Managing nitrogen to restore water quality in China.

The nitrogen cycle has been radically changed by human activities1. China consumes nearly one third of the world's nitrogen fertilizers. The excessive application of fertilizers2,3 and increased nitrogen discharge from livestock, domestic and industrial sources have resulted in pervasive water pollution. Quantifying a nitrogen 'boundary'4 in heterogeneous environments is important for the effective management of local water quality. Here we use a combination of water-quality observations and simulated nitrogen discharge from agricultural and other sources to estimate spatial patterns of nitrogen discharge into water bodies across China from 1955 to 2014. We find that the critical surface-water quality standard (1.0 milligrams of nitrogen per litre) was being exceeded in most provinces by the mid-1980s, and that current rates of anthropogenic nitrogen discharge (14.5 ± 3.1 megatonnes of nitrogen per year) to fresh water are about 2.7 times the estimated 'safe' nitrogen discharge threshold (5.2 ± 0.7 megatonnes of nitrogen per year). Current efforts to reduce pollution through wastewater treatment and by improving cropland nitrogen management can partially remedy this situation. Domestic wastewater treatment has helped to reduce net discharge by 0.7 ± 0.1 megatonnes in 2014, but at high monetary and energy costs. Improved cropland nitrogen management could remove another 2.3 ± 0.3 megatonnes of nitrogen per year-about 25 per cent of the excess discharge to fresh water. Successfully restoring a clean water environment in China will further require transformational changes to boost the national nutrient recycling rate from its current average of 36 per cent to about 87 per cent, which is a level typical of traditional Chinese agriculture. Although ambitious, such a high level of nitrogen recycling is technologically achievable at an estimated capital cost of approximately 100 billion US dollars and operating costs of 18-29 billion US dollars per year, and could provide co-benefits such as recycled wastewater for crop irrigation and improved environmental quality and ecosystem services.

Opportunities to produce food from substantially less land.

The vast majority of the food we eat comes from land-based agriculture, but recent technological advances in agriculture and food technology offer the prospect of producing food using substantially less or even virtually no land. For example, indoor vertical farming can achieve very high yields of certain crops with a very small area footprint, and some foods can be synthesized from inorganic precursors in industrial facilities. Animal-based foods require substantial land per unit of protein or per calorie and switching to alternatives could reduce demand for some types of agricultural land. Plant-based meat substitutes and those produced through fermentation are widely available and becoming more sophisticated while in the future cellular agricultural may become technically and economical viable at scale. We review the state of play of these potentially disruptive technologies and explore how they may interact with other factors, both endogenous and exogenous to the food system, to affect future demand for land.

A restatement of the natural science evidence base concerning grassland management, grazing livestock and soil carbon storage.

Approximately a third of all annual greenhouse gas emissions globally are directly or indirectly associated with the food system, and over a half of these are linked to livestock production. In temperate oceanic regions, such as the UK, most meat and dairy is produced in extensive systems based on pasture. There is much interest in the extent to which such grassland may be able to sequester and store more carbon to partially or completely mitigate other greenhouse gas emissions in the system. However, answering this question is difficult due to context-specificity and a complex and sometimes inconsistent evidence base. This paper describes a project that set out to summarize the natural science evidence base relevant to grassland management, grazing livestock and soil carbon storage potential in as policy-neutral terms as possible. It is based on expert appraisal of a systematically assembled evidence base, followed by a wide stakeholders engagement. A series of evidence statements (in the appendix of this paper) are listed and categorized according to the nature of the underlying information, and an annotated bibliography is provided in the electronic supplementary material.

Options for keeping the food system within environmental limits.

