Ecology

Loose coupling and human intervention promote collective foraging successWe first determined group search performance by assessing the average search time, consumption time, and total targets found in each movement condition with and without intervention.Results showed that search performance as measured by mean trial time was better with loose coupling and human intervention, as seen in the lowest average trial times in Fig. 3. Movement type had a reliable effect on performance without human intervention, F(1,59) = 27.65, p  More

Study areaKogi East located in Kogi State, North Central Nigeria. It is a geographical region comprising of nine (9) Local Government Areas (LGAs); Ankpa, Bassa, Dekina, Ibaji, Idah, Igalamela/Odolu, Ofu, Olamaboro and Omala. The region is located between latitude 6º32′33.8′′N to 8º02′44.8′′N and longitude 6º42′08.5′′E to 7º51′50.3′′E. It occupies an area of 26,197 square kilometres sharing boundaries with six (6) states of Nigeria28. The population of the region at 2006 is 1,479,144 with a projected population of 1,996,700 at 201629.Ethical approval and informed consentEthical clearance was obtained from Research Ethics Committee, Kogi State Ministry of Health (KSMoH), Lokoja with reference number MOH/KGS/1376/1/82 and permission was obtained from the State Universal Basic Education Board (SUBEB), Lokoja with reference number KG/SUBEB/GEN/04/’T’ which was conveyed to the Education Secretaries of the 9 LGAs and the Headmasters (mistress) of the schools.This study follows guidelines for the care and use of human samples established by the Human Care and Use Committee of the Ahmadu Bello University, Zaria, Kaduna State, Nigeria and the Research Ethics Committee, Kogi State Ministry of Health (KSMoH), Lokoja.Statement of consent from participantsWritten consents were obtained from the guardians/parents of study participants, informing them of their rights and granting permission for their children to participate in the study.Source of epidemiological dataThe epidemiological data used for this study were obtained from an earlier district-wide survey carried out in 2018 (Table 1)25 in rural communities of Kogi East, Kogi State, Nigeria. The study obtained samples from school-children of age 5 to 14 years. Samples collected were examined using formal ether sedimentation technique. The study was carried out in schools that did not receive anthelminthic drugs during the yearly periodic deworming exercise carried out by the State Ministry of Health. During the survey, the geographical coordinates of each school and community were captured within the school premises using a handheld Global Positioning system (GPS) device, Garmin 12XL (Garmin Corp, USA).Table 1 Epidemiological Data from District Wide Survey Conducted in 2018 by Yaro et al. (2020) in Kogi East, North Central Nigeria.Full size tableSpatial analysis of STHsCo-ordinate of schools sampled and the mean prevalence of each parasites from the baseline study for A. lumbricoides, Hookworms and S. stercoralis were computed in Microsoft Excel version 2013 and converted to comma delimited file (.csv). These files were further converted from text files to shapefiles using DIVA-GIS version 7.5.0 and were geo-referenced on the map of Kogi East, Nigeria. The prevalence of these parasites were categorized; 0.0–1.0,  > 1.0–5.0,  > 5.0–10.0,  > 10.0–20.0,  > 20.0–50.0 and  > 50.0 on the map (Figs. 1 and 2).Figure 1Source of Satellite Imagery: Image Google Earth: Landsat/Copernicus (Data SIO, NOAA, U.S. Navy, NGA, GEBCO. Maps were visualized on ArcMap 10.1. https://www.google.com/maps/place/Kogi/@7.3195959,7.2632804,189324m/data=!3m1!1e3!4m5!3m4!1s0x104f41e9d61f12dd:0xbdc9f94f2d58aafd!8m2!3d7.7337325!4d6.6905836.Spatial Distribution of STHs in Communities of Kogi East, North Central Nigeria.Full size imageFigure 2Source of Satellite Imagery: Image Google Earth: Landsat/Copernicus (Data