal meetings  between the MDT lead and a radiologist/MDT coordinator/deputy MDT lead (three MDTs); (2) single MDT member completing triage on their own (three MDTs); or (3) ad‐hoc triage where there was no apparent fixed process/time for triage (two MDTs).The additional forums available for case discussion outside of the full MDTM were also diverse across the MDTs. Only four of the eight MDTs had a separate MDTM for making decisions relating to further diagnostics  and the MDT would have limited communication with them outside of the meetings.3.3The variability in relation to referral processes, triage methods, and existence of other lung‐related MDTMs or forums within the hospital (in particular whether or not they had a separate diagnostic MDTM)—described above—meant that the types of cases discussed in full MDTMs also varied between the eight MDTs. Regardless of the models of triage or other MDTs/meetings outside of the full MDTM, most cases  listed for discussion in lung MDTMs were prior to lung cancer diagnosis  were pre‐diagnostic patients that had been discussed at least once previously in the full MDTM  there needs to be a culture change from discussing everybody multiple times before you make any decision about the patient into making decisions that are fairly straightforward outside the MDT [MDT lead]In most MDTs, there was a view that some patients were discussed unnecessarily in the full MDTM. A variety of reasons was given for this including that the MDTM provided the only opportunity in some MDTs for a benign case to be discussed with a surgeon, for example where the surgeon worked for a different hospital and was not easily accessible otherwise. Reference was also made to a team culture where it had become the norm to list patients for MDTM discussion, regardless of whether it was appropriate or not, and therefore to have patients discussed multiple times:the radiologists… for example, if we wanted a CT biopsy, they want to see the PET first, and discuss it in the meeting to agree that a CT biopsy is appropriate. (…) it's whether we can persuade them to, or somehow find a way of doing that outside of the meeting [MDT Lead]In some MDTs the availability of slots for diagnostic procedures and the radiology departments' preferred process of booking in patients affected the timeliness of diagnostic results and consequently caused difficulty in implementing diagnostic “bundles”  for patient referrals to the MDTM, but these were often poorly used/completed; the MDTs depended on GPs and their colleagues in other hospital departments to follow referral procedures and provide comprehensive and accurate information on referrals, but this was often not the case:it's up to the referring clinician to provide the data. I can't go back and say, “That's not there. I'm not listing it”, because I'm not clinical [MDT coordinator]One MDT coordinator expressed that they felt disempowered when dealing with poor quality referrals:my barriers are a lack of admin staff who are specifically trained to deal with lung cancer patients, because our cancer office all revolve and one day we have one co‐ordinator, and another day we have another co‐ordinator [MDT lead]It was also mentioned that in some MDTs there was insufficient or inadequately trained administrative staff to support collection of the required data/information for each listed case:So, if you have to gather information for a patient who has got a lung cancer, and you need the pulmonary function test, you need the CT, the PET CT but which normally is done by a radiologist. You need the histology, and you need the previous investigations from different places. For one patient that can take you like an hour and a half, two hours already. [surgeon]MDT members judged that they did not have sufficient time to prepare for MDTMs which would include assessing which referrals should be listed for discussion and which sent back for more information and providing comprehensive patient information for MDTM agendas. This was made more time consuming by having to access multiple systems for information:This was often perceived as difficult to resolve as the staff time required to prepare for MDTMs would need to be agreed across multiple departments in the hospital, which would not necessarily benefit directly from having a more efficient MDTMs.3.5Despite being the putative aim of MDTMs, only half 53%) of observed case discussions concluded with a recommendation for treatment (48%) or to discuss treatment options with a patient (5%). This varied across MDTs . It indicates that even within a relatively limited geographical area in the UK, there is considerable variation in terms of MDTM processes and ways of working, particularly the MDTs' structures and processes relating to referral and diagnostic pathway management. These differences are driven by contextual and organisational factors , which cannot be easily overcome, implying that streamlining MDTMs and improving efficiency cannot be achieved via a one‐size‐fits‐all approach. Despite this, there are several possible measures which could improve the efficiency of lung MDTMs.Considerable MDTM time is spent confirming diagnoses with multiple discussions per patient prior to initiating treatment. Data from this study indicates that if clinicians managed a proportion of cases individually or within a smaller team up to the point of diagnosis, it would reduce the number of cases discussed in the MDTM. This would require alterations in clinician job plans to provide