The food system is a major driver of climate change, changes in land use, depletion of freshwater resources, and pollution of aquatic and terrestrial ecosystems through excessive nitrogen and phosphorus inputs. Here we show that between 2010 and 2050, as a result of expected changes in population and income levels, the environmental effects of the food system could increase by 50-90% in the absence of technological changes and dedicated mitigation measures, reaching levels that are beyond the planetary boundaries that define a safe operating space for humanity. We analyse several options for reducing the environmental effects of the food system, including dietary changes towards healthier, more plant-based diets, improvements in technologies and management, and reductions in food loss and waste. We find that no single measure is enough to keep these effects within all planetary boundaries simultaneously, and that a synergistic combination of measures will be needed to sufficiently mitigate the projected increase in environmental pressures.

A restatement of the natural science evidence base regarding the source, spread and control of Campylobacter species causing human disease.

Food poisoning caused by Campylobacter (campylobacteriosis) is the most prevalent bacterial disease associated with the consumption of poultry, beef, lamb and pork meat and unpasteurized dairy products. A variety of livestock industry, food chain and public health interventions have been implemented or proposed to reduce disease prevalence, some of which entail costs for producers and retailers. This paper describes a project that set out to summarize the natural science evidence base relevant to campylobacteriosis control in as policy-neutral terms as possible. A series of evidence statements are listed and categorized according to the nature of the underlying information. The evidence summary forms the appendix to this paper and an annotated bibliography is provided in the electronic supplementary material.

Intraspecific variation in symbiont density in an insect-microbe symbiosis.

Many insects host vertically transmitted microbes, which can confer benefits to their hosts but are costly to maintain and regulate. A key feature of these symbioses is variation: for example, symbiont density can vary among host and symbiont genotypes. However, the evolutionary forces maintaining this variation remain unclear. We studied variation in symbiont density using the pea aphid (Acyrthosiphon pisum) and the bacterium Regiella insecticola, a symbiont that can protect its host against fungal pathogens. We found that relative symbiont density varies both between two Regiella phylogenetic clades and among aphid "biotypes." Higher density symbiont infections are correlated with stronger survival costs, but variation in density has little effect on the protection Regiella provides against fungi. Instead, we found that in some aphid genotypes, a dramatic decline in symbiont density precedes the loss of a symbiont infection. Together, our data suggest that the optimal density of a symbiont infection is likely different from the perspective of aphid and microbial fitness. Regiella might prevent loss by maintaining high within-host densities, but hosts do not appear to benefit from higher symbiont numbers and may be advantaged by losing costly symbionts in certain environments. The standing variation in symbiont density observed in natural populations could therefore be maintained by antagonistic coevolutionary interactions between hosts and their symbiotic microbes.

Income, consumer preferences, and the future of livestock-derived food demand.

In recent decades there has been a sustained and substantial shift in human diets across the globe towards including more livestock-derived foods. Continuing debates scrutinize how these dietary shifts affect human health, the natural environment, and livelihoods. However, amidst these debates there remain unanswered questions about how demand for livestock-derived foods may evolve over the upcoming decades for a range of scenarios for key drivers of change including human population, income, and consumer preferences. Future trends in human population and income in our scenarios were sourced from three of the shared socioeconomic pathways. We used scenario-based modeling to show that average protein demand for red meat (beef, sheep, goats, and pork), poultry, dairy milk, and eggs across the globe would increase by 14% per person and 38% in total between the year 2020 and the year 2050 if trends in income and population continue along a mid-range trajectory. The fastest per person rates of increase were 49% in South Asia and 55% in sub-Saharan Africa. We show that per person demand for red meat in high-income countries would decline by 2.8% if income elasticities of demand (a partial proxy for consumer preferences, based on the responsiveness of demand to income changes) in high-income countries decline by 100% by 2050 under a mid-range trajectory for per person income growth, compared to their current trajectory. Prices are an important driver of demand, and our results demonstrate that the result of a decline in red meat demand in high-income countries is strongly related to rising red meat prices, as projected by our scenario-based modeling. If the decline in the income elasticity of demand occurred in all countries rather than only in high-income countries, then per person red meat demand in high-income countries would actually increase in 2050 by 8.9% because the income elasticity-driven decline in global demand reduces prices, and the effect of lower prices outweighs the effect of a decline in the income elasticity of demand. Our results demonstrate the importance of interactions between income, prices, and the income elasticity of demand in projecting future demand for livestock-derived foods. We complement the existing literature on food systems and global change by providing quantitative evidence about the possible space for the future demand of livestock-derived foods, which has important implications for human health and the natural environment.

