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Experimental designOver 3 years, six paired organic British Blacktail flocks with intact beaks (i.e. not beak-trimmed) were visited between 1 and 40 weeks of age. Within each pair, one flock was ramp reared (RR) and one flock was control reared without ramps (CR). All flocks were kept on one farm which possessed two rearing houses and six laying sheds of approximately 2000 birds per flock. The site was multi-age, meaning that of the six laying sheds there were three different ages on the site at one time.The availability of this commercial facility enabled us to design an experiment whereby we allocated two rearing treatments, one with ramps provided to access elevated structures and a control with elevated structures but no ramps and to alternate these treatments between the two rearing houses available to avoid treatment x house confounds. Each rearing flock was moved independently to a laying house with no mixing, so we were able to continue data collection and examine any long-term effects of the rearing treatment during the laying period. Rearing flocks were systematically allocated so that each laying house received one RR flock and one CR flock during the experiment.Observations were made in the mornings at three time points during the rearing period at 1, 3 and 15–16 weeks, and three in the laying period at 16–17, 24 and 40 weeks of age. See Table 5 for a summary of experimental design, flock and housing information.Table 5 Experimental design for each ramp reared and control reared flock for the 6 replicates. There were two rearing sheds used, Rear1 (R1) and Rear2 (R2), with 6 different laying sheds named A1, A2, B1, B2, C1 and C2.Full size tableThe rearing sheds were static with 142.7 m2 of floor space covered with wood shavings. Rearing sheds were both set up with feed tracks giving mini pellet feed up to 11 weeks of age then pellet grower feed and 7 nipple drinker lines. The lighting schedule was 23 h light in the first day reducing gradually over the rearing period to 10 h light at 7 weeks of age. A minimum light intensity of 10 lx is required, but with windows and pop-holes light intensity was higher in the houses. The temperature was maintained at 30 °C during the first few days then slowly reduced to match the temperature in the laying sheds. Shed heating was provided by gas spot lamps, whole shed heating through hot pipes running along the length of the shed and hot air fans run by a biomass boiler. All flocks had access to the outside range by 10 weeks of age through two pop holes (each L: 2 m by H: 0.4 m). Flocks were moved the short distance from the rearing to the laying house at between 15 to 16 weeks of age in one night using transport modules.All rearing flocks had access to six elevated structures (ES) (see Fig. 3) from four days of age when the chicks were released from the brooding circles. Each ES comprised nine metal perches (length 302 cm, width 3.5 cm), with three perches (25 cm apart) at three different heights (43 cm, 73 cm and 103 cm). Two plastic grids (width 60 cm, length 115 cm) were fixed within the ES to provide platforms at different heights (Fig. 3). In each replicate, the RR flock had one ramp attached to each ES. Three of the ES were fitted with plastic grid ramps (width 60 cm, length 74 cm, angle 35.5°) leading up to the low perch and three ES had ramps (width 60 cm, length 115 cm, angle 40°) leading up to the middle perch. The CR flock had six ES without ramps.Figure 3Elevated structure dimensions used in the ramp reared sheds, (a) shows the high ramp (b) shows the low ramp. The control sheds elevated structures were identical to these but without ramps.Full size imageThe six single-tier laying houses on-site were mobile organic units with approximately 345m2 of floor space. See Fig. 4 for a schematic plan of their layout. All had a raised area comprising plastic slats over supports (approx. 70 cm from the litter) and a ground-level litter area covered with wood chip. Four of the sheds (Fig. 4a) were set up with the slatted area spanning the whole width of the shed and halfway down the length. In two of the sheds (Fig. 4b) the litter area was either side of the elevated slatted area. Nest boxes ran down the centre of the slatted area, dividing this into two sections. Intermittent ramps were installed at the level change, resulting in 4 m of ramp access and 4 m without ramps in the shed with litter at the end and 8 m of ramp access and 13 m without ramps in the shed with litter at the sides. In sheds A1 and A2 the height of the slatted area resulted in a steeper ramp angle of 45° compared to 30° in the other sheds. There were four pop holes at ground level with two on each side of the house (L: 2.35 m by H: 0.4 m) leading to the range from the litter area on both sides of the sheds. All sheds had aerial perches at 1 m high with 18 cm of perching space per bird resulting in approximately 360 m of perch length running the length of the slatted area. Feed tracks and drinker lines matched those in the rearing sheds. The lighting schedule was 16 h of light and varied between summer and winter with the lights set to turn off at the same time as natural dusk. The birds were fed on organic mini pellets throughout lay. Enrichment was provided to the flocks in the form of pecking objects such as buckets and boots. Replicates 5 and 6 were provided with pecking blocks and alfalfa hay nets hung on the litter area.Figure 4Plan view of the laying house layout (a) for replicates 1, 2, 4 and 5 and (b) for replicates 3 and 6. Images not to scale.Full size imageAssessments of behaviourObservations were made at three time points during the rearing period at 1, 3 and 15–16 weeks. On the first visit at 1 week of age, the total number of chicks on each ES was counted once in spot counts in the morning. At 3 and 15–16 weeks, observations of the movements up and down the ES were made. Three of the 6 structures were chosen at random in each shed. The number of chicks present on the different parts of the ES was counted at the beginning and end of the recording period to allow a comparison with the 1-week counts. The recordings involved 5-min continuous sampling where all movements down the ES were recorded and the area the chicks moved down from was noted. This was then repeated for movements up the ES. Focal bird recordings were taken at 3 and 15–16 weeks of age. Records were made for each of 3 randomly selected ES. When 10 focal birds had been observed (approximately 30 birds per flock), or 10 min had passed recordings stopped. A focal bird was chosen if it was performing orientation behaviour, indicating a downwards or upwards