dataesr/scientific-paragraphs-categorization huggingface dataset:
Eutrophication impairs lake ecosystems at a global scale. In this context, as benthic microalgae are well-established warnings for a large range of stressors, particularly nutrient enrichment, the Water Framework Directive required the development of diatom-based methods to monitor lake eutrophication. Here, we present the diatom-based index we developed for French lakes, named IBDL. Data were collected in 93 lakes from 2015 to 2020. A challenge arose from the discontinuous pressure gradient of our dataset, especially the low number of nutrient-impacted lakes. To analyze the data we opted for the so-called "Threshold Indicator Taxa ANalysis" method, which makes it possible to determine a list of "alert taxa". We obtained a multimetric index based on different pressure gradients (Kjeldahl nitrogen, suspended matter, biological oxygen demand and total phosphorous).
, 2010)  consistent with a potential subsequent combination of this index with the existing French macrophyte index IBML (Indice Biologique Macrophytique en Lac: Macrophyte Biological Index for Lakes,  Boutry et al. , 2015). We detail here how diatom data were sampled and analyzed and how we developed the IBDL. Finally, we discuss the relevance of this new index, comparing the results obtained with index scores based on macrophytes, and assessing its ability to reveal environmental gradients.
2-1 Data collection Samples were collected from 93 French lakes (Figure  1 ) during the summer period, each year from 2015 to 2020, according to  Morin et al. (2010). The lakes were classified into three (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted March 10, 2023. ; https://doi. org/10. 1101/2023. 03. 08. 531691 doi: bioRxiv preprint metatypes based on alkalinity, according to the European intercalibration exercise previously performed  (Kelly et al. , 2014b) : low alkalinity (LA, alkalinity ≤ 0. 2 meq. l -1 ), medium alkalinity (MA, 0. 2 meq. l -1 < alkalinity < 1 meq. l -  ), and high alkalinity (HA, alkalinity ≥ 1 meq. l -  ). Diatoms were collected from both mineral substrates and lakeshore macrophyte surfaces in observation units (OUs), whose number and location varied according to the lake surface area and the riparian zone types.
Such units are defined in the French macrophyte sampling protocol for lakes NF T90-328 (AFNOR, 2022). Figure  1 : Study sites, number of surveys per site, and lake alkalinity classes (LA: low alkalinity; MA: medium alkalinity; HA: high alkalinity)  (Kelly et al. , 2014a)  2-1-1 Biological data Samples from hard mineral substrates were taken from at least five boulders or cobbles selected at random for each OU. The total surface area sampled was equivalent to 100 cm², as defined in the NF T90-354 standard  (AFNOR, 2016). Selected substrates had to be submerged within the euphotic zone, at a maximum depth of 0. 5 m. Samples performed on macrophytes were taken from helophytes (mainly Phragmites australis (Cav. ) Trin. ex Steud. ). Green stem segments submerged for at least 4 to 6 weeks were collected from a minimum of 5 macrophytes chosen at random.
2-1-2 Physico-chemical data Parameter values were determined in summer at the deepest point of each lake, according to European standards. Data were obtained from national surveillance monitoring programs. Water quality analysis was not systematically performed each year: in a few cases, the most recent physicochemical data available were collected three years before the diatom samples. The following parameters were recorded: biological oxygen demand (BOD 5, mg. l -1 ), oxygen (O 2 , mg. l -1 ), oxygen saturation (% O 2 ), conductivity (Cond, µs. cm²), Kjeldahl nitrogen (NKJ, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted March 10, 2023. ; https://doi. org/10.
2-2 Data analysis and index settlement All analyses were performed with R version 4. 1. 2 (2021-11-01) (R Core Team, 2021) (Platform: x86_64-pc-linux-gnu (64-bit), Running under: Ubuntu 22. 04. 1 LTS). Considering that the final dataset revealed a discontinuous trophic gradient, we opted for the so-called "Threshold Indicator Taxa ANalysis" method (TITAN2 package, Baker et al. , 2020), which, based on bootstrapping and permutations, makes it possible to determine a list of "alert taxa". The presence and/or increasing abundance of alert taxa reveal the existence of anthropogenic pressures. TITAN replaces the community level response along a composite gradient with taxon specific responses towards single environmental variables  (Dufrêne and Legendre 1997). Negative and positive responses are distinguished, and cumulative decreasing or increasing responses in the community are tracked.