Modelling the suppression of a malaria vector using a CRISPR-Cas9 gene drive to reduce female fertility.

BACKGROUND: Gene drives based on CRISPR-Cas9 technology are increasingly being considered as tools for reducing the capacity of mosquito populations to transmit malaria, and one of the most promising options is driving endonuclease genes that reduce the fertility of female mosquitoes. In particular, there is much interest in constructs that target the conserved mosquito doublesex (dsx) gene such that the emergence of functional drive-resistant alleles is unlikely. Proof of principle that these constructs can lead to substantial population suppression has been obtained in population cages, and they are being evaluated for use in sub-Saharan Africa. Here, we use simulation modelling to understand the factors affecting the spread of this type of gene drive over a one million-square kilometre area of West Africa containing substantial environmental and social heterogeneity. RESULTS: We found that a driving endonuclease gene targeting female fertility could lead to substantial reductions in malaria vector populations on a regional scale. The exact level of suppression is influenced by additional fitness costs of the transgene such as the somatic expression of Cas9, and its deposition in sperm or eggs leading to damage to the zygote. In the absence of these costs, or of emergent drive-resistant alleles that restore female fertility, population suppression across the study area is predicted to stabilise at ~ 95% 4 years after releases commence. Small additional fitness costs do not greatly affect levels of suppression, though if the fertility of females whose offspring transmit the construct drops by more than ~ 40%, then population suppression is much less efficient. We show the suppression potential of a drive allele with high fitness costs can be enhanced by engineering it also to express male bias in the progeny of transgenic males. Irrespective of the strength of the drive allele, the spatial model predicts somewhat less suppression than equivalent non-spatial models, in particular in highly seasonal regions where dry season stochasticity reduces drive efficiency. We explored the robustness of these results to uncertainties in mosquito ecology, in particular their method of surviving the dry season and their dispersal rates. CONCLUSIONS: The modelling presented here indicates that considerable suppression of vector populations can be achieved within a few years of using a female sterility gene drive, though the impact is likely to be heterogeneous in space and time.

Impact of mosquito gene drive on malaria elimination in a computational model with explicit spatial and temporal dynamics.

The renewed effort to eliminate malaria and permanently remove its tremendous burden highlights questions of what combination of tools would be sufficient in various settings and what new tools need to be developed. Gene drive mosquitoes constitute a promising set of tools, with multiple different possible approaches including population replacement with introduced genes limiting malaria transmission, driving-Y chromosomes to collapse a mosquito population, and gene drive disrupting a fertility gene and thereby achieving population suppression or collapse. Each of these approaches has had recent success and advances under laboratory conditions, raising the urgency for understanding how each could be deployed in the real world and the potential impacts of each. New analyses are needed as existing models of gene drive primarily focus on nonseasonal or nonspatial dynamics. We use a mechanistic, spatially explicit, stochastic, individual-based mathematical model to simulate each gene drive approach in a variety of sub-Saharan African settings. Each approach exhibits a broad region of gene construct parameter space with successful elimination of malaria transmission due to the targeted vector species. The introduction of realistic seasonality in vector population dynamics facilitates gene drive success compared with nonseasonal analyses. Spatial simulations illustrate constraints on release timing, frequency, and spatial density in the most challenging settings for construct success. Within its parameter space for success, each gene drive approach provides a tool for malaria elimination unlike anything presently available. Provided potential barriers to success are surmounted, each achieves high efficacy at reducing transmission potential and lower delivery requirements in logistically challenged settings.

Modelling the potential of genetic control of malaria mosquitoes at national scale.