transition. This was described as the bird rotating its head to look in the direction of movement. Behaviours performed after the orientation behaviour were tallied, thus recorded as counts per behaviour (see Table 6). Recordings were stopped if birds completed a transition or moved away from transitioning.Table 6 An ethogram of behaviours of focal birds during up and down movements.Full size tableAt 15–16 weeks of age, three types of interactions were recorded for feather pecking. These included severe feather pecking (SFP), gentle feather pecking (GFP) and aggressive pecks (AP)28. A quadrat area 2 m by 2 m was randomly selected, with the number of birds in each quadrat counted at the beginning and end of the recording period. The number of SFP, GFP and AP were recorded over three minutes of continuous recordings in three different areas of the house, selected randomly at each end and the middle of the shed. Feather pecks and aggressive pecks were recorded as bouts: a series of pecks not separated by more than 5 s28. Rates of pecking were calculated as the number of pecks per bird per second.In the laying shed around 16–17 and at 24 weeks of age 3-min continuous sampling and focal bird recordings were taken for transitions between the slats and litter. Four recordings were made at 2-m lengths along the elevated slatted area: two areas with ramps (RA) and two areas without ramps (NRA) were selected. Separate recordings were taken for upwards and downwards movements and the number of birds in the recording area were counted at the start and end of the scans. At 16–17 and 24 weeks of age, feather pecking observations were taken using the same procedure as for the 15–16-week observations during rear.Welfare assessments and production data rearing phaseFeather scores of 20 birds per flock were recorded at 16–17 weeks of age by walking in a straight line down the centre of the shed, selecting a bird at random then counting two birds to the left of this and visually feather scoring that bird. Birds were not handled to minimise disturbance and plumage was scored using the method from Bright et al.33. The neck, back, rump, tail and wings were scored using a four-point scale 0 (best) to 4 (worst). Data were obtained from the farm records for percentage cumulative mortality and body weight.Welfare assessments and production data laying phaseAt 16–17 and 24 weeks of age, the attitude of the flocks was assessed using the approach distance and reactions to novel objects methodology developed by Whay et al.34. Distance to approach birds before they moved away was recorded by walking through the house selecting a bird at random and counting two birds to the left. The bird had to be standing up and facing the researcher, who approached the bird at a steady pace and recorded the distance before the bird moved away. This was repeated on 20 birds in each flock. Reactions to a novel object (blue folder at 17 weeks of age and a white and blue tub at 24 weeks of age) were assessed by placing a novel object on the ground and recording the time taken for the first bird to interact with it and then how many birds were within a 30 cm radius after 60 s. The novel object test was repeated in 4 areas per flock. Range use was recorded by counting the number of birds near to the house (5 m) in the middle range (5–20 m) and far (the rest of the range). Feather scores of 20 birds per flock were recorded at 17 and 24 weeks of age using the same procedure as for the 16-week assessment for birds at rear.At 40 weeks of age, feather cover and keel bone fractures were scored. Up to 100 birds per shed were caught from four different locations (25 litter, 25 slats, 25 perches, 25 nest boxes). In four sheds only 50 birds were caught as the birds were fearful and showed signs of distress. Feather cover was scored by picking the bird up and scoring the body and flight feathers separately using a the AssureWel three-point scale 0 (best) to 2 (worst)35. The keel damage was then scored using a 0 (no damage) to 2 scale based on the technique used by Wilkins et al.36. Validation for keel bone palpations was conducted. A score of 94% matched scores compared to an experienced gold standard assessor and 85% match at dissection for scoring a break. At 24 weeks of age, the number of floor eggs were counted over 1 day.Data were collected from the farm records on laying house percentage of daily eggs, average egg weight (grams), average hen body weight and feed conversion ratio.During the 16 week recordings in the final rearing flocks, the lighting inside the shed was considerably reduced compared to previous flocks. This resulted in poor visibility for feather cover and feather pecking observations, so these were not taken during this visit. Data were not obtained on keel fractures and feather cover scores at 40 weeks for the first laying flocks visited as their sheds were destroyed by strong winds.Statistical analysisData were analysed using SPSS 24 (IMB) or MLwiN 3.0. The statistical package MLwiN was chosen as it is designed for multilevel modelling and can therefore accommodate data nested within levels with repeated measures. Such models account for dependence between responses caused by grouping of birds within sheds, and repeated measures taken from the same sheds on different visits within and between replicates. Including visit and replicate as nested effects ensures that dependences (e.g. due to differing times of year when data were collected) are accounted for. All residuals were checked for normal distributions using a Shapiro-Wilks test or plotted graphically and no transformations were needed to meet the assumptions of the tests. All results are reported in the format mean ± SD unless when stated as the percentage of birds performing a behaviour during transitions.Assessments of behaviourAt rear, from the counts of chicks on structures and counts of transitions up and down the structures, a normal model (generalised linear model) was used with a four-level hierarchy (bird within shed within visit within replicate). The same normal model and four level hierarchy were used for the counts of transitions in the laying shed.For the focal bird behaviours of birds transitioning at rear and lay, the data were presented as the percentage of each behaviour calculated for the birds in the recording session for the two rearing treatments. The direction (up or down) was analysed separately. For the focal birds at lay, all were included in the analysis for the pre-transitioning behaviours, only birds that attempted a transition were included for analysis of the transitioning behaviours. Pre-transition behaviours for birds that moved-away and did not transition