adequate time to triage cases referred to the MDTM and to discuss patient diagnostics in other appropriate forums. Adequate governance must exist around patient tracking lists and associated meetings, as it has been previously found that some clinicians are reluctant to streamline due to concerns about the detrimental impact this may have on the quality and safety of patient care, including clinical governance process.Furthermore, data from this study reinforce the need to prioritise addressing workforce shortages, both in clinical and administrative roles, although it is recognised that the COVID‐19 pandemic placed a substantial burden on staff during the data collection period. This is particularly evident for radiology and pathology expertise,A large proportion of MDTM case discussions did not include key clinical information about the patient. Although use of MDT referral proformas has been recommended to ensure information is available to the MDT5This study highlights the complexity of factors influencing the ways that lung MDTs in the UK are structured and the differing ways in which they work. Improving efficiency of the MDT pathway will therefore require contextualised approaches that take into account these differences. However, based on our findings we recommend that MDTs: (1) work with stakeholders to standardise the referral process and improve the quality of clinical information on referrals; (2) commit adequate clinical and administrative resources to support managing caseloads more effectively outside of MDTMs; and (3) examine potential for improvement regarding internal and external input from pathology, oncology and surgery. Finally, local, regional and national support are needed for lung MDTs to assist them in developing their diagnostic workforce and facilities. The findings from this research have implications for cancer multidisciplinary working in a variety of contexts, with similar challenges to efficient MDTMs noted in research outside of the UK and in other tumour types.Magdalena Zasada: Formal analysis (supporting); investigation ; project administration ; writing – original draft (lead); writing – review and editing (lead). Jenny Harris: Formal analysis (lead); methodology ; writing – review and editing . Johanna Groothuizen: Investigation ; project administration ; writing – review and editing . Eunice Aroyewun: Investigation ; project administration ; writing – review and editing . Jeewaka Mendis: Formal analysis (supporting); methodology ; writing – review and editing . Cath Taylor: Conceptualization ; funding acquisition ; methodology ; project administration ; supervision ; writing – review and editing . Madeleine Hewish: Conceptualization ; funding acquisition ; methodology ; supervision ; writing – review and editing .All authors have declared there are no financial conflicts of interest with regards to this work.This study was reviewed and approved by the UK Health Research Authority (IRAS 270697) following approval from the NHS London Fulham Research Ethics Committee (ref. 19/LO/1699) and Confidentiality Advisory Group (ref. 19/CAG/0211). Study researchers held: (1) Fully completed and signed Research passport document; (2) Letter of Access from each participating organisation; (3) Additional confidentiality agreements as required by individual organisations depending on their own requirements for CAG supported studies. Participants consented to the study by returning informed consent forms.Data S1.Click here for additional data file.


The timing of seasonal activity, or phenology, is an adaptive trait that maximizes individual fitness by timing key life events to coincide with favorable abiotic factors and biotic interactions. Studies on the biotic interactions that determine optimal phenology have focused on temporal overlaps among positively‐interacting species such as mutualisms. Less well understood is the extent that negative interactions such as parasitism impact the evolution of host phenology. Here, we present a mathematical model demonstrating the evolution of host phenological patterns in response to sterilizing parasites. Environments with parasites favor hosts with shortened activity periods or greater distributions in emergence timing, both of which reduce the temporal overlap between hosts and parasites and thus reduce infection risk. Although host populations with these altered phenological patterns are less likely to mature and reproduce, the fitness advantage of parasite avoidance can be greater than the cost of reduced reproduction. These results illustrate the impact of parasitism on the evolution of host phenology and suggest that shifts in host phenology could serve as a strategy to mitigate the risk of infection. Environments with parasites favor hosts with shifted phenology to reduce the temporal overlap between hosts and parasites and thus reduce infection risk. Although host populations with altered phenological patterns are less likely to mature and reproduce, the fitness advantage of parasite avoidance can be greater than the cost of reduced reproduction. These results illustrate the impact of parasitism on the evolution of host phenology and suggest that shifts in host phenology could serve as a strategy to mitigate the risk of infection. The phenological pattern that maximizes individual fitness is impacted by many abiotic environmental factors that determine when activities such as breeding, foraging, and migration are favorable  than early‐flowering plants  and the length of