BACKGROUND: The persistence of malaria in large parts of sub-Saharan Africa has motivated the development of novel tools to complement existing control programmes, including gene-drive technologies to modify mosquito vector populations. Here, we use a stochastic simulation model to explore the potential of using a driving-Y chromosome to suppress vector populations in a 106 km2 area of West Africa including all of Burkina Faso. RESULTS: The consequence of driving-Y introductions is predicted to vary across the landscape, causing elimination of the target species in some regions and suppression in others. We explore how this variation is determined by environmental conditions, mosquito behaviour, and the properties of the gene-drive. Seasonality is particularly important, and we find population elimination is more likely in regions with mild dry seasons whereas suppression is more likely in regions with strong seasonality. CONCLUSIONS: Despite the spatial heterogeneity, we suggest that repeated introductions of modified mosquitoes over a few years into a small fraction of human settlements may be sufficient to substantially reduce the overall number of mosquitoes across the entire geographic area.

A restatement of the natural science evidence base on the effects of endocrine disrupting chemicals on wildlife.

Endocrine disrupting chemicals (EDCs) are substances that alter the function of the endocrine system and consequently cause adverse effects to humans or wildlife. The release of particular EDCs into the environment has been shown to negatively affect certain wildlife populations and has led to restrictions on the use of some EDCs. Current chemical regulations aim to balance the industrial, agricultural and/or pharmaceutical benefits of using these substances with their demonstrated or potential harm to human health or the environment. A summary is provided of the natural science evidence base informing the regulation of chemicals released into the environment that may have endocrine disrupting effects on wildlife. This summary is in a format (a 'restatement') intended to be policy-neutral and accessible to informed, but not expert, policy-makers and stakeholders.

Analysis and valuation of the health and climate change cobenefits of dietary change.

What we eat greatly influences our personal health and the environment we all share. Recent analyses have highlighted the likely dual health and environmental benefits of reducing the fraction of animal-sourced foods in our diets. Here, we couple for the first time, to our knowledge, a region-specific global health model based on dietary and weight-related risk factors with emissions accounting and economic valuation modules to quantify the linked health and environmental consequences of dietary changes. We find that the impacts of dietary changes toward less meat and more plant-based diets vary greatly among regions. The largest absolute environmental and health benefits result from diet shifts in developing countries whereas Western high-income and middle-income countries gain most in per capita terms. Transitioning toward more plant-based diets that are in line with standard dietary guidelines could reduce global mortality by 6-10% and food-related greenhouse gas emissions by 29-70% compared with a reference scenario in 2050. We find that the monetized value of the improvements in health would be comparable with, or exceed, the value of the environmental benefits although the exact valuation method used considerably affects the estimated amounts. Overall, we estimate the economic benefits of improving diets to be 1-31 trillion US dollars, which is equivalent to 0.4-13% of global gross domestic product (GDP) in 2050. However, significant changes in the global food system would be necessary for regional diets to match the dietary patterns studied here.

Modelling the persistence of mosquito vectors of malaria in Burkina Faso.

BACKGROUND: Populations of the Anopheles gambiae complex are found during the rainy season throughout West Africa, even in arid areas with long dry seasons during which mosquitoes appear to be absent. Several hypotheses have been proposed to explain this apparent paradox, including aestivation, dispersal between neighbouring settlements, and long distance migration using high-altitude wind currents. METHODS: An individual-based, spatially explicit model of mosquito populations was developed for a region of West Africa centred on, and including all of, Burkina Faso. Populations associated with human settlements were linked by dispersal and the model incorporated geospatial data on the distribution of settlements, water bodies and rainfall. RESULTS: Local dispersal (at rates consistent with experimental data) was necessary to explain observed patterns of rainy season populations across all of the simulation area, but by itself failed to account for the presence of populations in the arid North (the Sahel). The presence of rare dry-season larval sites could explain these northern populations, but seems inconsistent with field surveys. Aestivation by female mosquitoes explained rainy-season populations in all but the very sparsest and driest areas of human habitation, while long-distance migration based on annual wind patterns could account for all observed populations. CONCLUSIONS: Modelling studies such as this can help assess the potential validity of different hypotheses and suggest priority areas for experimental study. In particular, the results highlight a shortage of empirical research on mosquito dispersal between neighbouring settlements, which may be critically important to the continued presence of many mosquito populations in West Africa. Further research that establishes the extent to which mosquitoes aestivate, and migrate using high altitude winds, is also much needed to understand Sahelian mosquito populations.