were analysed separately. Owing to the low occurrence of behaviours during the focal recordings for transitions up and down the ramps, data were coded as yes or no, and a Binomial model was used for analysis for both the rear and lay focal transition data with four hierarchical levels (bird within shed within visit within replicate).Welfare and production dataFor the Novel object test, human approach, feather pecking and feather cover data a normal model was used in MLwiN with four-levels (Bird within Shed within visit within replicate). Floor eggs were analysed using a two-tailed t-test in SPSS, due to limited data. Ordinal data such a keel bone fracture scores and feather cover recorded at 40 weeks of age were converted to binomial data due to a lack of data for some scores, these were therefore analysed using a binomial model in MLwiN with two levels (Bird within shed).Production data at rear (body weight in grams) and lay (% eggs daily, egg weight in grams, body weight in grams and feed conversion ratio) were obtained from farm records and analysed in SPSS using a general linear model with treatment (CR and RR) as a fixed factor and age (3, 8 and 14 weeks at rear and 20, 30 and 70 weeks at lay) as a random factor to account for repeated results. Cumulative percentage mortality was analysed at 14 weeks of age using a t-test to compare the treatment groups.Ethical approvalEthical approval for this project was granted by the University of Bristol’s Animal Welfare and Ethical review body under UIN: UB/16/040 and all methods were conducted in accordance with the review body and UK legislation. More

In the case of the first studied facility—Facility (I), thermo-visual examinations did not demonstrate any signs of thermal activity in the majority of the area. Above all, the new layered sections of the facility are free from thermal phenomena. However, the central section of the dump is characterized by rather intense thermal phenomena. The cone is also thermally active, yet in this section the thermal activity is not very intensive (see Fig. 1). In the section with the strongest thermal activity, i.e. the southern part of the top, the recorded surface temperature exceeded 600 °C (see Fig. 2). Outside the thermally active zone, the surface temperature did not exceed 25–35 °C.Figure 1Thermo-visual examination of the extractive waste dump (Facility I); October, 2017.Full size imageFigure 2Subsidence (the so-called crater) at the top of Facility I; October, 2017.Full size imageThe tests conducted on the premises of Facility II demonstrated the lack of thermal activity in the majority of the area. Only in the central section, on the top of the older part of the dump, the measurements showed the occurrence of thermal anomalies (see Fig. 3). These were minor areas located along the edge of the scarp on the northern and southern sides where the measured surface temperature was above 80 °C. It is worth mentioning that in 2010, the recorded temperatures for this section were at the level of several hundred degrees Celsius. Outside the sections, the temperature did not exceed 25–40 °C. The relatively high temperature in the area without thermal activity may be explained by the fact that in the morning hours it was exposed to the sun.Figure 3Thermo-visual examination of the extractive waste dump (Facility II); June, 2017.Full size imageDuring the course of the thermo-visual examination of the extractive waste dump in Facility III, no thermal activity was observed in the majority of the area. Only in the central section of the Facility, in the area which is still in operation, the measurements demonstrated the occurrence of thermal anomalies (see Fig. 4). These were minor areas where the local measured surface temperature was above 200 °C. Outside these areas, the temperature did not exceed 20–30 °C.Figure 4Thermo-visual examination of the extractive waste dump (Facility III); October, 2017.Full size imageThe simplest method of analyzing the differences and similarities between the studied objects is their visualization in the space of measured parameters. When two or three parameters are measured, such visualization is not problematic. However, in our study there are nine measured parameters. The graphic presentation of a 9-dimensional space is not possible. This is the rationale of applying the HCA which enables to analyze the similarities between the studied objects (three different dumping facilities in different periods of time) as well as the similarities between the parameters in object space. Nevertheless, the HCA does not allow to simultaneously analyze the relationships between the objects and the measured parameters. This problem was solved by the use of a color map of the experimental data, which enabled an in-depth interpretation of the data structure. In addition, the application of the color map facilitates highlighting the differences and similarities among the clusters showed in the dendrograms, and, in consequence, it helps to distinguish the facilities which are characterized by the highest or the lowest values of the measured parameters. Figure 5 demonstrates the dendrogram for 21 objects representing the studied dumping facilities in different periods of time in the space of 9 measured parameters, the dendrogram for the measured parameters in the object space as well as a color map presenting the values of the measured parameters for particular objects.Figure 5Dendrograms for (a) 21 objects representing the studied dumping facilities (see Table 1) in the space of 9 measured parameters; (b) parameters in the object space; (c) a color map presenting the values of measured parameters for particular dumping facilities.Full size imageBased on the dendrogram presented in Fig. 5a, a clear distinction of the examined objects representing the discussed dumping facilities into two clusters—A and B can be observed. Cluster A includes all samples representing Facility II in the whole period of the monitoring as well as samples representing Facility I taken in Quarter 3, 2017 and in Quarters 1–4, 2018 (objects nos. 1 and 3–6). All samples representing Facility III as well as two samples representing Facility I taken in Quarter 4, 2017 and in Quarter 4, 2018, respectively (objects nos. 2 and 6) are collected in Cluster B. In addition, within each of the clusters certain sub-clusters may be distinguished. Within Cluster A, the following sub-groups can be observed:

sub-group A1 collecting all samples taken from Facility II during the whole period of the monitoring (objects nos. 8–14);

sub-group A2 including samples taken from Facility I in Quarter III, 2017 and in Quarters 1–4, 2018 (objects nos. 1 and 3–6).