time over which s^n emerge (tl). Hosts have nonoverlapping generations and are alive for one season. The parasite, vn, infects juvenile hosts, sn, who are susceptible to infection, analogous to, for example, baculoviruses of forest Lepidoptera , which determines the number of rounds of infection the parasite completes within a season. The parasite completes one round of infection per season if the parasite has a long latency period (long τ) while the parasite completes multiple rounds of infection per season if the parasite has a short latency period (short τ). Juvenile hosts mature into the adult developmental stage, an, that is resistant to infection during the season. Adult hosts reproduce in between‐seasons to give rise to next season's host cohort (s^n+1). We assume infected sn hosts cannot reproduce and thus ignore their progression to the reproductively active an stage.The model describes the transmission dynamics of a free‐living, sterilizing parasite that infects seasonally available juvenile hosts Figure . The sust=0 to t=T. Time units are not specified in order to maintain the generality of the model across disease systems. It is expected that the relevant time unit will be in months for many disease systems, corresponding to spring and summer  in season n−1 and the probability that those parasites survive between‐seasons (γ). The transmission dynamics in season n are given by the following system of delay differential equations . We make the common assumption for free‐living parasites that the removal of parasites through transmission (α) is negligible  for simplicity, although other distributions are expected to have qualitatively similar results , given byϵ is the probability that hosts survive to the next season, σ is host reproduction, and ρ is the density‐dependent parameter.The function τ) by the end of the season do not release progeny. Background mortality arises from predation or some other natural cause. We assume that infected hosts that die from background mortality do not release parasites because the parasites are either consumed or the latency period corresponds to the time necessary to develop viable progeny   produced by a single invading mutant host  introduced in season n in the environment set by the resident host at equilibrium density s^*. That is, mutant invasion fitness is equivalent to s^mn+1 that were produced in season n by sn,m who successfully matured by the end of the season an,mT. Following the same approach as in previous analyses  are those that invade and replace populations with alternative trait values when rare and also prevent invasion, while at equilibrium, from mutants with different trait values. We are only able to solve the current model analytically when hosts are challenged by parasites that complete one round of infection per season  and the host emergence period duration (tl) when hosts are in an environment with a parasite with a defined latency period length. Optimal trait values for t0 and tl thus balance the cost of parasite infection with the cost of not reproducing. This study followed the evolution of t0 or tl in isolation, rather than the simultaneous evolution of both traits. Parameter values are listed in Table The invasion analyses predict the evolution of the timing that host emergence begins , although the impacts are quantitatively different . Nevertheless, host populations with greater proportions of later‐emerging individuals can have a selective advantage and invade systems with shorter emergence periods.Host populations with widely distributed emergence periods can also reduce the average risk of infection with long latency period parasites Figure . That isIntermediate host emergence period lengths optimize host evolutionary fitness in the presence of short latency period parasites Figure . Specifi4The selection pressure created by parasites can be sufficiently strong to drive evolutionary shifts in host phenology Figures  and 5. ESilene alba could be an adaptation to avoid Ustilago violacea infection  could represent several activity types that impact host contact with parasites in natural systems. For example, this model could apply to host species that undergo diapause during unfavorable environmental conditions, as in many disease systems with insect hosts , host fecundity σ, and the strength of the host density‐dependent parameter (ρ) in Appendix Similarly, parameter ranges that increase infections would likely select for stronger shifts in host phenology  while parameter ranges that decrease the density of susceptible hosts would likely select for weaker shifts in host phenology by decreasing the number of infections  We explore the impact of changing transmission rates ; formal analysis (lead); investigation (lead); methodology (lead); writing – original draft (lead); writing – review and editing . Dustin Brisson: Funding acquisition (lead); writing – original draft (supporting); writing – review and editing .This work was supported by the National Institutes of Health (T32AI141393 (HM) R01AI142572 (DB), R01AI097137 (DB)); the National Science Foundation (DEB‐1354184 (DB)); and the Burroughs Wellcome Fund (1012376 (DB)).The authors declare no conflicts of interest.https://github.com/hanneloremac/Parasite‐mediated‐selection‐on‐host‐phenology].This article has earned Open Data, Open Materials and Preregistered Research Design badges. Data, materials and the preregistered design and analysis plan are available at [None.Appendix S1.Click here for additional data file.


