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    Contribution of conspecific negative density dependence to species diversity is increasing towards low environmental limitation in Japanese forests

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    Presence and biodistribution of perfluorooctanoic acid (PFOA) in Paracentrotus lividus highlight its potential application for environmental biomonitoring

    Samples collectionThree sampling campaigns were carried out at the two sample sites (A and B) on the coast of north-western Sicily (Fig. 1a) chosen for this study. The main features of the sites and sampling details are summarized in Table S1 (Supplementary Information). A total of 90 specimens of sea urchins Paracentrotus lividus (45 specimen per each site), 30 l of seawater (15 per site), 40 samples (20 per site) of sea grass Posidonia oceanica (less than 5 cm leaf fragments, according to the institutional and national ethical guidelines) were collected and analyzed together with 30 l of brackish water from site B (15 l from each creek).Figure 1Map of the sampling site. (a) Geographic area, (b) bathymetric chart and (c) relative distance between sample sites; (d, e) close ups of sampling sites. (Images obtained by courtesy of Google Earth Pro and map.openseamap.org).Full size imageThe samplings activity was authorized by the Capitaneria di Porto of Palermo with protocol number: 0029430. In the absence of data about PFOA contamination in the most recent report about chemical contamination in the coastal region subjected to this study31, the choice of sample sites was based on supposedly different status of pollution based on the site position or proximity to human activities (e.g. restaurants, pipeline, sewages, etc.).Site A (see Fig. 1d), was chosen assuming a lower state of pollution based on its position in proximity to Capo Zafferano, at the northern extremity of S. Elia’s bay, with an average depth of 11 m and rocky seabed (see Fig. 1b and Supplementary Information: Table S1). Conversely, Site B (see Fig. 1e was chosen in the same coastal area (only 4.7 km away from Site A) assuming a higher state of pollution due to its position located on the southern side of Solanto promontory, nearby a pipeline and the mouths of two small creeks from inland, with a shallow (3 m) sandy seabed and where a bathing prohibition order is in place32 (see Fig. 1b, c and Supplementary Information: Table S1).The biodistribution of PFOA in the various matrices was evaluated by analyzing sea urchin’s coelomocytes (CC) (90 samples) and coelomic fluid (CF) (90 samples), as well as gonads (G) (63 samples from 32 sea urchins collected in site A and 31 sea urchins collected in site B), or mixed organs (MIX) (27 samples from 13 sea urchins collected in site A and 14 sea urchins collected in site B) consisting of a homogenized mixture of urchin’s inner matrices when gonads were not developed enough for sampling. Due to their mutually exclusive nature the latter two datasets (G and MIX) were merged and labelled as “Gonads or Mixed organs” (GoM) for statistical analysis and graphical representations that needed a uniform dataset of 45 items per site. Further details on the collection of matrices and their labelling are described in the Supplementary Information.The size of the sea urchins (horizontal diameter without spines) ranged between 30 and 51 mm indicating specimen that have lived in their respective site approximately from 3 to 5 years25.PFOA extraction and analysisMaterials, equipment and software are described in the Supplementary Information.PFOA extraction procedures were adapted33 to the type of matrix to