In turn, Cluster B contains the following three sub-groups:

sub-group B1 collecting two samples taken from Facility I in Quarter 4, 2017 and 2018, respectively (objects nos. 2 and 6) as well as samples representing Facility III in Quarter 3, 2017, Quarters 1–3, 2018 and Quarter 1, 2019 (objects nos. 15, 17–19 and 21);

sub-group B2 encompassing the remaining two samples taken from Facility III in Quarter 4, 2017 and in Quarter 4, 2018 (objects nos. 16 and 20).

The dendrogram obtained by means of Ward’s linkage method for the measured parameters in the space of 21 objects (see Fig. 5b) enables to distinguish the three principal clusters of parameters listed below:

class A collecting parameters nos. 2, 3 and 4 (describing the concentrations of acenaphtene, fluorene and phenanthrene);

class B containing parameters nos. 1 and 9 (describing the concentrations of naphthalene and chrysene);

class C including the remaining parameters nos. 5, 6, 7 and 8 (describing the concentrations of anthracene, pyrene, fluoranthene and B(a)anthracene).

The PAHs emissions from a burning mine waste dump must be carefully monitored due to their potential toxicity and genotoxicity38,39. PAHs have two different roots; one is incomplete combustion of organic matter, whereas the other one is their production in the geological formation when organic sediments were chemically transformed into fossil fuels. In our study, the first path, namely spontaneous coal waste combustion is observed40,41. It is also worth mentioning that PAHs pose significant human health hazards. The exposure to PAHs may result in skin, lung or stomach cancers in the human organism28.As mentioned before, a considerable drawback of the Hierarchical Clustering Analysis lies in that it does not allow for simultaneous interpretation of the dendrograms describing objects which represent particular samples taken from the extractive waste facility in different periods in the space of measured parameters and parameters in the object space. The lack of the possibility of interpreting the above relationships significantly limits the knowledge of the studied phenomena because the purpose of the analysis is to determine not only the differences among particular samples but also the underlying cause of such differences. Therefore, the dendrogram presenting the examined samples taken from the three selected facilities in different periods of time (see Fig. 5a) was juxtaposed with a color map of the experimental data (see Fig. 5c) demonstrating the values of the measured parameters arranged according to the order of the objects and parameters organization. The juxtaposition enables to determine the reason why the examined samples were distributed in such a way. In addition, the interpretation of the dendrogram for the objects in parameters space complemented with the color map of experimental data allows distinguishing samples which are characterized by the highest values of the measured parameters.Analyzing the dendrogram presented in Fig. 5a together with the color map of the experimental data, it can be observed that all samples within Cluster A were characterized by relatively lower values of the measured parameters. Moreover, sub-group A1 identified within Cluster A was characterized by relatively lowest concentrations of acenaphtene, fluorene, phenanthrene and pyrene (parameters nos. 2, 3, 4, and 7) among all of the examined samples taken from the three selected facilities in the whole period of the monitoring. It confirms the observations which were made previously with the use of a thermo-visual camera which demonstrated that there were no thermal phenomena in the majority of the waste dump areas. The observed sparse emissions of gases result from certain thermal anomalies occurring in the central section of Facility II as well as some small areas located along the northern and southern side of the scarp; however, in any of the places the temperature did not exceed 80 °C (see Fig. 3). The thermo-visual examinations of the remaining sections of the facility showed that the surface temperature did not exceed the level of 25–40 °C and it is a result of sun exposure rather than the occurrence of any thermal phenomena.Sub-group A2 which includes all samples taken from Facility I in Quarter 3, 2017 and Quarters 1–4, 2018 (objects nos. 1 and 3–6) differs from A1 objects in terms of relatively higher concentrations of acenaphtene, fluorene, phenanthrene and pyrene (parameters nos. 2, 3, 4, and 7). Additionally, within sub-group A2, the uniqueness of sample 1 taken in Quarter 3, 2018 (object no. 5) can be observed; it was characterized by the lowest concentration of chryzene (parameter no. 9) of all the examined samples.Despite the fact that the results of the thermo-visual analysis of Facility III in general did not show any major signs of thermal activity, the Hierarchical Clustering Analysis by means of which the samples were examined in terms of the emission of the 9 parameters indicated that some thermal phenomena take place at this dump.Furthermore, the analysis of the color map of the experimental data for the objects grouped in Cluster B makes it abundantly clear that thermal activity takes place in the majority of the monitored areas located at Facility III. What is more, for two samples taken from Facility I in Quarter 4, 2017 and Quarter 4, 2018 (objects nos. 2 and 6), thermal activity was observed. For the two objects, the concentrations of acenaphtene and fluorene are the highest among all of the examined samples (parameters nos. 2 and 3); similarly, the concentrations of phenanthrene, pyrene and B(a)anthracene (parameters nos. 4, 7, and 8) are also high. It confirms the observations made by means of a thermo-visual camera which demonstrated that there were no thermal phenomena if one considers the facility in its entirety. However, there are certain areas in the central section of the facility where thermal phenomena occur.In the case of the samples taken from Facility III in Quarter 3, 2017 as well as in Quarter 2 and Quarter 3, 2018 (objects nos. 15, 18, and 19) within sub-group B1, the values of all measured parameters are only slightly increased, which can indicate the occurrence of thermal phenomena. Yet, it must be kept in mind that the concentrations are decidedly lower than for the remaining samples taken from Facility III. It may be also an indication that self-heating of the coal extractive waste takes place. The other two samples taken from Facility III in Quarter 1, 2018 and Quarter 1, 2019 (objects nos. 17 and 21) are characterized by relatively highest concentrations of pyrene and B(a)anthracene (parameters nos. 7 and 8) as well as high concentrations of anthracene and fluorene (parameters nos. 5 and 6), which can result from an intense thermal activity.A similar observation can be made for the two samples taken from Facility III in Quarter 4, 2017 and Quarter 4, 2018 (objects nos. 16 and 20) which are characterized by the highest concentrations of naphthalene, anthracene, fluoranthene and chrysene of all the examined samples (parameters nos. 1, 5, 6 and 9) as well as high concentrations of phenanthrene (parameter no. 4). Although the results of the thermo-visual analysis of the whole area of Facility III did not demonstrate signs of thermal activity in most of its sections, there exist certain spots with intense thermal processes, which is confirmed by relatively high values of all the measured parameters. More