This study aims to analyze the impact of Framework of Convention of Tobacco Control (FCTC) on regulatory contents of trade agreements from 2001 to 2019.A search of trade agreements from‘WTO Regional Free Trade Agreement Database’ using keywords including “tobacco”, “cigarette”, “smoking” and “FCTC” from May to August 2020 resulted in a total sample of 268 trade agreements, from which 69 trade agreements were coded and analyzed. Provisions in trade agreements, identified via the aforementioned keywords, were categorized into 6 trade measures. The word counts of the provisions containing; FCTC; were calculated. Chi-square tests were applied to analyze the differences of regulatory patterns between different time frames. The import and export values (USD) of tobacco products under trade agreements containing the term “FCTC” were further collected from the “International Trade Statistics 2001–2020” for understanding the impact of the provision on the trade flow.Among 69 agreements, the percentage of trade agreements containing keyword as “FCTC” increased significantly from 0% to 2011 to 12% after 2011. A significant decrease of using trade measures as “the exclusion list” was found after 2011 (from 10% to 0). The word counts of provisions containing; FCTC; increased from 24 words in 2011 to 164 words in 2018, and the content of the provisions became more concrete over time. There are six trade agreements containing “FCTC”, and all these 6 agreements were ratified by European Union (EU). Despite EU ratified trade agreements with “FCTC”, the import and export values of tobacco products between EU and the other party countries increased with time. But the gap of average trade values between trade agreements with and without “FCTC” being widened with time.As a first study evaluated the impact of FCTC on regulatory contents of trade agreements, our study results showed that after countries signed trade agreements containing keyword FCTC, the regulatory contents changed significantly. Further studies are recommended to understand the reason and criteria for incorporating FCTC provisions into trade agreements, especially in the EU. Tobacco smoking causes approximately 8 million deaths worldwide every year , 2. SmokFCTC has contributed to significant and rapid progress in implementing national tobacco control measures like regulating smoke-free public places and health warnings on cigarette package in many countries , 8. The The World Trade Organization (WTO) plays a key role in regulating the international trade of tobacco products. As an organization aims to promote trade liberalization by reducing and eliminating tariffs and other trade barriers , WTO encThe FCTC noticed the possible harmful impact trade liberalization may have on tobacco control and public health. Mamudu et al. 2010)’s important research indicated that the lack of consensus between trade and health caused the absence of explicit FCTC trade provision. He also argued that the public health community should become more involved in trade and health issues at all levels of governance and press the FCTC Conference of the Parties (COP) for clarification of this critical issue . In addi10’s impoPrevious studies pointed out that the negotiation process before FCTC ratification can accelerate the pace of regulation in countries , 26. TheTo our knowledge, there has not yet been a study evaluating the impact the FCTC has had on trade agreements. For understanding the issue, the objectives of this study are:  Compare We collected all the trade agreements with a date of entry into force between 1 and 1995 to 31 July 2020 in the “WTO Regional Free Trade Agreement Database (FTA/RTA Database)” from May to August 2020. Among these 25 years, there were 268 trade agreements in total. And all these 268 trade agreements were collected for further analysis.Then trade agreements that were not written in English and did not contain the keywords “tobacco”, “cigarette”, “smoking”, or “Framework Convention of Tobacco Control (FCTC)” were excluded. And irrelevant trade agreements, referred to trade agreements that did not substantially address tobacco control were further excluded after content review. For example, ‘tobacco’ only present in the title ‘Section IV Prepared foodstuffs; beverages, spirits and vinegar; tobacco and manufactured tobacco substitutes’ in the annex of the “Canada – Israel Trade Agreement”. With the fact that the keyword only presented as an item on the general list without further impact on regulatory content, it is considered as irrelevant trade agreement in our study. Two researchers independently screened all the trade agreements to exclude irrelevant ones. The inter agreement rate was 100%.The process was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . The selInformation collected from the trade agreements included the date of entry into force, which countries were party to the treaty, and the text sections containing the keywords.The keywords were shown in the provisions of 69 trade agreements. The provisions containing keywords were collected and categorized into six trade measures, including “tariffs”, “the exclusion list”, “rules of origin”, “quantitative restrictions”, “non-tariff measures”, and “other exclusion measures” (see supplements). Based on the reason that these six trade measures are the most common measures used in trade agreements for explaining trade barriers between contracted