The use of driving endonuclease genes to suppress mosquito vectors of malaria in temporally variable environments.

BACKGROUND: The use of gene drive systems to manipulate populations of malaria vectors is currently being investigated as a method of malaria control. One potential system uses driving endonuclease genes (DEGs) to spread genes that impose a genetic load. Previously, models have shown that the introduction of DEG-bearing mosquitoes could suppress or even extinguish vector populations in spatially-heterogeneous environments which were constant over time. In this study, a stochastic spatially-explicit model of mosquito ecology is combined with a rainfall model which enables the generation of a variety of daily precipitation patterns. The model is then used to investigate how releases of a DEG that cause a bias in population sex ratios towards males are affected by seasonal or random rainfall patterns. The parameters of the rainfall model are then fitted using data from Bamako, Mali, and Mbita, Kenya, to evaluate release strategies in similar climatic conditions. RESULTS: In landscapes with abundant resources and large mosquito populations the spread of a DEG is reliable, irrespective of variability in rainfall. This study thus focuses mainly on landscapes with low density mosquito populations where the spread of a DEG may be sensitive to variation in rainfall. It is found that an introduced DEG will spread into its target population more reliably in wet conditions, yet an established DEG will have more impact in dry conditions. In strongly seasonal environments, it is thus preferable to release DEGs at the onset of a wet season to maximize their spread before the following dry season. If the variability in rainfall has a substantial random component, there is a net increase in the probability that a DEG release will lead to population extinction, due to the increased impact of a DEG which manages to establish in these conditions. For Bamako, where annual rainfall patterns are characterized by a long dry season, it is optimal to release a DEG at the start of the wet season, where the population is growing fastest. By contrast release timing is of lower importance for the less seasonal Mbita. CONCLUSION: This analysis suggests that DEG based methods of malaria vector control can be effective in a wide range of climates. In environments with substantial temporal variation in rainfall, careful timing of releases which accounts for the temporal variation in population density can substantially improve the probability of mosquito suppression or extinction.

Consequences of symbiont co-infections for insect host phenotypes.

Most animals host communities of symbiotic bacteria. In insects, these symbionts may have particularly intimate interactions with their hosts: many are intracellular and can play important roles in host ecology and evolution, including protection against natural enemies. We investigated how interactions between different species or strains of endosymbiotic bacteria within an aphid host influence the outcome of symbiosis for both symbiont and host. We first asked whether different combinations of facultative symbiont species or strains can exist in stable co-infections. We then investigated whether the benefits that facultative bacteria confer on their hosts (protection against natural enemies) are enhanced, reduced or unaltered by the presence of a co-infecting symbiont. We asked this both for co-infecting symbionts that confer different phenotypes on their hosts (protection against fungal pathogens vs. parasitoid wasps) and symbionts with overlapping functions. Finally, we investigated the additional survival costs to aphids of carrying multiple infections of symbiont species or strains, and compared symbiont titres in double and single infections. We found that stable co-infections were possible between all of the combinations of facultative symbiont species (Regiella insecticola + Hamiltonella defensa, Regiella + Rickettsiella sp., Regiella + Spiroplasma sp.) and strains (Hamiltonella) that we studied. Where symbionts provided protection against different natural enemies, no alteration in protection was observed in the presence of co-infections. Where symbionts provided protection against the same natural enemy, the level of protection corresponded to the higher of the two symbionts present. In some instances, aphid hosts suffered additional survival costs when hosting double infections. In the case of Hamiltonella, however, infection with multiple strains of the same symbiont led to lower symbiont titres than single infections, and actually improved aphid survival. We conclude that the long-term maintenance of symbiont co-infections in aphids is likely to be determined primarily by costs of co-infections and in some instances by redundancy of symbiont benefits.