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In this study we successfully applied a DNA metabarcoding approach to identify consumed food items of plant and animal origin in human stomach content samples, even when digestion was advanced and macroscopic inspection no longer possible. A wide panel of common and less common edible food items were found, including meat, fish, legumes, cereals, nuts, fruits and spices. So far, gastric content analyses in a forensic context are typically based on microscopic and macroscopic identification of food items (reviewed e.g. in1). However, this approach is characterised by low taxonomic resolution, low sensitivity, and proves ineffective when meal leftovers are rendered unidentifiable due to chewing and digestive processes. In the field of molecular ecology, studies on animals have shown that morphological identification of prey items in the stomach underestimates prey diversity, which is particularly true when digestion is advanced (e.g.25). The only study to date applying DNA metabarcoding to infer human diet was based on faecal samples and did not assess any animal components of diet, although including a controlled feeding trial of an animal-based diet12. The comparison of the obtained plant DNA sequences to self-reporting indicated that, while some items were not reported but detected by DNA metabarcoding, all but one self-reported items were detected (the only exception being coffee), thus highlighting the sensitivity of the method. The present study, based on a random sampling of 48 human stomach contents collected during routine autopsies, includes a higher number of vegetal items and shows for the first time the successful detection of dietary items of animal origin. We found no correlation between the diversity of species detected and the time since death or digestion degree, which advocates for the utility of this methodology. The Vert01 primer set, highly specific to vertebrates, enables to distinguish between commonly eaten animal taxa and is clearly advantageous over morphological identification. In line with regional eating habits and previously published diet surveys26, we found within the 48 samples mainly pig, cattle/dairy and OTUs assigned to the plant families Poaceae, Rosaceae and Asteraceae (likely cereals, fruits, lettuces; Fig. 1). We did not detect coffee (Coffea spp.) in any of the stomach content samples, in line with12, which might be due to a degrading effect of roasting procedures on DNA, the absence of this popular beverage in all of the stomach samples being unlikely. Similarly, although common in Swiss eating habits, we also did not detect potato, which is usually eaten boiled or baked. Note that additional edible plant species, not listed in Fig. 2 since not constituting at least 10% of RRA but with 100% match with the database, were also detected (e.g. buckwheat, citrus fruits, flax, mangoes, sesame; Supplementary Table S2). Because we could obviously not compare our results to self-reported diets, we applied very stringent filtering parameters to avoid the occurrence of false positives (see Bioinformatic data treatment). It is beyond the approach of this study to distinguish between the animal source and a final processed food item (e.g. dairy or egg products) based on the obtained DNA sequences. However, this could be achieved by complementing the primer set with a bacterial marker (to e.g. identify the presence of a particular cheese27) or using proteomics (see below).Overall, the Vert01 metabarcode is able to discriminate well among commonly eaten genera. However, owing to its limited taxonomic resolution (72.4% at the species level, based on in silico testing11), species-level distinction is not always possible (e.g. between perch and pikeperch) or between potentially-eaten wild species and their conspecific domestic counterparts (e.g. wild boar and pig). In Fig. 2, we present the taxonomical assignation done using ObiTools together with a common name, selected after manually inspecting each sequence using BLAST and only considering 100% matches with edible species. In some cases, the common name refers to a group of species because the barcode was not specific enough to distinguish between genera or species. This is more relevant concerning plants, as the Sper01 metabarcode length ranges from 10 to 220 bp, implying that some items with shorter metabarcode and/or closely related phylogenetically could not be distinguished to genus or species level due to limited resolutive power. This is related to the nature of this universal plant marker, which has been designed to target a region of the trnL intron of chloroplast DNA which lacks taxonomic resolution within several plant families (only 21.5% resolution at the species level9,11) but has wide taxonomic coverage. This trade-off meant for our study that we could genetically not distinguish between some close species which are clearly different morphologically (e.g. stone fruits, cucurbits). To overcome this issue and increase the taxonomic resolution of the results, it is possible to envisage multiplexing within the same PCR of additional primers specifically targeting groups of species that cannot be identified at the species level by the P6 loop of the trnL intron. Such a strategy has already been implemented to distinguish between Carpinus betulus and Corylus avellana in bison diet28. Furthermore, it must be outlined that by using these primer sets only, diet assessment is not comprehensive as it does not target all possibly present food products. Even so-called universal primers may result in preferential amplification of some taxa over others and non-amplification of target taxa29,30. For this pilot study, we chose to use two universal PCR primer pairs with wide taxonomic coverage but limited specific resolution, in order to detect a broad range of items. To gain resolution for specific vertebrate or plant taxonomic groups (e.g. fish, birds, cereals) or target taxa not covered