countries, we used these six trade measures to classify the measures used in the trade agreements . TariffsIn addition, there are other measures that have not traditionally been included trade agreements or considered in the literature that could be used in trade agreements for tobacco control, such as restrictions on duty-free entry, distribution wholesale, and retail sale of tobacco products. The purpose of these trade measures is to exclude tobacco products from easy access to the market; we refer to these measures as “other exclusion measures”.Among 69 trade agreements, there are 6 agreements containing the keyword as “FCTC”. All these 6 agreements were ratified by European Union (EU). With the finding, we additionally collected import and export values (USD) of tobacco products between EU and party countries from 2001 to 2019 from the WTO database under agreements for further analysis.For the consistency of trade data, tobacco and manufactured tobacco substitutes were classified as Item 24 under the Harmonized System Code (HS Code). Hence, we collected Item 24 data from the database of “International Trade Statistics 2001–2020” in the “International Trade Centre” (ITC) website from May to August 2020. The yearly trade flow data available in ITC is mainly based on “UN Comtrade Database” and covers more than 90% of world trade data .To capture the influence of the FCTC on trade agreements, the time scale of analyzed trade agreements were divided into three phases by two crucial events: the date of the FCTC coming into force (27 February 2005) and the ratification date of the first trade agreement containing provisions from the FCTC (1 July 2011).Word counts were calculated and content analysis was conducted to compare regulatory contents included in trade agreements before and after FCTC ratification.Comparing trade agreements negotiated before and after 2005 captures the influence of the FCTC on trade agreements. Comparing trade agreements enforced before and after 2011 allows us to examine if there is a mutual learning effect (or imitation effect) between trade agreements. The two sets of comparisons reveal changes in normative contents of trade agreements concerning the 6 trade measures and FCTC contents.Chi-square test was used to analyze differences between categorical variables. In this study, chi-square test was used to compare the differences in trade measures used in agreements between different time frames. In detail, chi-square test was used to compare the regulatory content of trade agreements that coded into the six categories between pre-2005 and post-2005 agreements. Also, chi-square test was used to compare the regulatory content of trade agreements between pre-2011 and post-2011 agreements. Import and export values (USD) of tobacco products in countries party to the included trade agreements were calculated. The statistical analysis was performed using IBM SPSS Statistics 20.Among the 69 trade agreements included in this study, there were 19 trade agreements that came into force before 2005 (27.5%), 20 trade agreements that came into force between 2005 and 2011 (28.89%), and 30 trade agreements that entered into force after 2011 (43.47%).Additionally, 99 countries entered into 69 trade agreements. Among them, 91 countries (91.92%) are party to the FCTC; 84 countries are WTO members and 6 countries are WTO Observers (90.91%). 85 countries (85.86%) are both party to the FCTC and WTO member states or observers.Comparisons of trade measures in trade agreements between different time frames by chi-square test are shown in Table The differences in the types of trade measures before and after 2011 were showed in Table The text of provisions that were taken directly from the FCTC in the six trade agreements included in this study are shown in Table The word counts of provisions taken directly from the FCTC increased with time. For the articles listed in a public health related chapter, there were 24 words regarding FCTC in the trade agreements ratified in 2011 and 28 words in agreements enforced in 2013. The trade agreements ratified in 2018 included 164 FCTC related words, five more times than before ” shown in our study as regular agreement with keywords in its provisions of “Rules of Origin (ROO)” and “Other exclusive measures”. Since the TPP has provided a new possibility as the first regional trade agreement including tobacco carve-out measure by containing related language in the “general exceptions” chapter, further study on the issue is recommended.Given that the EU is the exception in terms of trade agreements making explicit reference to FCTC and its implementation, the finding of this study is limited.As a first study evaluated the impact of the FCTC on trade agreements, we found that the FCTC first appeared in trade agreements in 2011. The usage of trade measures as “the exclusion list” for tobacco products in trade agreements significantly decreased after 2011. In addition, the EU is involved in all the trade agreements containing FCTC. Although the EU takes the lead in including FCTC contents in trade agreements, the flow of tobacco trade between the EU and its countries remains on an upward trend.Our study results showed the possible impact of FCTC on trade agreements and trade flow on tobacco products. Further studies are recommended to better understand how FCTC contents are incorporated into trade agreements, especially in the EU.