SIO, NOAA, U.S. Navy, NGA, GEBCO. Maps were visualized on ArcMap 10.1. https://www.google.com/maps/place/Kogi/@7.3195959,7.2632804,189324m/data=!3m1!1e3!4m5!3m4!1s0x104f41e9d61f12dd:0xbdc9f94f2d58aafd!8m2!3d7.7337325!4d6.6905836.Spatial Distribution of STHs in Local Government Areas of Kogi East, North Central Nigeria.Full size imageEnvironmental data collectionClimatic and elevation variablesRemotely sensed environmental data for altitude, temperature and precipitation were obtained from Worldclim database30. The climatic variables such as temperature and precipitation are at global and meso scales and topographic variables such as elevation and aspect likely affect species distributions at meso and topo-scales31. Hence, the use of the climatic and topographic variables in the prediction of distributions of soil transmitted helminths in Kogi East, Nigeria. Also, temperature was considered in the analysis because A. lumbricoides, hookworms and S. stercoralis have thermal thresholds of 38 °C, 40 °C and 40 °C respectively outside of which the survival of the infective stages in the soil decline32,33.In this study, a total of 19 bioclimatic factors of present climate for Nigeria were downloaded at 1 km spatial resolution (Table 2) from Worldclim database30 and were used in the prediction of soil transmitted helminths distribution in Kogi East. Elevation data derived from the Shuttle Radar Topography Mission (SRTM) (aggregated to 30 arc-seconds, “1 km”) were also downloaded from WorldClim database30.Table 2 Characteristics of Environmental Variables Used in Predicting the Distribution of STHs in Nigeria.Full size tableEdaphic variableThe influence of edaphic factors on the distribution of STHs have been reported by several researchers globally34,35,36 as important factors in the biology of STH parasites. In view of this, data for soil pH, soil moisture content, soil organic carbon and soil clay content for Africa continent were downloaded from International Soil Reference Centre (ISRIC) soil database as spatial layers (Table 2)37.File conversions and resamplingThe 19 bioclimatic factors downloaded from WorldClim data are in geographic coordinates of latitudes and longitudes which comes as .bil files were extracted into a folder. These data were transformed into predefined geographic coordinate system (GCS_WGS_1984), this projection was done on ArcMap 10.1 and were converted to asci files on DIVA-GIS 7.5. These files were transferred back to ArcMap and assigned a projected coordinate system of Universal Transverse Mercator (UTM) Zone 32 N (Nigeria is located on UTM Zone 31, 32 and 33). Also, the edaphic factors obtained were also assigned a projected coordinate system. The projected raster files (i.e. climatic, elevation and edaphic) were all clipped into a layer using the administrative boundary map of the study area, this was downloaded on DIVA-GIS database38.Prior to modelling, all variables were resampled from their native resolution to a common resolution of 1 km spatial resolution using the nearest neighbour technique on ArcMap 10.1 to enable overlaying of variables. The resampled raster files were converted to float files on ArcMap 10.1 and transferred to DIVA-GIS 7.5. Float files were converted to grid files and then to asci files on DIVA-GIS 7.5 and were used on MaxEnt tool for modelling the distribution of STHs in Kogi East.Ecological niche modellingThe potential distribution of STHs were modelled using maximum entropy (MaxEnt) software version 3.3.3k39. MaxEnt uses environmental data at occurrence and background locations to predict the distribution of a species across a landscape31,40. This modelling tool was selected based on the reasons of Sarma et al.41, they stated that this tool allows the use of presence only datasets and model robustness is hardly influenced by small sample sizes. It has been shown to be one of the top performing modelling