by these primers and which could be of forensic interest (e.g. marine crustaceans and molluscs, algae, fungi), it is possible to complement Vert01 and Sper01 with additional, taxonomically-restricted PCR metabarcoding primers described in the literature (e.g.31; examples reviewed in11). Taxonomic assignation of an unknown DNA sequence strongly depends on the exhaustiveness and quality of a reference database, either public as e.g. GenBank or custom-made/local (reviewed in32). In case of a priori knowledge of the overall consumed diet in samples, local databases may be restrained to the expected DNA sequences, which subsequently improves taxonomic assignment. For this study we in silico compiled databases containing all possible sequences amplified by our markers, but restricted these to vertebrates and spermatophytes (i.e. seed plants), respectively.The duration of stomach emptying has been estimated by the percentage of a meal present in a stomach3, but this process is influenced by several variables including the type and volume of consumed food, lifestyle and health, and can therefore last from few hours to days2. While one could argue that plant items usually remain longer in the stomach, our findings do not allow to draw robust conclusions about correlations of certain food items and digestion times. In order to establish hypotheses useful for time-frame estimations, additional experiments are necessary. In a controversial case of death, MS-based proteomics provided additional information through the analysis of food-derived proteins and peptides in the gastric content sampled at autopsy, indicating a last breakfast of milk and bread. While this method is certainly promising, it might reveal difficult if digestion is in an advanced stage, and has a less comprehensive scope than a DNA metabarcoding assay33. Furthermore, the effect of food processing techniques on DNA quality must be taken into account since cooking denatures e.g. proteins which in turn renders DNA amplification preferential to immunological approaches1. Different cooking treatments (variable duration of boiling, frying, baking) of tomato seeds showed that DNA extraction yielded in good quality DNA only for fresh seeds34, while digestion did not destroy DNA21. Hence, there might be an implicit bias of DNA metabarcoding to preferentially detect non-processed food (i.e. raw versus cooked). Another issue of environmental DNA-based methods is that it is not possible to distinguish between different states of food products based on DNA sequences. As mentioned before, we could not discriminate between e.g. grapes/wine, fruits/juices, beef meat/dairy products or chicken meat/eggs, since the DNA sequence of a derived product is identical to the DNA sequence of its source. While it is less common to encounter such biases for plants, mainly in cereal-derived products, it has to be taken into account when extrapolating diet patterns from DNA metabarcoding results.Stomach content sampling is invasive, but advantageous or even required with certain animal species and in particular circumstances, including definitely the human forensic context. An advantage of stomach content over faecal samples is that food is in an early stage of digestion before passing through the pyloric sphincter into the intestines, thus the effects of inhibition by bacteria or enzymes and degradation of DNA are less significant11,18. While some food particles such as seeds sometimes remain identifiable, even morphologically, after passing through the digestive system21, others do not and the same applies to DNA which is degraded by the digestive processes taking place in the intestinal tract. In a controlled feeding experiment on insects, the detectability of food DNA in different types of dietary samples showed that regurgitates and entire animals (including stomach content) outperformed faeces regarding detectability of prey DNA13. While food journals in dietary surveys may contain errors or deliberate omissions12, they are a comprehensive and easily accessible method of human diet assessment. However, in case of deceased persons that option is no longer available.Stomach content analyses provided crucial information for criminal investigations about cases of sudden and unexplained death on numerous occasions in recent years, enabling investigators to interpret perimortem events in detail (case examples reviewed in2). The results of this pilot study show that human stomach content analyses by DNA metabarcoding can be used as a complementary tool to traditional forensic macro- and microscopic approaches, with clear advantages such as an almost unlimited flexibility in terms of nature and range of taxa targeted, as well as high sensitivity and taxonomic resolution. Consequently, information that might otherwise remain undetected can be revealed, highlighting timings and circumstances surrounding the last hours of a person and his/her food intake. In a broader perspective, taking into account the potential improvements and refinements described above, and the growing amount of research literature available for wildlife species (i.e. environmental DNA-based studies), our results open up promising and novel prospects in the broader framework of human biomedical investigations of dietary patterns, based on partially or fully digested food found in the gastrointestinal tract or in faecal samples. More

The δ-MAPS analysis is performed onto monthly mean SST anomalies from the Mediterranean Sea Physical Reanalysis (CMEMS MED-Physics25) over the period 1987–2017. The advantage of using a reanalysis resides in the availability of a velocity field consistent with the SSTs that allows us to confirm the coupling between network domains and ocean currents within the euphotic layer.Validation of the δ-MAPS frameworkThe proposed ecoregionalization is first applied to the 2007–2010 period, when the domains, representing ecoregions, can be compared to those identified by Ref.2 using Lagrangian methods. The details of this validation are reported below and relevant figures can be found in the Supplementary Information.The 2007–2010 ecoregions in Figure S1 are consistent with the ones derived in Ref.2 through computationally intensive simulations. The name of each domain corresponds with those used in Ref.2 to ease comparison. It is worthwhile remarking that this work and the one of Ref.2 not only use very different methods to define connectivity, but also different data sources. Our study uses velocity and SST output fields from CMEMS MED Physics reanalysis, while Ref.2 uses the configuration PSY2V3 of the operational system MERCATOR OCEAN with a resolution of 8 km in the horizontal downscaled to a connectivity grid of 50 × 50 