Despite well‐documented benefits of rehabilitation and therapy services for children with disabilities, long waiting lists to access these services are common. There is a growing body of evidence, primarily from mixed or adult services, demonstrating that waiting times can be reduced through strategies that target wasteful processes and support services to keep up with demand. However, providers of rehabilitation and therapy services for children face additional complexities related to the long‐term nature of many developmental conditions and the need to consider timing of interventions with developmental milestones and education transition points. This review aimed to synthesise available evidence on service redesign strategies in reducing waiting time for paediatric therapy services. We conducted a systematic review of studies conducted in outpatient paediatric rehabilitation or therapy settings, including physical and mental health services, evaluating a service redesign intervention and presenting comparative data on time to access care. Two reviewers independently applied inclusion criteria, assessed risk of bias and extracted data. Findings were analysed descriptively and the certainty of evidence was synthesised according to criteria for health service research. From 1934 studies identified, 33 met the criteria for inclusion. Interventions were categorised as rapid response strategies, process efficiency interventions or substitution strategies . Reductions in waiting time were reported in 30 studies. Evidence is limited by study designs with high risk of bias, but this is mitigated by consistency of findings and large effect sizes. There is moderate‐certainty evidence that service redesign strategies similar to those used in adult populations can be applied in paediatric rehabilitation and therapy settings to reduce waiting time. Delays to access paediatric therapy and rehabilitation services are a major problem.Service redesign interventions can improve access, but previous reviews have focussed on services for adults or mixed populations.Evidence generated in other settings may not be directly applicable to children's services.Service redesign initiatives—including process improvement approaches, rapid response interventions and strategies that use trained allied health in place of medical specialists for some assessments—can reduce waiting lists in paediatric settings.These findings are similar to those observed in adult/mixed settings.Evidence is limited by low‐quality studies; future studies using more rigorous designs are required to guide service providers on the best use of limited resources to reduce waiting times.1Children with disabilities and chronic health conditions require timely access to services to minimise impairments, activity limitations and/or participation restrictions, and maximise their quality of life. The right to rehabilitation and therapy interventions is recognised in the United Nations Convention on the Rights of Persons with Disabilities, and should ideally be timed to take advantage of the optimal periods for intervention during a child's growth and development Novak, .Long waiting lists for speech and language therapy, occupational therapy, physiotherapy, psychology and other services that offer rehabilitation or therapy for children with disabilities and chronic health conditions are common Feldman, . This isWaiting times for health services can be reduced by implementing strategies that target wasteful processes and alter models of care delivery to help services to keep up with demand  whether service redesign strategies are effective in reducing waiting time for paediatric therapy services and (2) whether some types of strategies are more effective than others in achieving this goal.2http://prisma‐statement.org/prismastatement/Checklist.aspx).This systematic review was registered prospectively with PROSPERO (CRD42019140963) and reported according to the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines , conceptual terms associated with these services such as disability, mental health and rehabilitation, and common health conditions seen in these services such as cerebral palsy, autism and developmental delay. Due to the broad nature of the concept of ‘waiting’, searches were done using the MeSH terms ‘waiting list’ and ‘appointments and schedules’ and terms related to ‘wait’ and ‘time’ were combined using proximity operators. For example, “Wait adj5 time” was used to identify “time spent waiting” or “waiting time” and “wait* adj5 number” to find “number of patients waiting” or “number on the waitlist”. An example of the search strategy can be found in Appendix Reference lists of selected studies were scanned for relevant articles, and citation tracking of included papers completed in Google Scholar.2.2Studies were included from settings that provided rehabilitation or therapy services to children within the community, investigated the effectiveness of a service redesign intervention and reported data on an outcome related to timeliness of care, such as time to first appointment or the size of the waiting list.For the purpose of the review, service redesign was defined as any intervention that changes the process by which a service is delivered to achieve timely and effective care. This could include acceptance of referrals, appointments and scheduling, the personnel delivering the intervention, the style of intervention  or location of intervention . Paediatric rehabilitation or therapy was broadly defined as a service provided by members of the allied health professions, such as physiotherapy, occupational therapy, speech and language therapy or psychology, either