2-2-2 Building metrics and selecting the relevant ones For each environmental variable, a metric was calculated at the OU scale according to (1): ‫ܿ݅ݎݐ݁ܯ‬ ெ ൌ 1 െ ቀ ௧ ೌೣೌ ூௗ௫ ೌೣೌ ቁ (1) Where: Alert taxa is the number of alert taxa and Index taxa is the number of index taxa in the sample. The metric value is bounded between 0 and 1. The lowest value (0) corresponds to a species list entirely composed of alert taxa (determined for the environmental variable considered). To build our index, we then selected the most relevant metrics, i. possible to obtain a single response pattern for a metric whatever the lake metatype and substrate type considered. ‫ܵܧܵ‬ ெ ൌ ൬ ெ௧ ಾ ିெ ೠ ௦ௗ ೠ ൰ (2) Where: Metric M is ‫ݎ݊ܵܧܵ‬ ெ ൌ ሺௌாௌ ಾ ିெሻ ሺெ௫ିெሻ (3) Where: SES M is the observed value of SES for a given metric, Min its minimum value and Max its maximum value in the whole dataset (values of Min and Max are given in  Table 2 S1 ).
2-3 Comparing IBDL and IBML scores We compared IBDL and IBML scores, based respectively on diatom and macrophyte communities, to evaluate their complementarity or redundancy. IBML scores were computed with the online application https://seee. eaufrance. fr/api/indicateurs/IBML/1. 0. 1 and the "httr" package  (Wickham, 2022). We built a multiple linear regression model ("mass" package) to test which index correlated best with Pt values: IBML, IBDL or a combination of both (mean value). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted March 10, 2023. ; https://doi. org/10. 1101/2023. 03. 08. 531691 doi: bioRxiv preprint.
2-4 Preparing intercalibration Considering a future intercalibration exercise, we analyzed the relationships between IBDL scores and P t for each lake metatype. A good correlation of the candidate metric with Pt constitutes a key criterion for considering the index ready for integration into the intercalibration process  (Kelly et al. , 2014b). We also plotted IBDL against CM scores (intercalibration Common Metric, i. e. , the Trophic Index developed by  Rott et al. , 1998) , to check their compliance. The CM was calculated with Omnidia 6 software.
3-Results Our data revealed discontinuous pressure gradients (Table  1 ), with a clear lack of impacted conditions and an over-representation of lakes characterized by low eutrophication levels. Biotic and abiotic data were obtained for 958 samples. Considering the data validation criteria, 99% of the samples were included in the analysis. Data from both substrate types were available for 552 OUs. Seven hundred eighty taxa were recorded, 8% of which were identified to the genus level. One hundred and twenty-one alert taxa were determined out of 590 index taxa (S2). Metric values (in EQR) calculated from the lists of taxa sampled on mineral substrates and macrophytes for a single OU did not differ significantly (p-value =0. 65). IBDL scores at the lake level were calculated from the selected metrics following the aggregation rules proposed.
4-Discussion As required by the WFD, we developed a diatom index for the assessment of the ecological status of French lakes. We obtained very good correlations between IBDL and key environmental variables. One major challenge arose from the discontinuous pressure gradient of our dataset, especially the low available number of nutrient-impacted lakes. The scarcity of impacted lakes in the datasets used to build diatom indices is not rare and has already been pointed out by some authors  (Bennion et al. , 2014). This lack makes it impossible to capture the entire trophic gradient or to build reliable species' ecological profiles. However, the majority of existing indices are calculated as an abundance-weighted average of the ecological profiles of every taxon from a sample, according to the Zelinka & (which was not certified by peer review) is the author/funder.
All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted March 10, 2023. ; https://doi. org/10. 1101/2023. 03. 08. 531691 doi: bioRxiv preprint Marvan formula  (Zelinka and Marvan, 1961). This method is far from optimal for datasets showing discontinuous or very specific environmental conditions  (Carayon et al. , 2020). In such cases, the identification of alert taxa seems more appropriate than considering diatom communities as a whole. This has made the TITAN algorithm increasingly popular for detecting specific taxa providing reliable signals of a specific stress  (Khamis et al. , 2014; Costas et al. , 2018; Gieswein et al. , 2019; Carayon et al. , 2020; Gonzalez-Paz et al. , 2020). Using this method, we built a multimetric index based on different pressure gradients (NKJ, SP, BOD 5 and P t ).