tools42.Probability of presence of each of the STH was estimated by MaxEnt using the prevalence of each of the STH parasites obtained for 45 sampled communities in the 9 LGAs of Kogi East during the district-wide survey carried out in 201825 served as the presence records to generate background points were used41. Regularization of the prevalence was performed to control over-fitting. This modelling tool uses five different features to perform its statistics; linear, quadratic, product, threshold and hinge features to produce a geographical distribution of species within a define area. The MaxEnt produces a logistic output format used in the production of a continuous map that provides a visualization with an estimated probability of species between 0 and 1. This map distinguish areas of high and low risk for STH infections41.The 19 bioclimatic factors, elevation data and the edaphic factors obtained were used for the ecological niche modelling. The level of significance of contribution of the altitude and 19 bioclimatic factors was used to calculate the area under the receiver operating characteristics curve (AUC) was used to evaluate the model performance. The AUC values varies from 0.5 to 1.0; an AUC value of 0.5 indicates that model predictions are not better than random, values  0.9 indicates high model performance43.Model validation was performed as follows41, using the ‘sub-sampling’ procedure in MaxEnt. 75% of the parasites prevalence data were used for model calibration and the remaining 25% for model validation. Ten replicates were run and average AUC values for training and test datasets were calculated. Maximum iterations were set at 5000. Sensitivity, which is also named the true positive rate, can measure the ability to correctly identify areas infected. Its value equals the rate of true positive and the sum value of true positive and false negative. Specificity, which is also named the true negative rate, can measure the ability to correctly identify areas uninfected. Its value equals the rate of true negative and the sum value of false positive and true negative.Ethics approvalThis study follows guidelines for the care and use of experimental animals established by the Animal Care and Use Committee of the Ahmadu Bello University, Zaria for the purpose of control and supervision of experiments on animals and ethical permission for the study was obtained from the ethical Board of Kogi State Ministry of Health, Lokoja with reference number: MOH/KGS/1376/1/82. More

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Endolysosomal patch-clamp experimentsEndolysosomal patch-clamp experiments were performed as previously described6,14,23,25,29,30. In brief, for whole-LE/LY manual patch-clamp recordings, cells were treated with 1 μM vacuolin (HEK293 cells: overnight) in an incubator at 37 °C with 5% CO2. Compound was washed out before patch-clamp experimentation. Currents were recorded using an EPC-10 patch-clamp amplifier (HEKA, Lambrecht, Germany) and PatchMaster acquisition software (HEKA). Data were digitized at 40 kHz and filtered at 2.8 kHz. Fast and slow capacitive transients were cancelled by the compensation circuit of the EPC-10 amplifier. All recordings were obtained at room temperature and were analyzed using PatchMaster acquisition software (HEKA) and OriginPro 6.1 (OriginLab). Recording glass pipettes were polished and had a resistance of 4–8 MΩ. For all experiments, salt-agar bridges were used to connect the reference Ag–AgCl wire to the bath solution to minimize voltage offsets. Liquid junction potential was corrected. For the application of small molecules, compounds were added directly to the patched endolysosomes to either evoke or inhibit the current. The cytoplasmic solution was completely exchanged by cytoplasmic solution containing compound. The current amplitudes at −100 mV were extracted from individual ramp current recordings. Unless