Intrinsic pre-zygotic reproductive isolation of distantly related pea aphid host races.

Human activities may weaken or destroy reproductive isolation between young taxa, leading to their fusion with consequences for population and community ecology. Pea aphid host races are adapted to different legume taxa, providing a degree of pre-mating isolation mediated by habitat choice. Yet, all races can feed and reproduce on the broad bean (Vicia faba), a major crop which represents a 'universal host plant', which can promote hybridization between races. Here, we ask if pea aphid host races have reproductive barriers which prevent or reduce gene flow when they co-occur on the universal host plant. We observed mating behaviour, female survival, number of eggs and egg fertilization rates for three types of crosses: among individuals of the same host race, between closely related host races and between distantly related host races. We did not find significant differences in mating behaviour and female survival among the three types of crosses. However, we observed a drastic reduction in the number of eggs laid, and in the number of fertilized eggs, in distant crosses. We conclude that widespread broad bean cultivation in agriculture may predispose closely related-but not distantly related-host races to hybridize, disrupting reproductive isolation between incipient species.

How driving endonuclease genes can be used to combat pests and disease vectors.

Driving endonuclease genes (DEGs) spread through a population by a non-Mendelian mechanism. In a heterozygote, the protein encoded by a DEG causes a double-strand break in the homologous chromosome opposite to where its gene is inserted and when the break is repaired using the homologue as a template the DEG heterozygote is converted to a homozygote. Some DEGs occur naturally while several classes of endonucleases can be engineered to spread in this way, with CRISPR-Cas9 based systems being particularly flexible. There is great interest in using driving endonuclease genes to impose a genetic load on insects that vector diseases or are economic pests to reduce their population density, or to introduce a beneficial gene such as one that might interrupt disease transmission. This paper reviews both the population genetics and population dynamics of DEGs. It summarises the theory that guides the design of DEG constructs intended to perform different functions. It also reviews the studies that have explored the likelihood of resistance to DEG phenotypes arising, and how this risk may be reduced. The review is intended for a general audience and mathematical details are kept to a minimum.

Global and regional health effects of future food production under climate change: a modelling study.

BACKGROUND: One of the most important consequences of climate change could be its effects on agriculture. Although much research has focused on questions of food security, less has been devoted to assessing the wider health impacts of future changes in agricultural production. In this modelling study, we estimate excess mortality attributable to agriculturally mediated changes in dietary and weight-related risk factors by cause of death for 155 world regions in the year 2050. METHODS: For this modelling study, we linked a detailed agricultural modelling framework, the International Model for Policy Analysis of Agricultural Commodities and Trade (IMPACT), to a comparative risk assessment of changes in fruit and vegetable consumption, red meat consumption, and bodyweight for deaths from coronary heart disease, stroke, cancer, and an aggregate of other causes. We calculated the change in the number of deaths attributable to climate-related changes in weight and diets for the combination of four emissions pathways (a high emissions pathway, two medium emissions pathways, and a low emissions pathway) and three socioeconomic pathways (sustainable development, middle of the road, and more fragmented development), which each included six scenarios with variable climatic inputs. FINDINGS: The model projects that by 2050, climate change will lead to per-person reductions of 3·2% (SD 0·4%) in global food availability, 4·0% (0·7%) in fruit and vegetable consumption, and 0·7% (0·1%) in red meat consumption. These changes will be associated with 529,000 climate-related deaths worldwide (95% CI 314,000-736,000), representing a 28% (95% CI 26-33) reduction in the number of deaths that would be avoided because of changes in dietary and weight-related risk factors between 2010 and 2050. Twice as many climate-related deaths were associated with reductions in fruit and vegetable consumption than with climate-related increases in the prevalence of underweight, and most climate-related deaths were projected to occur in south and east Asia. Adoption of climate-stabilisation pathways would reduce the number of climate-related deaths by 29-71%, depending on their stringency. INTERPRETATION: The health effects of climate change from changes in dietary and weight-related risk factors could be substantial, and exceed other climate-related health impacts that have been estimated. Climate change mitigation could prevent many climate-related deaths. Strengthening of public health programmes aimed at preventing and treating diet and weight-related risk factors could be a suitable climate change adaptation strategy. FUNDING: Oxford Martin Programme on the Future of Food.