, 2014; Lainé et al. , 2014). Considering the generally high abundance of A. minutissimum in samples, this tends to blur the overall pressure-response relationship between index scores and environmental variables  (Potapova and Hamilton, 2007). TITAN provides a means to avoid this pitfall, as such complexes are not selected as alert taxa, given that their abundance dynamics do not show clear response patterns to environmental gradients. Indeed, A. minutissimum, although highly abundant in our dataset (22% of total species abundances), was not considered an alert taxon. The fact remains that IBDL tends to be less stringent than IBML, despite better relationships with Pt. In consequence, we have to explain why we think that the use of diatom-based indices to assess lake ecological status is justified.
, 2014b). The first step consisted in testing the correlation between IBDL scores and total phosphorus in our dataset. Only HA and MA typologies were considered here but, in any case, the last intercalibration exercise could not be performed for LA lakes. We obtained very good correlations that are clearly an (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint this version posted March 10, 2023. ; https://doi. org/10. 1101/2023. 03. 08. 531691 doi: bioRxiv preprint improvement compared to the non-significant relationship previously obtained between BDI (diatom index used for the assessment of rivers) and Pt, and even better than the pressureimpact relationships observed at a pan-European scale (R² between national methods and Pt ranged from 0.
org). For this purpose, genes of ResFinder, VirulenceFinder, and PlasmidFinder database were manually searched after download on the assembly genome using BLAT (BLAST-Like Alignment Tool) software with 80% of coverage and 90% identity thresholds  [28]. Resfinder databases identified genes encoding ESBL and pAmpC along with the resistance determinants to all antimicrobials (S4 and S5 Tables). Core genome multilocus sequence typing (cgMLST) was calculated from allelic comparison of genes and the sequence types (STs) were identified with an in-house tool (github. com/ bvalot/pyMLST). We built phylogenetic trees from the distances calculated on the core genome genes using Neighbor-Joining algorithm with Grapetree software  [29]. Phylogenetic tree figure were built on the iTOL (Interactive Tree Of Life) website  [30].
coli, independent of the animal source and species. Then, we frequently identified bla CTX-M-14 and bla CTX-M-15 (11. 5% for each) among the 3GC-R E. coli collection. More specifically, this ranking differed between HA and DA reservoirs, independent of the animal species. We found all the same ranking for the HA reservoir, with CTX-M-1 as the most identified (n = 13, 38. 0%) followed by both SHV-12 and CMY-2 (n = 7, 21. 0% each). Regarding the DA reservoir, although CTX-M-1 (n = 14, 32%) dominated, CMY-2 and SHV-12 were in fourth and fifth rank (n = 4, 9% and n = 3, 7%, respectively), surpassed by CTX-M-15 (n = 7, 16%) and CTX-M-14 (n = 6, 14%). We confirmed previous European data findings showing that CTX-M-1, SHV12, and CMY-2 dominated in HA, whereas CTX-M-1, CTX-M-14, and CTX-M-15 dominated mainly in DA  [20, 35].
Acknowledgments The authors gratefully thank the national coordinators, the different slaughterhouses and the national microbiological laboratories involved in the sampling and isolation procedures. Other members of the EASSA and VetPath Study Groups are  Pascal Butty  ( CEVA, France ),  Rebecca Klee , ( Boehringer-Ingelheim, Germany ),  Ulrich Klein  ( Huvepharma, Belgium ),  Herve Marion  ( CEESA, Belgium ),  Sylvie Meschi  ( Virbac, France ),  Markus Rose  ( MSD, Germany ),  Guido Schiffer  ( Elanco, Germany ),  Shabbir Simjee  ( Elanco, United Kingdom ),  Beata Truszkowska  ( Fatro, Italy ), and  Clèlia Stevenin  ( Boehringer-Ingelheim, France ).