otherwise stated, cytoplasmic solution contained 140 mM K-MSA, 5 mM KOH, 4 mM NaCl, 0.39 mM CaCl2, 1 mM EGTA and 10 mM HEPES (pH was adjusted with KOH to 7.2). Luminal solution contained 140 mM Na-MSA, 5 mM K-MSA, 2 mM Ca-MSA 2 mM, 1 mM CaCl2, 10 mM HEPES and 10 mM MES (pH was adjusted with methanesulfonic acid to 4.6). In all experiments, 500-ms voltage ramps from − 100 to + 100 mV were applied every 5 s. All statistical analysis was completed using OriginPro9.0 and GraphPadPrism software.Cell cultureHEK293 cells stably expressing hTPC2-YFP or hTRPML1-YFP were used for patch-clamp experiments. Cells were maintained in DMEM supplemented with 10% FBS, 100 U penicillin/mL, and 100 μg streptomycin/mL. Cells were plated on glass cover slips 24–48 h before experimentation. Cells were transiently transfected with Turbofect (Fermentas) according to the manufacturer’s protocols and used, e.g. for confocal imaging or patch-clamp experiments 24–48 h after transfection. Cells were treated with compounds at 37 °C and 5% CO2. MNT-1 WT and TPC2−/− KO cell lines were grown in high glucose DMEM, supplemented with 20% FBS, 10% AIM-V, 1% sodium pyruvate (Thermo Fisher), and 1% penicillin–streptomycin (Sigma-Aldrich). B16F10 cells were grown in high glucose DMEM, supplemented with 10% FBS (Thermo Fisher), 1% L-glutamin, and 1% penicillin–streptomycin (Sigma-Aldrich). Cell lines were maintained at 37 °C in a 5% CO2 incubator.Melanin screening in B16F10 mouse melanoma cellsMelanin content determination was performed as described previously with some modifications31. In brief, B16F10 cells at density of 5 × 103 cells/well in 96-well plate were cultured and incubated with various plant extracts or flavonoids at a concentration of 20 µg/ml or 20 µM, respectively, for 4–5 days. Melanin content was measured using a microplate reader (Anthros, Durham, NC, USA) and calculated based on the OD ratio between treated and untreated cells.Melanin content and tyrosinase activity assaysMNT-1 WT and TPC2−/− KO cell lines were grown as described in the cell culture section. After reaching 80–90% confluency, cells were subcultured (every 2–3 days). Forskolin (Sigma-Aldrich Cas Nr. 66,575,299) was used as positive control and 4-Butyl-resorcinol (TYR-inh., Sigma-Aldrich, Cas Nr.18979-61-8) as negative control. For experiments, cells were plated in 6-well plates with 200,000 cells per well. Cells were incubated for 72 h at 37 °C and 5% CO2. After removing cell culture media, cells were washed in DPBS twice, then cells were collected using a cell scraper. Cells were centrifuged at 3000 rpm for 5 min. Pellets were lysed with RIPA buffer, supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich) and 1% phosphatase inhibitor (Sigma-Aldrich) at 4 °C (on ice) for 45 min. Cells were centrifuged at 12.000 rpm for 15 min (4 °C), supernatant was subsequently removed and protein content determined using a protein dye reagent assay (Bio-Rad; protein standard curve (BSA) 0, 1, 3, 5, 8, 10, 12, 15 μg/mL). Cell pellets were dissolved in 250 μL 1 N NaOH/10% DMSO and incubated at 80 °C for 2 h. After centrifugation at 12.000 rpm for 10 min, supernatants were removed to a 96-well plate. Absorbance was measured (in triplicates, each) at 405 nm using a microplate reader (Tecan, Infinite M200 PRO). Melanin content was normalized to total protein content.To measure tyrosinase activity 100 μg protein from the supernatant after RIPA lysis were transferred into a 96-well plate and 50 μL of 15 mM L-DOPA (Sigma) were added (total volume was adjusted to 100 μL using PBS, pH 6.8 (adjusted with 1 N HCl)). After 30 min incubation at 37 °C, dopachrome formation was determined by measuring the absorbance at 475 nm using a microplate reader (Tecan, Infinite M200 PRO). Tyrosinase activity (%) was calculated as follows: OD475 (sample) × 100 / OD475 (control).Cell proliferation assayCell proliferation assay was performed in 