alone or within the context of a multi‐disciplinary team, to reduce impairments, activity limitations, and/or participation restrictions for children with disabilities, mental health conditions, developmental delays or chronic health conditions.Any research design was acceptable, provided quantitative data were reported that compared either waiting time from referral to first appointment or the size of a waiting list under different service designs. For example, eligible designs could include randomised trials allocating patients to different models of care, studies presenting data before and after an intervention or comparisons of different sites with different models of care. Only peer‐reviewed papers were included, published in either English or French. Inclusion and exclusion criteria are presented in more detail in Appendix 2.3http://app.covidence.org). Full‐text articles were retrieved for all studies that could not be excluded based on screening of titles and abstracts, and these were assessed against inclusion criteria. Discrepancies were resolved either by consensus or with the assistance of a third reviewer if required. Data were extracted using a form designed within Covidence, which included study characteristics , nature of intervention and comparison condition, outcomes reported, waiting time outcomes and any secondary outcomes reported .Two of a pool of five reviewers independently reviewed the title and abstracts of each retrieved study against selection criteria using the online platform Covidence  were consistently reported.An overall evaluation of the certainty of evidence for the effectiveness of different groups of interventions for reducing waiting time was made following guidelines published by the Evidence‐based Practice Centre (EPC) supported by the Agency for Healthcare Research and Quality , no service redesign was described , the setting was not outpatient therapy and/or paediatrics , the publication type was a conference abstract or otherwise ineligible for inclusion (n = 8) or the full text was unavailable (n = 2). A total of 33 studies were selected for inclusion.The search yielded 1934 studies after removal of duplicates, including an additional two articles identified through citation and abstract checking , USA (n = 8), Canada (n = 6), Australia (n = 3) and Ireland (n = 2). Almost half of the studies (n = 14) were conducted in mental health services. Autism services made up the next biggest group (n = 8), with the remaining studies conducted in occupational therapy services (n = 2), services integrating physiotherapists and orthopaedic specialists (n = 3) and services broadly described as rehabilitation, early intervention or child development services (n = 5). All but two of the included studies described observational studies comparing waiting time for patients seen before and after an intervention, or reporting the number of people on waiting lists at two or more time points before, during or after a service redesign intervention. The exceptions were one trial involving concurrent comparison of two clinic models  used any inferential statistics to test the hypothesis that the redesign intervention was associated with a change in waiting time. Only five studies provided any information about the characteristics of the groups being compared and therefore about confounding variables, . Eleven studies were identified Table  that evaAll but one (Ahlers et al., 3.2.4Two studies (Vallerand & McLennan, 3.3Nine of the 33 studies reported on consumer satisfaction (Ahlers et al., Rates of missed appointments were reported in two studies using rapid response interventions (Clemente et al., Two studies measured clinical outcomes, both conducted in a child and adolescent mental health services. Introduction of a rapid response model found no reduction in effectiveness of treatment following the intervention, and some improvement measured against the intervention goals (Fuggle et al., Some additional observational data were reported that provided some additional insights into the impact of the service redesign strategies. Qualitative data regarding staff satisfaction were reported in two studies in the rapid response category; both reported the changes to have had benefits in relation to collaboration, but noted concerns about increased time pressures from some participants (Clemente et al., 3.4Using the EPC guidelines (Berkman et al., 4This review of 33 studies investigated the effectiveness of service redesign interventions to reduce waiting time in paediatric rehabilitation and therapy settings. The interventions were classified into three major categories: models of care promoting rapid access to a first appointment; general redesign initiatives that sought to increase efficiency or modify care pathways; and enabling/substituting allied health or other professionals to deliver services previously provided by medical specialists. The vast majority of included studies reported more timely access to care following the intervention, with many reporting reductions in either time to first appointment or the number of people on a waiting list by more than 50%.The consistent evidence with large estimates of effect in reducing waiting time from more than 30 studies provides moderate‐certainty evidence that reductions in waiting time can be achieved with service redesign interventions in paediatric settings. Long waiting lists for these services have been reported in multiple countries (Harding et al., Despite these positive findings, conclusions are limited by the high risk of bias in the majority of included studies. Most of the studies were observational studies, with comparisons made with