Measurement of DNA methylation Cohorts assessed cord blood DNA methylation data individually, using their own laboratory methods, quality control, and normalisation. DNA methylation data was sampled using the lllumina Infinium® HumanMethylation450 (486,425 probes), except for BiB, which used the Illumina EPIC BeadChip array (866,553 probes). Probes on single nucleotide polymorphisms (SNPs), cross-hybridizing probes  (26) , and probes on the sex chromosomes were excluded. In the final meta-analysis, only probes that were available in both arrays (maximum 364,678) were included. Methylation was measured using normalised beta values ranging from 0 to 1, representing 0 to 100% methylation.
Cohort-specific statistical analyses Probe-level analysis The analysis plan and R-script is available on GitHub (https://github. com/ammegandchips/Prenatal_Caffeine). Cohorts were asked to exclude multiple pregnancies (e. g. , twins) and siblings so that each mother was only included once in the dataset. If cohorts included more than one major ethnic group, they were asked to run the EWAS analysis separately for each group. The EWAS R script included: a function to remove probes classified as an outlier according to the Tukey method of outlier removal (valuesR<R25th percentileR-R3*interquartile range and valuesR>R75th percentileR+R3*interquartile range)  (37) , a function to generate surrogate variables using the R package SVA  (38) , and a function to run an EWAS of each model using the R package Limma  (39).
Differentially methylated regions (DMR) The probe level approach was complemented using a regional analysis, which considers DNA methylation at clusters of neighbouring CpG sites throughout the epigenome. This approach is more statistically powerful and arguably more biologically plausible; neighbouring CpG sites are assumed to exert similar biological functions. We used the DMRff method  (41)  to identify differentially methylated regions (DMRs). Cohorts were supplied with an R script to conduct the DMR analysis using their own data (https://github. com/ammegandchips/Prenatal_Caffeine/blob/master/dmrff. mat. caff. E WAS. cohorts. r). Probes were annotated to the human reference genome version 19, build 37h using the annotation data available from the R-package meffil  (42).
Meta-analysis Probe level meta-analysis Results were meta-analysed with fixed effect estimates weighted by the inverse of the variance using the software METAL  (43). Multiple testing was accounted for using a 5% false discovery rate (FDR)  (44). Meta-analysed results were scrutinised in a similar manner as the individual cohort results and leave-one-cohort-out analysis using the R package metafor  (45)  was performed on the CpGs that showed evidence to be associated with maternal caffeine consumption. Results were deemed to be driven by a single cohort (and therefore to "fail" the leave-one-out test), if the meta-analysis effect estimate changed direction, moved towards the null by more than 20%, or had a confidence interval that included 0 after removal of a single cohort. DMR meta-analysisR DMR cohort results were meta-analysed using an inverse-variance weighted fixed effects approach using the dmrff.
Summary & interpretation of findings We investigated the association of self-reported maternal caffeine consumption during pregnancy with offspring cord blood DNA methylation using data from six international birth cohorts. For the EWAS meta-analysis, probe-level and regional DMR analyses were applied as hypothesis-free approaches to detect associations between maternal caffeine phenotypes and differential methylation levels in cord blood. We compared caffeine consumption during pregnancy across countries and caffeinated drinks, which reduced the potential for cultural confounding, and analysed the CpG sites of the maternal caffeine associated DMRs for their biological function. Results of these analyses show little converging evidence between the associations of the different sources of caffeine, indicating that the associations that we observed are most likely explained by other factors than caffeine exposure.
| Skeletal abnormalities, growth and survival At 20 dpf, a random sample of 50 individuals from each group and replicate was acquired to estimate skeletal abnormalities and fish size. Individuals were euthanatized (overdose of 2-phenoxyethanol, 0. 3-0. 5 mL L -1 ), fixed in 5% phosphate-buffered formalin (pH = 7. 2) and stained for cartilage and mineralised bone  (Walker & Kimmel, 2007). Stained samples were photographed under a stereo-microscope (Olympus SZ61) and measured for total length (TL) using tpsDig2 software  (Rohlf, 2010). Cranial, vertebral and fin abnormalities were examined following the classification methodology and terminology of  Koumoundouros (2010). When an individual presented more than one abnormality, multiple abnormality scores were recorded. Following  Harder (1975) , the terms prehaemal and haemal were used to describe the vertebrae with and without a haemal spine, respectively (corresponding to the pre-caudal and caudal vertebrae of  Bird & Mabee, 2003).