96-well, flat-bottom microtiter plates (Sarstedt), in triplicates, and at a 5 × 103 cell density per well. Cells were seeded overnight, including cells measured as day zero control. Proliferation rate was assessed by incubation with CellTiter-Blue (Ctb, Promega, Mannheim, Germany) reagent for 3 h. Fluorescence was measured using a microplate reader at 560Ex/600Em (Tecan, Infinite M200 PRO).Wound healing/migration assayWound healing assay was performed using 12-well plates (Sarstedt) at a density of 120,000 cells/well. Cells were incubated overnight, and a scratch was performed using a yellow pipet tip. Pictures were taken at 0, 24, 48, and 72 h with an inverted microscope (Leica DM IL LED) and using a microscope camera (Leica DFC 3000 G). The wounded cell area was quantified using ImageJ 1.52a software and was subtracted from 0 h values.Invasion assayTranswell chambers in 24-well permeable support plates (Corning, #3421) were coated with Corning Matrigel basement membrane matrix (Corning, #354234) for 1.5 h. A total of 3 × 104 MNT-1 cells were seeded on top of the chambers in serum-free medium, and direct stimulation with compounds was performed. The lower compartment contained the chemotactic gradient, medium with 10% FBS. Cells were allowed to migrate for 24 h, and were then fixed and stained with crystal violet containing methanol. Non-invaded cells were removed with Q-tips and pictures were taken of the bottom side of the membrane using an inverted microscope (Olympus CKX41) and an Olympus SC50 camera (Olympus). The number of invaded cells was quantified using ImageJ 1.52a software.Western blottingWestern blot experiments were performed as described previously32. Briefly, cells were washed twice with 1 × PBS and pellets were collected. Total cell lysates were obtained by solubilizing in TRIS HCl 10 mM pH 8.0 and 0.2% SDS supplemented with protease and phosphatase inhibitors (Sigma). Protein concentrations were quantified via Bradford assay. Proteins were separated via a 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE; BioRad) and transferred to polyvinylidene difluoride (PVDF; BioRad) membranes. Membranes were blocked with 5% bovine serum albumin (Sigma) or milk diluted in Tris Buffered Saline supplemented with 0.5% Tween-20 (TBS-T) for 1 h at room temperature (RT), then incubated with primary antibody at 4 °C overnight. Then, membranes were washed with TBS-T and incubated with horseradish peroxidase (HRP) conjugated anti-mouse or anti-rabbit secondary antibody (Cell Signaling Technology) at RT for 1 h. Membranes were then washed and developed by incubation with Immobilon Crescendo Western HRP substrate (Merck) and by using an Odyssey imaging system (LI-COR Biosciences). Quantification was carried out using unsaturated images on ImageJ 1.52a software. The blots were cropped prior to hybridisation with antibodies against vinculin, GAPDH, or actin. The following antibodies were used: Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, 1:1000, cat. #9106), p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology, 1:1000, cat. #9102), Phospho-Akt (Ser473) (Cell Signaling Technology, 1:1000, cat. #4058), Akt (Cell Signaling Technology, 1:1000, cat. #9272), MITF (Santa Cruz Biotechnology, 1:1000, cat. #Sc-71588), MITF (Cell Signaling Technology, 1:1000, cat. #97800), MITF (Abcam, 1:1000, cat. #ab12039), GSK-3β (Cell Signaling Technology, 1:1000, cat. #9832), CREB and pCREB (Cell Signaling Technology, 1:1000, cat. #9197S and #9198S), ß-Actin (Santa Cruz Biotechnology, 1:1000, cat. #Sc-47778), Vinculin (Cell Signaling Technology, 1:1000, cat. #4650), GAPDH (Cell Signaling Technology, 1:1000, cat. #5174S), Anti-Mouse (Cell Signaling Technology, 1:10,000, cat. #7076), and Anti-Rabbit (Cell Signaling Technology, 1:10,000, cat. #7074).RNA isolation and quantitative PCRTotal RNA was isolated from the cells using the RNeasy Mini Kit (Qiagen). Reverse