km. The data assimilation and clustering algorithms are different and Ref.2 employs a cut-off in addition to the clustering grid downscaling. These differences unavoidably translate into slightly different shapes and patterns of the domains inferred. For example, the D + V area in panel (a) of Figure S1 is effectively two separate ecoregions in Ref.2, in which the Messina Strait is not resolved at the connectivity grid level. However, this separation appears inconsistent with the surface kinetic energy (K.E.) of panel (b) in Figure S1, computed from the horizontal currents, e.g. zonal (u) and meridional (v) velocity components, as K.E. = 1/2 |V|2 where |V|= (u2 + v2)0.5. Indeed, there is no clear separation between the regions north and south at Messina Strait in our dataset. Having detailed this example and acknowledged that some differences should be expected, the overall basin eco-regionalization using δ-MAPS is consistent with that in Ref.2. The spatial accuracy is enough to well separate the main ecological areas, despite small-scale differences (i.e. some km, due to resolution choices).By and large, the SST anomaly domains in Figure S1 are bounded by ocean currents, in agreement with Ref.2. This is due to the dominance of advective forcing by ocean currents on the SSTs at equatorial and mid latitudes, on monthly timescales and spatial scales of few hundreds kilometers24. This link, which is foundational to the proposed methodology, is further quantified as follows: First, we calculate the surface K.E. per unit mass averaged over the time slot of interest; second, we select the points in the validation period (2007–2010) that exceed the 50th percentile of surface K.E. computed for the entire basin over 1987–2017 (e.g. 0.004 m2/s2); third, we compute the domain-boundary matrix augmented by 1 grid point in each direction; finally, we count which fraction of the domain boundaries computed in the boundary matrix overlaps with the K.E. fronts (above the 50th percentile threshold). The fraction obtained is high and equal to 0.73, and remains elevated when increasing the threshold to the 60th percentile (0.66). This procedure was repeated for all the time slots with Δ = 7 years used next in this study, obtaining high and very stable values in each case (mean ± variance = 0.73 ± 0.01 for the 50th percentile threshold, and 0.65 ± 0.01 for the 60th percentile threshold).Additionally, the correlation between the surface K.E. and K.E. at 50 m, 100 m, and 150 m over the whole 1987–2017 period (Figure S2) remains positive and significant, with coefficients for the whole domain (field mean c.c.  ± variance) of 0.83 ± 0.04 at 50 m, 0.68 ± 0.05 at 100 m, and 0.54 ± 0.06 at 150 m, indicating that the link extends to the whole euphotic layer.Mediterranean Sea ecoregions: long-term changesThe space-averaged (e.g. averaged on the whole basin) SSTs over the 1987–2017 period are characterized by a linear warming trend of about 0.04 °C per year, stronger in the eastern portion of the basin (Figure S3 in Supplementary Information). Over the same period, the K.E. per unit mass is characterized by different trends over decadal or quasi-decadal periods (Fig. 2, shown for surface only but the trend extends similarly to 50 m and 100 m depths) and no clear east–west contrast. A positive trend is found in the first part of the curve (1987–2001, 2.3 × 10–4 m2/s2 per year, green line in figure), followed by a central decade without statistically significant changes (2001–2010, blue line), and a steep negative trend afterward (2010–2017, – 4.1 10–4 m2/s2 per year, red line). We refer to 1987–2001, 2001–2010 and 2010–2017, as the UP, MAX and DOWN periods. The dynamical changes associated with the strengthening and weakening of ocean currents are hypothesized to coincide with a reshaping of the sub-basin ecoregions and reciprocal connectivity. The ecoregionalization inference is therefore performed considering time slots of varying length, so that yrend = yrini + Δ with yrini = y0 + n, n = 0,1,…,N, where y0 is the initial year of the dataset (1987) and N is the total number of time slots, each of duration Δ years, between 6 and 8. Time slots overlapping by more than one year among different trends periods are excluded. The choice of Δ = 7 years represents the best trade-off for having enough time slots to quantify the evolution of ecoregions and a sufficiently large number of data points in each time slot for statistical inference. We will focus on this case, but results are verified also for the other Δ values (see Supplementary Information).Figure 2Mean surface kinetic energy timeseries. Monthly time series of deseasonalized surface kinetic energy per unit mass (m2/s2), averaged over the whole Mediterranean Sea between 1987 and 2017. The shaded areas indicate the 1987–1993 (during the UP period), 2004–2010 (during MAX) and 2011–2017 (during DOWN) time slots used in Fig. 3.Full size imageStrength maps for three representative time slots are presented in Fig. 3a,c,e while maps of domain strengths for all Δ = 7 time slots can be found in Figure S4. The mean surface kinetic energy averaged within each timeslot is next compared to the number of ecoregions in corresponding timeslots. The fragmentation level, or the total number of ecoregions, and the mean surface kinetic energy content are highly correlated (Figure S5b in Supplementary Information), with a Pearson’s coefficient of 0.79 for the whole Mediterranean Sea, and 0.8 (0.65) for the eastern (western) basin. The fact that time slots are not independent does not invalidate the analysis, and a large correlation (c.c = 0.73) is retained even when using four non-overlapping time slots. A higher fragmentation occurs whenever the upper ocean layer is more energetic, and this relationship is robust to changes of Δ (see Supplementary Information). The domain strength is next compared to the mean K.E. content. For each timeslot, the domain strength is spatially averaged over the eastern and western basin separately. The correlations between the averaged strengths and the corresponding time slot mean surface K.E. values, both varying as the time slots change, are then calculated for eastern and western basins separately. No linkage is found in the western basin, but a strong anticorrelation describes the relationship in the eastern Mediterranean (c.c. − 0.74). This anticorrelation remains high (− 0.73) also when the eastern basin strengths are related to the whole basin surface K.E. averaged over each timeslot.Figure 3Domains and connectivity networks for the domain containing the Suez Canal. The three 7-year timeslots selected as representative of the UP (a,b), MAX (c,d) and DOWN (e,f) periods. The color of the domains represents their