Field Monitoring and Data Analyses Starting in 2000, we monitored seven freshwater sites (ponds and ditches with slow streams) in Monza Park (Figure  1 ) to assess the abundance of breeding R. latastei females by counting the number of egg clutches laid during the reproductive season. Field surveys were conducted in 10 different years, from 2000 to 2023:  2000, 2001, 2004, 2005, 2006, 2011, 2017, 2019, 2022, and 2023. Surveys were conducted at daytime, during the peak of the breeding season of the Italian agile frog (February-April) in order to perform visual counts of the egg clutches present at each site, following Dalpasso, Ficetola, Giachello, Lo Parrino, Manenti, Muraro, and Falaschi  [20]. For each year, each site was surveyed multiple times (range: 3-7) between February and April. During surveys, we carefully inspected the whole surface of the sites and counted the number of egg clutches seen.
g. , particularly dry periods). Samples were aggregated as follows: 2000/2003, 2004/2007, 2016/2019, 2020/2023; surveys were also performed during 2011, but during this year, we did not follow the standard protocol for counting clutches. Therefore, for 2011, we only reported the occurrence of clutches, without providing estimates of the overall abundance of the species. During 2020-2021, performing complete surveys was not possible due to travel restrictions related to COVID-19. We calculated the maximum clutch count per year and then the minimum and maximum abundance for each 4-year period, that is, the highest and lowest yearly counts, respectively. Counts were aggregated over 4-year periods only for graphical representation, while statistical analyses were performed with non-aggregated (year by year) data in which year was a continuous variable.
Since the species was never found in the last period (see results), we ran a generalized linear mixed model (GLMM) to assess whether the population was showing any significant decline in abundance before its putative extinction in 2022. GLMM was run following Barker et al. [34]. Simulations and real-world analyses showed that GLMMs using observed abundances can provide good estimates of population trends even for species with imperfect detection  [34]. In the GLMM, we used the maximum yearly clutch count in each site as a dependent variable, year as an independent variable, and site identity as a random effect. Before running the model, the year variable was scaled to mean = 0 and SD = 1 to improve model convergence. The number of clutches per site showed strong overdispersion; thus, GLMM was run in a Bayesian framework using the brms R package (version 2.
Assessing the Factors Related to Changes in Occupancy Given the small amount of data available, we used occupancy instead of abundance data to assess the factors related to the decline of the study species. We used a binomial GLMM to assess the relationship between the presence/absence of clutches and four variables representing climate and habitat suitability. For climate, we used mean temperature and total precipitation of March, which is the month corresponding to the peak of the breeding season of the species in the study area  [37]. Climatic data were retrieved from the ERA5 database  [38]. We downloaded the hourly temperature and precipitation for all the days of March of sampled years and, for each year, calculated the average temperature and the total precipitation of the month. Additionally, we considered two factorial variables related to human-mediated pressures on breeding sites: the occurrence of the invasive red swamp crayfish (0/1) and water management.
Continuous independent variables (temperature and precipitation) were scaled (mean = 0 and variance = 1) before analyses, and sub-population identity was included as a random factor. We used first-order autoregressive models to take into account temporal autocorrelation. Before running the analysis, we tested the correlation among independent variables and found no strong correlations (all Pearson's correlation coefficients <|0. 44|). The analysis was performed in the R environment using the INLA package  [40]  (version 23. 04. 24).
Trees are an integral part in European landscapes, but only forest resources are systematically assessed by national inventories. The contribution of urban and agricultural trees to national-level carbon stocks remains largely unknown. Here we produced canopy cover, height and above-ground biomass maps from 3-meter resolution nanosatellite imagery across Europe. Our biomass estimates have a systematic bias of 7. 6% (overestimation; R = 0. 98) compared to national inventories of 30 countries, and our dataset is sufficiently highly resolved spatially to support the inclusion of tree biomass outside forests, which we quantify to 0. 8 petagrams. Although this represents only 2% of the total tree biomass, large variations between countries are found (10% for UK) and trees in urban areas contribute substantially to national carbon stocks (8% for the Netherlands).