be analyzed. Recovery percentages (R %) were checked per each batch of analyses by spiking blank samples with different amounts of PFOA analytical standard before the extraction procedure33.Spiked samples underwent the same extraction procedure of unspiked samples and the percentage of recovery R was calculated according to Eq. 1, where Cspike is the known concentration of spiked PFOA, Dspiked is the instrumental (LC–MS) analytical response of the spiked sample (i.e. the “detected” concentration), Dunspiked is the analytical response of the unspiked sample. R was then used in Eq. 2 to calculate the actual values, [PFOA], of PFOA concentrations in unspiked analyzed samples.$$ {text{R}} = 100 times left( {{text{D}}_{{{text{spiked}}}} – {text{D}}_{{{text{unspiked}}}} } right)/{text{C}}_{{{text{spike}}}} $$
    (1)
    $$ left[ {{text{PFOA}}} right] = 100 times {text{D}}_{{{text{unspiked}}}} /{text{R}} $$
    (2)
    With the exception of [PFOA]seawater and [PFOA]creek, which are expressed as nanograms per liter (ppt), all other PFOA concentrations are expressed in nanograms per gram of matrix (ppb).The PFOA standard was used for calibration before each batch of analyses and a linear response (R2  > 0.99) was recorded in the concentration range from 0.1 to 1000 ppb. The RSDs on three replicates were below 10%. LOD (0.1 ppb) and LOQ (1.0 ppb) were quantified by IUPAC method. LC–MS analyses were performed in the negative ion-monitoring mode (see Supplementary Information).For the analysis of P. lividus specimens, an estimate of the total PFOA concentration, [PFOA]TOT in ng/g, in each sea urchin has been calculated considering the sampled weight (W) in grams of each matrix (Eq. 3):$$ left[ {{text{PFOA}}} right]_{{{text{TOT}}}} = left( {{text{W}}_{{{text{CF}}}} left[ {{text{PFOA}}} right]_{{{text{CF}}}} + {text{W}}_{{{text{CC}}}} left[ {{text{PFOA}}} right]_{{{text{CC}}}} + {text{W}}_{{{text{GoM}}}} left[ {{text{PFOA}}} right]_{{{text{GoM}}}} } right)/left( {{text{W}}_{{{text{CF}}}} + {text{W}}_{{{text{CC}}}} + {text{W}}_{{{text{GoM}}}} } right) $$
    (3)
    Water analysisDuring each one of the 3 sampling campaigns, 2 samples of seawater (5 l from Site A and 5 l from site B) and 2 samples of brackish water (5 l from each creek mouths in site B) were collected for a total of 6 seawater samples and 6 brackish water samples.Samples were checked for the presence of PFOA by solid phase extraction (SPE) (see Supplementary Information) followed by LC–MS analysis34.The percentage of recovery, calculated according to Eq. 1, was R = 120%. [PFOA]seawater and [PFOA]creek concentrations (ng/L) were determined from analytical data according to Eq. 2.
    Posidonia oceanica analysisA total of 40 samples of leaves were collected from different individuals of P. oceanica (20 samples from site A and 20 samples from site B). Each sample was cut in tiny pieces and homogenized using an agate mortar and pestle, weighed (0.5 g) and transferred to a glass tube for extraction (see Supplementary Information).The percentage of recovery, calculated according to Eq. 1, was R = 70%. [PFOA]P. oceanica concentrations (ng/g) were determined from analytical data according to Eq. 2.Coelomocytes and coelomic fluid analysisThe coelomic fluid, containing also the coelomocyte population, was taken from all the ninety collected specimens (45 per site) by inserting an ultrathin and sharp needle (32G 0.26 mm × 12 mm) of a 1 mL syringe through the peristomal membrane35. All samples were centrifuged at 4 °C and 1500 rpm for 5 min in a 5804R refrigerated centrifuge (Eppendorf, Germany) thus separating the supernatant coelomic fluid (CF) from the coelomocytes (CC). CF and CC were then weighed and placed in different glass tubes for subsequent PFOA extractions (see Supplementary Information).The percentage of recovery, calculated according to Eq. 1, was R = 28% for CF and R = 68% for CC. [PFOA]CF and [PFOA]CC concentrations (ng/g) were determined from analytical data according to Eq. 2.Gonads analysisThe extraction of PFOA from 63 samples of gonads (32 from Site A and 31 from Site B) was performed with LC–MS grade methanol following the same procedure used for extraction from CF and CC (5 mL for samples greater than 0.5 g samples; 2.5 mL for samples between 0.1 g and 0.5 g). In case of undetected PFOA (considered as zero-values in graphics and statistical data treatment), analyses were repeated for confirmation on concentrated sample extracts.Twenty spiked samples were prepared from the most abundant samples of gonads (10 spiked samples per site), by adding 25 µL of an aqueous 1 mg/L stock solution of PFOA to 0.25 g of gonads samples. The percentage of PFOA recovery from gonads, calculated according to Eq. 1, was R = 73%. [PFOA]G concentrations (ng/g) were determined from analytical data according to Eq. 2.Mixed organs analysisIn 27 specimens of sea urchins (13 from Site A and 14 from Site B), the developmental status was not sufficient to collect at least 0.1 g of gonad sample. For these individuals, organs remaining after CF and CC collection, mainly intestine and undeveloped gonads, were mixed together and extracted similarly to the other matrices.Spiked samples were prepared by adding 25 µL of an aqueous 1 mg/L stock solution of PFOA to 0.25 g of mixed organs (MIX) samples. The percentage of PFOA recovery from MIX, calculated according to Eq. 1, was R = 20%. [PFOA]MIX concentrations (ng/g) were determined from analytical data according to Eq. 2.Statistical analyses and graphical data representationThe distribution of PFOA concentrations in all the sampled matrices from collected sea urchins is graphically represented by box and jitter plot (Fig. 2) where the 25–75 percentiles are drawn using a box; minimum and maximum are shown at the end of the thin lines (whiskers), while the median is marked as a horizontal line in the boxfitting. Statistical tests and linear fittings were used to evaluate data significance and correlations (see Supplementary Information).Figure 2Box and jitter plot showing the concentrations of PFOA found in the Coelomic Fluid (CF) Coelomocytes (CC) and Gonads or Mixed organs (GoM), as well as the total PFOA concentration (TOT), in 45 specimens of P. lividus collected from Site A (left side) and in 45 specimens of P. lividus collected from Site B (right side).Full size imageA permutational multivariate analysis of variance PERMANOVA36 was performed to evaluate the differences in the PFOA concentrations between the two groups of sea urchins collected from site A and site B. The experimental design comprised of one factor (Site) two levels (fixed and orthogonal) and four variables corresponding to the concentrations of PFOA in each type of sample analysed (coelomocytes, coelomic fluid, gonad or mixed organs) including the estimated total PFOA concentration. Each term in the analysis was tested by 999 random permutations.Finally, Principal Component Analysis (PCA) (see Supplementary Information: PCA tables and graphs) was performed on a dataset, containing five variables. Specifically sea urchin’s size and PFOA concentrations in each type of sample (CF, CC, and GoM) as well as in the entire sea urchin (TOT), to verify the multivariate nature of data in a relatively small number of dimensions, thus limiting the loss of information. More