A restatement of the natural science evidence base concerning the health effects of low-level ionizing radiation.

Exposure to ionizing radiation is ubiquitous, and it is well established that moderate and high doses cause ill-health and can be lethal. The health effects of low doses or low dose-rates of ionizing radiation are not so clear. This paper describes a project which sets out to summarize, as a restatement, the natural science evidence base concerning the human health effects of exposure to low-level ionizing radiation. A novel feature, compared to other reviews, is that a series of statements are listed and categorized according to the nature and strength of the evidence that underpins them. The purpose of this restatement is to provide a concise entrée into this vibrant field, pointing the interested reader deeper into the literature when more detail is needed. It is not our purpose to reach conclusions on whether the legal limits on radiation exposures are too high, too low or just right. Our aim is to provide an introduction so that non-specialist individuals in this area (be they policy-makers, disputers of policy, health professionals or students) have a straightforward place to start. The summary restatement of the evidence and an extensively annotated bibliography are provided as appendices in the electronic supplementary material.

Species-Level Para- and Polyphyly in DNA Barcode Gene Trees: Strong Operational Bias in European Lepidoptera.

The proliferation of DNA data is revolutionizing all fields of systematic research. DNA barcode sequences, now available for millions of specimens and several hundred thousand species, are increasingly used in algorithmic species delimitations. This is complicated by occasional incongruences between species and gene genealogies, as indicated by situations where conspecific individuals do not form a monophyletic cluster in a gene tree. In two previous reviews, non-monophyly has been reported as being common in mitochondrial DNA gene trees. We developed a novel web service "Monophylizer" to detect non-monophyly in phylogenetic trees and used it to ascertain the incidence of species non-monophyly in COI (a.k.a. cox1) barcode sequence data from 4977 species and 41,583 specimens of European Lepidoptera, the largest data set of DNA barcodes analyzed from this regard. Particular attention was paid to accurate species identification to ensure data integrity. We investigated the effects of tree-building method, sampling effort, and other methodological issues, all of which can influence estimates of non-monophyly. We found a 12% incidence of non-monophyly, a value significantly lower than that observed in previous studies. Neighbor joining (NJ) and maximum likelihood (ML) methods yielded almost equal numbers of non-monophyletic species, but 24.1% of these cases of non-monophyly were only found by one of these methods. Non-monophyletic species tend to show either low genetic distances to their nearest neighbors or exceptionally high levels of intraspecific variability. Cases of polyphyly in COI trees arising as a result of deep intraspecific divergence are negligible, as the detected cases reflected misidentifications or methodological errors. Taking into consideration variation in sampling effort, we estimate that the true incidence of non-monophyly is ∼23%, but with operational factors still being included. Within the operational factors, we separately assessed the frequency of taxonomic limitations (presence of overlooked cryptic and oversplit species) and identification uncertainties. We observed that operational factors are potentially present in more than half (58.6%) of the detected cases of non-monophyly. Furthermore, we observed that in about 20% of non-monophyletic species and entangled species, the lineages involved are either allopatric or parapatric-conditions where species delimitation is inherently subjective and particularly dependent on the species concept that has been adopted. These observations suggest that species-level non-monophyly in COI gene trees is less common than previously supposed, with many cases reflecting misidentifications, the subjectivity of species delimitation or other operational factors.