strength (left column), and the red dot shows the location of the Suez Canal. Links in the connectivity nets (right column) are colored according to the correlation between (the domain containing) the Suez Canal and other domains as labeled. Only correlations stronger than 0.35 are plotted. (Domains maps visualization produced with Matlab R2018a, https://www.mathworks.com/).Full size imageWe hypothesize that the amount of K.E. associated with semi-permanent jets, currents or large mesoscale eddies, grouped here together and named KE fronts, can be used as an indicator of their role as connectivity modulators. We identify KE fronts applying a pattern recognition algorithm on the K.E. fields for each time slot. The resulting pictures are processed by an image segmentation technique, based on K-means clustering, to separate the K.E. in four clusters of increasing energy content. The maximum-intensity group is selected as indicators for KE fronts and the number of pixels contained in each cluster is counted and used to estimate the size or abundance of each one. The maximum-intensity cluster well represents the energy-containing structures as measured by the correlation between the mean surface K.E. content in each time slot and the pixels within the corresponding cluster (c.c.  > 0.99). The more pixels reside within each cluster, the larger the KE fronts-populated areas that this cluster approximates. This estimation is carried out for the whole basin, and separately in the eastern and western parts. The number of pixels is then correlated to the number of inferred ecoregions for the whole Mediterranean (c.c. = 0.81), and for eastern (c.c. = 0.81) and western (c.c. = 0.69) basins. Figure S6 in the Supplementary Information compares the clustering maps of a low energy time slot (1987–1993, in panel (a)) and a higher one (2004–2010 in panel (b)), for the whole Mediterranean Sea for the maximum cluster. The number of ecoregions is highly correlated with the KE fronts everywhere and especially in the eastern Mediterranean Sea. The higher level of fragmentation found in the MAX period is thus associated with more abundant and/or larger surface KE fronts, acting as eco-dynamical barriers.To further strengthen this assessment, we consider that energy fronts can act as modulators for SSTa-derived domains. The ecoregionalization over a certain time slot characterizes that time range in one single ecoregion-map but stems from data known at several time points (i.e. monthly SSTa in our case). The resulting domains account therefore for the inherent physical variability of the system over time. A higher (lower) ecoregions fragmentation may therefore by associated with dynamical fronts occurring at different times and not necessarily in the same place, over a certain time range. If this is plausible, we expect to count more (less) occurrences of higher energy in broad areas where the domains are more (less) fragmented. For each time slot, the number of occurrences of a front in each pixel is therefore counted. Specifically, having defined a front as a K.E. realization above the 50th percentile of the overall (1987–2017) time varying surface K.E., we count how many times a front appears in the considered time slot at each pixel. In Figure S7 pixels are colored according to the number of occurrences in each time slot. The result is consistent with the domain fragmentation evolution. The higher fragmentation occurring in timeslots from 2001 to 2010 in the eastern basin is associated with more frequent fronts. Similar considerations hold for the other sub-basins, clearly distinguishing low energy periods from higher ones.Mediterranean ecoregions connectivity networksChanges in functional networks or connectivity among ecoregions can be assessed by comparing a network from each energy period (UP: 1987–1993, MAX: 2004–2010 and DOWN: 2011–2017) (Fig. 3b,d,f for the eastern basin and Figure S8 in the Supplementary Information for the western basin).In 1987–1993 the western basin was characterized by a high mean positive correlation of 0.73, with a strong, non-directional connectivity among the Tyrrhenian and Ligurian-Algero Provençal domains. In 2004–2010 the connectivity was overall weaker, and in particular reduced among Tyrrhenian waters. The connectivity between the Balearic domain (Bal) and the Tyrrhenian ones was also reduced. In 2011–2017 the connectivity was mostly recovered, especially in Tyrrhenian waters. In this period, the Algero-Provençal domain separated from the Ligurian Sea (Lig), enforcing its connectivity with the Balearic and the Alboran ecoregions.In the eastern basin we focus our attention on the ecoregion immediately offshore the Suez Canal (Fig. 3), the major anthropogenic corridor for the introduction of non-indigenous marine species in the Mediterranean Sea, the so-called Lessepsian immigrants32. According to δ-MAPS, connectivity from the domain surrounding Suez was high in the first decade, decreased approaching MAX, remained small until about 2010–2011 with fewer statistically significant links, and increased again in the more recent time slot considered. During the UP and DOWN periods, the strongest connections were with the eastern Levantine (domain N), followed by that with the Aegean, Ionian and Tunisian Seas. During UP the connectivity extended to the Provençal and Algerian Seas, in the western basin, while in DOWN these links were absent and replaced by a connection with the Adriatic Sea.The 1987–1993 and 2011–2017 periods, while not too dissimilar in energy levels, differed indeed for the phase of the Ionian-Adriatic Bimodal Oscillating System or BiOS33,34. The BiOS is a mode of variability characterized by a decadal reversal of the Northern Ionian Gyre (NIG) from cyclonic to anticyclonic, and vice versa. In its anticyclonic spinning the NIG deviates the inflowing Modified Atlantic Water (MAW) from the Sicily Channel towards the northern Ionian, entering the Adriatic Sea and decreasing its salinity and temperature. This prevents a portion of the MAW from reaching the Levantine basin, and enhances the outflow of Levantine waters into the western basin, along a pathway that follows the African coastline. The anticyclonic NIG co-occurs with higher concentrations of Atlantic and Western Mediterranean organisms in the Adriatic Sea. When the NIG is cyclonic, on the other hand, Levantine waters enter the Adriatic Sea, whereas the MAW preferably flows toward the Levantine35 and Lessepsian migrations influence the Adriatic Sea at various latitudes, affecting also phytoplankton phenology33,36,37. The corresponding regions and connectivity networks in the two opposite NIG periods are detailed in Figure S9 in the Supplementary Information. More