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    Electric field detection as floral cue in hoverfly pollination

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    Sustainable irrigation based on co-regulation of soil water supply and atmospheric evaporative demand

    Field measurementsWe used two sets of field measurements of soil moisture, VPD, and stomatal conductance of maize at the daily scale to illustrate a proof-of-concept for the co-regulation of soil moisture and VPD on stomatal conductance.The first set was measurements from greenhouse experiments of maize (seed: Dekalb hybrid DKC52-04) at Colorado State University during the 2013 growing season (planted on June 10, 2013)49. There were two treatments (well-watered, WW, and water-stressed, WS) with five plants per treatment. The soil of the greenhouse experiments was the air-dried soilless substrate (8.8 kg) consisting of a 1:1.3 by volume ratio of Greens GradeTM, Turface® Quick Dry® and Fafard 2SV in 26 L pots49. The soil moisture measurements came from soil moisture sensors (Decagon5TM sensors) installed in the middle of the pots (~6 inches from top). The greenhouse measurements of leaf-level stomatal conductance and soil moisture were performed in approximately 2-week intervals beginning in the vegetative stage and continuing until plant senescence (DOY 198–199, 210–211, 217–218, 233–234, 247), with 11 replicates for each plant under two treatments (WW and WS). The environmental variables, such as relative humidity and air temperature, were continuously measured in minutes. Other detailed experimental setups can be found in Miner and Bauerle (2017)49.The second set was eddy-covariance measurements of maize cropping systems (seed: Pioneer 33P67/33B51) from 2001 to 2012 at three AmeriFlux sites (US-Ne1, Ne2, and Ne3). US-Ne1 and Ne2 were irrigated sites, with a continuous maize cropping system during 2001–2012 for US-Ne1 and with a maize-soybean rotation cropping system during 2001-2009 and then a continuous maize cropping system during 2010-2012 for US-Ne2. US-Ne3 was rainfed with a maize-soybean rotation cropping system during 2001–2012. The soil at the three AmeriFlux sites was a deep silty clay loam consisting of four soil series: Yutan, Tomek, Filbert, and Filmore. There are three replicates with the soil moisture sensors (theta probes: ML2, Dynamax Inc.) installed horizontally with the profile of soil depth (10, 25, 50, and 100 cm) in the US-Ne1 and US-Ne2, and four replicates with soil moisture sensors (theta probes: ML2, Dynamax Inc.) installed horizontally with the profile of soil depth (10, 25, 50, and 100 cm) in the US-Ne3 (http://csp.unl.edu/public/G_moist.htm). The soil moisture data used here was from the top soil layer (10–25 cm). The canopy-level stomatal conductance (Gs) was derived by inverting the Penman-Monteith equation50 (Equations 1 and 2) from the eddy-covariance measurements at the hourly scale18,24,51, and the averaged value near midday (from 12:00 to 14:00) was applied as the daily canopy-level stomatal conductance to remove the diurnal cycle. This inversion was only conducted during peak growing season (July and August) to avoid the impact of LAI24. The impact of evaporation from canopy interception and of low incoming shortwave radiation was removed by data filtering24, i.e., excluding the data within 2 days following every precipitation and irrigation event, and periods of low incoming shortwave radiation conditions ( More

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    Comprehensive mineralogical and physicochemical characterization of recent sapropels from Romanian saline lakes for potential use in pelotherapy

    Mineralogy and thermal propertiesThe bulk mineral composition of sapropels is detailed in Table 1. The XRD analysis indicates that Amara and Tekirghiol sapropels are enriched in silicates, i.e., quartz (30.8% and 29.1% respectively), plagioclase-albite (10.1% and 8.9%), carbonates, mainly calcite (6.8%) and aragonite (13.1%) in Amara, and calcite (8.7%) in Tekirghiol (Fig. 2). By contrast, Ursu sapropel contains lower concentrations of silicates, mainly quartz (15.4%), plagioclase (5.5% albite and 8% andesine), sulfides, i.e., pyrite (1.5%) and is enriched in halite (34.5%). The major clay components in the sapropels were 2:1 dioactahedral and 2:1 trioctahedral clays, representing 28.9%, 23.6% and 20.8% of clay minerals in Tekirghiol, Amara and Ursu samples, respectively. Muscovite was detected in similar concentrations in Tekirghiol (4.5%) and Amara (4.2%). Quantitative mineralogical clay composition of the fraction  90% in each sample), and kaolinite and chlorite as minor fractions (Table 2; Fig. 3).Table 1 Quantitative bulk mineralogical compositions of saline sapropels collected from Tekirghiol, Amara and Ursu lakes.Full size tableFigure 2X-ray diffraction patterns on the raw mud samples (upper image) collected from the three lakes. The main minerals that contribute to the most important reflections are indicated. Chl: Chlorite, M: Muscovite, K: Kaolinite Group minerals, Q: Quartz, A: Anatase, 2:1: 2:1 phyllosilicate (e.g., illite and smectite), Ca: Calcite, Pl: Plagioclase/Albite/Andesine, R: Rutile, P: Pyrite, Ar: Aragonite, H: Halite.Full size imageTable 2 Quantitative mineralogical clay composition of the fraction  More

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    Stronger temperature–moisture couplings exacerbate the impact of climate warming on global crop yields

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