historical controls. Many were designed as quality improvement initiatives, intended to achieve local results rather than demonstrate internal validity. This was reflected in the reporting: 85% failed to report on the characteristics of the sample, and only 45% conducted any statistical analysis to provide an estimate of confidence around the reported result. Of those that did report inferential statistics, parametric statistics were commonly used to test differences in waiting time without reference to the distribution of the data. This is potentially problematic given that waiting time data are often positively skewed (Siciliani et al., The three categories of interventions identified in this review were favoured by different types of services. Strategies that provided access to a rapid initial assessment appointment were used mainly by mental health services. Substitution services were trialled mainly in autism and orthopaedic services. The group of studies testing interventions that involved reducing inefficiencies and/or modifying aspects of the patient pathway were featured in a more diverse range of settings. None of the three categories of interventions showed clear benefit over the others, and heterogeneity within each category suggests that were also likely contextual factors such as the size of the original waiting list, the type of service provided, characteristics of the local population or organisational factors that influenced the outcome. However, findings suggest that all three categories of intervention are feasible in paediatric services.While the impact of redesign strategies on waiting times was the primary focus of this review, many of the included studies also considered other outcomes. Once again, the evidence is limited by the nature of the studies, but no adverse outcomes were reported over a very broad range of secondary outcomes. Under a variety of circumstances, benefits were reported in reducing failure‐to‐attend rates and decreased costs. Several studies checked for adverse impacts on factors such as time to second appointment, accuracy of assessment, patient satisfaction, clinical outcomes and impact on staff not involved in the intervention, but none reported any cause for concern as a result of the interventions. These findings suggest that interventions that reduce waiting time do not necessarily require trade‐offs with other aspects of care delivery, and may have additional benefits. However, the studies in this review primarily focussed on time to the first appointment, and two of the three studies that did measure time to review appointments noted a modest increase following the intervention (Clark et al., Paediatric rehabilitation and therapy services were the subject of this review because this population has unique features that we hypothesised could influence the choice or effectiveness of interventions to reduce waiting time. For example, services for children may need to accommodate timing around specific developmental stages, integration with the education system or the involvement of families in the care of the child. However, findings of this review suggest that the strategies trialled to reduce waiting time in paediatric settings are similar to those published in services for adult populations. Success in reducing health service waiting times has been reported for adult populations from substitution services (Desmeules et al., This review has some limitations. Developing a successful search strategy for the concept of waiting time was challenging, due to the broad nature of terms such as ‘wait’ and ‘time’. The matrix approach used in this review, based on proximity searches, is imperfect and may have missed some relevant papers. However, citation and reference list checking resulted in only two additional included articles, suggesting the strategy achieved its purpose and was sufficiently sensitive. Determining what should be included under the definition of ‘therapy’ used in this review was not always straightforward. In particular, autism services varied considerably in the extent to which they provided a medical diagnostic service only versus ongoing support and therapy. Given that the nature of these services was not always well documented, we used the presence of allied health professionals in a multi‐disciplinary team as the indicator for inclusion.The lack of robust studies with a low risk of bias is a clear limitation of this body of literature, and may be explained by a perception that these types of interventions, usually implemented at a service level, are not appropriate for testing with randomised controlled trial designs. While it is true that individual randomisation may not be possible or practical due to factors such as the risk of contamination, other trial designs such as cluster trials or interrupted time series approaches are viable alternatives, and have been successfully applied to these types of interventions in other settings (Handley et al., 5There is moderate‐certainty evidence that service redesign interventions, including substitution services, rapid access to a first appointment and process redesign initiatives, can improve timely access to care in paediatric rehabilitation and therapy services.All authors contributed to the conceptualisation and design of the study. Harding conducted the search of bibliographic databases for the review. All authors contributed to screening and selection of studies, and interpretation and validation of findings. Harding prepared the original draft of the manuscript, and all authors contributed to editing and review.The authors have no interests to declare.Appendix S1.Click here for additional data file.