Transcription was performed using the Revert First Strand cDNA Synthesis Kit (Thermo Fisher). Real-time quantitative Reverse Transcription PCR (qPCR) was performed in triplicates for each sample using the LightCycler 480 SYBR Green I Master and using the LightCycler 480 II machine (Roche Life Science), following the recommended parameters. HPRT was used as the housekeeping gene. The following human primer sets were used: Tyrosinase primers set A: fw: 5′-GTCTGTAGCCGATTGGAGGA -3′; rev: 5′- TGGGGTTCTGGATTTGTCAT -3′. Tyrosinase primers set B: fw: 5′-TGACAG TATTTTTGAGCAGTGG -3′; rev: 5′- GGTGCATTGGCTTCTGGATA-3′.Plant materialCommercially available heartwood of Dalbergia parviflora was purchased from “Chao Krom Poe” herbal medicine dispensary in Bangkok in 2004. The samples were identified as wild Dalbergia parviflora at Princess Sirindhorn Wildlife Sanctuary, known as “Pa Phru To Daeng” which is a peat swamp forest in Mueang Narathiwat, Tak Bai, Su-ngai Kolok, and Su-ngai Padi districts of Narathiwat Province in Southern Thailand (06° 04′ 33.8″ N, 101° 57′ 49.3″ E). Data collection in the area was carried out with the authorization and guidelines of the National Research Council of Thailand (NRCT), and complied with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Conservation (1989) and the Convention on International Trade in Endangered Species of Wild Fauns and Flora (CITES, 1975). The plant was identified by Dr. Chawalit Niyomdham of the Forest Herbarium, National Park, Wildlife and Plant Conservation Department, Bangkok, Thailand. Its voucher specimen (number 68143)33,34 was deposited at The Forest Herbarium, Bangkok, Thailand.Extraction and isolation of flavonoidsThe dried heartwood of D. parviflora (2 kg) was extracted three times with MeOH (3 × 20 L) at room temperature. The extracts were combined and concentrated under reduced pressure at 60 °C to yield 910 g of a viscous mass. A part of this concentrated extract (150 g) was chromatographed on a silica gel column (12 × 40 cm) and fractionated using chloroform-MeOH (98:2, 96:4, 94:6, 90:10, 15 L each). Fractions of 500 mL were collected and pooled by TLC analysis to yield a total of 26 combined fractions. Purification of these fractions as reported previously33,34 gave various flavonoid compounds as summarized in Fig. S1. Purification of fraction 14 (8.9 g) using HPLC on a Develosil- Lop-ODS column (5 × 100 cm, flow rate, 45 mL/min with detection at 205 nm), with MeCN-H2O (30:70) as the eluent gave MT-8 (pratensein) (715 mg) (tR = 220 min). Purification of fraction 6 (3.1 g) using HPLC on a Develosil-Lop-ODS column (5 × 100 cm, flow rate: 45 mL/min with detection at 205 nm), with MeCN-H2O (32:68) as the eluent, gave UM-9 (duartin) (39 mg) (tR = 240 min). Both compounds were identified by comparison of their spectroscopic data with published values35,36.NMR analytical dataNMR spectra were measured on an JEOL alpha 400 (1H-NMR: 400 MHz, 13C-NMR: 100.4 MHz) spectrometer33,34. NMR-Spectra were measured in deuterated solvents and chemical shifts are reported in δ (ppm) relative to the internal standard tetramethylsilane (TMS) or the solvent peak at 35 °C, respectively. J values are given in hertz. Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Signal assignments were carried out based on 1H, 13C, HMBC, HMQC and COSY spectra. Inverse-detected heteronuclear correlations were measured using HMQC (optimized for 1JC-H = 145 Hz) and HMBC (optimized for 3JC-H = 8 Hz) pulse sequences with a pulsed field gradient. FABMS spectra were obtained on a JEOL JMS-700 using a m-nitrobenzyl alcohol matrix. Optical rotation was measured on a JASCO DIP-360 digital polarimeter. Column chromatography (CC) was performed with powdered silica gel (Kieselgel 60, 230–400 mesh, Merck KGaA, Darmstadt, Germany) and styrene–divinylbenzene (Diaion HP-20, 250–800 µm particle size, Mitsubishi Chemical Co., Ltd.). Precoated glass plates of silica gel (Kieselgel 60, F254, Merck Co., Ltd., Japan) and RP-18 (F254S, Merck KGaA) were used for TLC analysis. The TLC spots were visualized under UV light at a wavelength of 254 nm and sprayed with dilute H2SO4, followed by heating. HPLC separation was mainly performed with a JASCO model 887-PU pump, and isolates were detected by an 875-UV variable-wavelength detector. Reversed-phase columns for preparative separations (Develosil Lop ODS column, 10—20 µm, 5 × 50 × 2 cm; Nomura Chemical Co. Ltd., Aichi, Japan; flow rate 45 mL/min with detection at 205 nm) and semi-preparative separations (Capcell Pak ODS, 5 µm, 2 × 25 cm, Shiseido Fine Chemiacls Co. Ltd, Tokyo, Japan; flow rate 9 mL/min with detection at 205 nm) were used. MT-8 (pratensein): Amorphous powder; 1H-NMR (400 MHz, (CD3)2CO) δ (ppm) = 13.03 (s, 1H, 5-H), 8.18 (s, 1H, 2-H), 7.13 (d, J = 2 Hz, 1H, 2′-H), 7.04 (dd, J = 9, 2 Hz, 1H, 6′-H), 6.99 (d, J = 9 Hz, 1H, 5′-H), 6.41 (d, J = 2 Hz, 1H, 8-H), 6.28 (d, J = 2 Hz, 1H, 6-H), 3.87 (s, 3H, 4′-OCH3). 13C-NMR (100.4 MHz, (CD3)2CO) δ (ppm) = 181.6 (C-4), 165.0 (C-7), 164.0 (C-5), 159.1 (C-9), 154.5 (C-2), 165.0 (C-7), 148.6 (C-4′), 147.3 (C-3′), 125.0 (C-1′), 121.3 (C-6′), 124.0 (C-3), 112.3 (C-5′), 106.3 (C-10), 99.9 (C-6), 94.5 (C-8), 56.4 (C-4′ OCH3). FABMS m/z 323 [MNa] + (calcd for C16H12O6Na). UM-9 (duartin): morphous powder; 1H-NMR (400 MHz, (CD3)2CO) δ (ppm) = 6.70 (d, J = 9 Hz, 1H, 5′-H), 6.65 (d, J = 9 Hz, 1H, 6′-H), 6.64 (d, J = 9 Hz, 1H, 5-H), 6.40 (d, J = 9 Hz, 1H, 6-H), 4.29 (ddd, J = 10, 3, 2 Hz, 1H, 2 eq-H), 3.96 (t, J = 10 Hz, 1H, 2ax-H), 3.47 (dddd, J = 11, 10, 5, 3 Hz, 1H, 3-H), 2.91 (dd, J = 16, 11 Hz, 1H, 4ax-H), 3.47 (ddd, J = 16, 5, 2 Hz, 1H, 4 eq-H), 3.87 (s, 3H, 2′-OCH3) , 3.81 (s, 3H, 4′-OCH3) , 3.77 (s, 3H, 8-OCH3). C-NMR (100.4 MHz, (CD3)2CO) δ (ppm) = 149.4 (C-7), 148.5 (C-9), 148.3 (C-4′), 146.5 (C-2′), 140.2 (C-3′), 136.6 (C-8), 128.0 (C-1′), 124.5 (C-6), 117.2 (C-6′), 115.4 (C-10), 108.4 (C-6), 107.9 (C-5′), 70.8 (C-2), 32.5 (C-2), 32.1 (C-3), 60.7 (C-8 OCH3), 60.5 (C-2′ OCH3), 56.4 (C-4′ OCH3). [α]D + 15.4° (c 1.0, CHCl3). FABMS m/z 355 [MNa] + (calcd for C18H20O6Na). More

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In Europe, bumblebees pollinate the flowers called foxgloves, but foxgloves that spread to the Americas are also pollinated by hummingbirds and have evolved as a result. Credit: Getty

Ecology
16 April 2021
Flowers adapt to welcome the birds — but not the bees

Once in the Americas, foxgloves swiftly evolved under pressure by pollinating hummingbirds.

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Evolution can forge new relationships between plants and pollinators in fewer than 85 generations.The showy purple flowers called common foxgloves (Digitalis purpurea) are native to Europe, where they are pollinated by bumblebees. When admiring humans took the foxglove to the Americas, it was enthusiastically embraced by a new guild of nectar-drinkers — the hummingbirds.Maria Clara Castellanos at the University of Sussex in Brighton, UK, and her colleagues tallied visitors to foxgloves in the United Kingdom, Colombia and Costa Rica during more than 2,000 3-minute study periods. They found that hummingbirds pollinate up to 27% of foxgloves in Colombia and Costa Rica, where the flowers’ corollas — the long purple tubes that gardeners love so much — are 13% and 26% longer, respectively, than those of UK foxgloves.So why would foxgloves with longer corollas do better? Plants with corollas too long for bumblebees to reach their nectar are guaranteed to be pollinated by hummingbirds, which are more effective than bees at depositing pollen on the next flower. The longer corolla also creates a more comfortable fit for a hovering hummingbird, perhaps improving pollination rates.Hummingbirds can travel further between flowers than can bees, which might reduce plant inbreeding.

J. Ecol. (2021)

Ecology More