Philippa Kaur

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Study areaThe study was conducted on two rivers in north-eastern Amazonia sensu lato, including the Guiana Shield and the Amazon River drainage (Fig. 2). The climate of the entire study area is homogeneous and the region is covered by dense, uniform lowland primary rainforest51. The altitude is in the range of 0–860 m a.s.l. The regional climate is equatorial, and the annual rainfall ranges from 3600 mm in the northeast to 2000 mm in the southwest. The Maroni River is 612 km long from its source to its estuary, and its watershed covers a surface of >68,000 km2 in Suriname and French Guiana. The Oyapock River (length, 404 km; area, 26,800 km2) is located in the state of Amapa in Brazil and in French Guiana.The foregoing river basins host nearly 400 freshwater fish species and more than 180 mammal species52,53. Most of the mammal species have a large distribution range, covering the entire study area53. The fish species have a less homogeneous distribution and a distinct upstream-downstream composition gradient54,55. Here, only large rivers were considered and most fish species were widespread over the whole study area. As habitat availability increases with river size, species richness is expected to increase upstream to dowsntream31,32. The Oyapock and Maroni river basins are among the last remaining wilderness areas on Earth17. Nevertheless, ecological disturbances are increasing there because of a growing human population and the development of small-scale gold mining activity. These disturbances have caused limited but diffuse deforestation23,56. The deforested areas currently comprise 0.67% of all Maroni and Oyapock catchments.SamplingEnvironmental DNA (eDNA) was collected from water samples at 74 locations (hereafter, sites) along the main channel and the large tributaries of the Maroni and Oyapock rivers (Fig. 2). Thirty-seven sites were sampled at each river basin. The minimum and maximum distances between adjacent sites were 1.07 and 50.20 km, respectively. The mean and median distances between adjacent sites were 10.18 and 9.14 km, respectively, and the standard deviation (SD) was 7.79 km. The sites were located from sea level to 157 m a.s.l. At all sites, the river was wider than 20 m and deeper than 1 m (Strahler orders 4–8; Supplementary Fig. 5). The physicochemical properties of the water slightly varied among sites. The temperature, pH, and conductivity were in the ranges of 28.4–33.2 °C, 6.5–7.6, and 16.9–54.6 µS/cm, respectively, at all sites except two estuarine locations where the conductivity was relatively high because of seawater incursion (Supplementary Data 2).The eDNA samples were collected during the dry seasons (October–November) of 2017 and 2018 for Maroni and Oyapock, respectively. At both rivers, the sites were sequentially sampled from downstream to upstream at a rate of 1–4 sites per day depending on the distance and travel time between sites. Following the protocol of ref. 45, we collected the eDNA by filtering two replicates of 34 L of water per site. A peristaltic pump (Vampire Sampler; Buerkle GmbH, Bad Bellingen, Germany) and single-use tubing were used to pump the water into a single-use filtration capsule (VigiDNA, pore size 0.45 μm; filtration surface 500 cm2, SPYGEN, Bourget-du-Lac, France). The tubing input was placed a few centimetres below the water surface in zones with high water flow as recommended by Cilleros et al.43. Sampling was performed in turbulent areas with rapid hydromorphologic units to ensure optimal eDNA homogeneity throughout the water column. To avoid eDNA cross-contamination among sites, the operator remained on emerging rocks downstream from the filtration area. At the end of filtration, the capsule was voided, filled with 80 mL CL1 preservation buffer (SPYGEN), and stored in the dark up to one month before the DNA extraction. No permits were required for the eDNA sampling and the access to all sites was legally permitted. The study complies with access and benefit permits ABSCH-IRCC-FR-246820-1 and ABSCH-IRCC-FR-245902-1, authorizing collection, transport and analysis of all environmental DNA samples used in this study.Laboratory procedures and bioinformatic analysesFor the DNA extraction, each filtration capsule was agitated on an S50 shaker (Ingenieurbüro CAT M. Zipperer GmbH, Ballrechten-Dottingen, Germany) at 800 rpm for 15 min, decanted into a 50 mL tube, and centrifuged at 15,000 × g and 6 °C for 15 min. The supernatant was removed with a sterile pipette, leaving 15 mL of liquid at the bottom of the tube. Subsequently, 33 mL of ethanol and 1.5 mL of 3 M sodium acetate were added to each 50 mL tube, and the mixtures were stored at −20 °C for at least one night. The tubes were then centrifuged at 15,000 × g and 6 °C for 15 min, and the supernatants were discarded. Then, 720 µL of ATL buffer from a DNeasy Blood & Tissue Extraction Kit (Qiagen, Hilden, Germany) was added. The tubes were vortexed, and the supernatants were transferred to 2 mL tubes containing 20 µL proteinase K. The tubes were then incubated at 56 °C for 2 h. DNA extraction was performed using a NucleoSpin Soil kit (Macherey-Nagel GmbH, Düren, Germany) starting from step six of the manufacturer’s instructions. Elution was performed by adding 100 µL of SE buffer twice. After the DNA extraction, the samples were tested for inhibition by qPCR following the protocol in ref. 57. Briefly, quantitative PCR was performed in duplicate for each sample. If at least one of the replicates showed a different Ct (Cycle threshold) than expected (at least 2 Cts), the sample was considered inhibited and diluted 5-fold before the amplification.For the fish, the “teleo” primers58 (forward: 3ʹ-ACACCGCCCGTCACTCT-5ʹ; reverse: 3ʹ-CTTCCGGTACACTTACCATG-5ʹ) were used as they efficiently discriminated local fish species43,45. For the mammals, the 12S-V5 vertebrate marker59 (forward: 3ʹ-TAGAACAGGCTCCTCTAG-5ʹ; reverse: 3ʹ-TTAGATACCCCACTATGC-5ʹ) was used as it also effectively distinguishes local mammal species44,60. The DNA amplifications were performed in a final volume of 25 μL containing 1 U AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA, USA), 0.2 μM of each primer, 10 mM Tris-HCl, 50 mM KCl, 2.5 mM MgCl2, 0.2 mM of each dNTP, and 3 μL DNA template. Human blocking primer was added to the mixture for the “teleo”58 (5′-ACCCTCCTCAAGTATACTTCAAAGGAC-C3-3′) and the “12S-V5” primers61 (5′-CTATGCTTAGCCCTAAACCTCAACAGTTAAATCAACAAAACTGCT-C3-3′) at final concentrations of 4 μM and 0.2 μg/μL bovine serum albumin (BSA; Roche Diagnostics, Basel, Switzerland). Twelve PCR replicates were performed per field sample. The forward and reverse primer tags were identical within each PCR replicate. The PCR mixture was denatured at 95 °C for 10 min, followed by 50 cycles of 30 s at 95 °C, 30 s at 55 °C for the “teleo” primers and 50 °C for the 12S-V5 primers, 1 min at 72 °C, and a final elongation step at 72 °C for 7 min. This step was conducted in a dedicated room for DNA amplification that is kept under negative air pressure and is physically separated from the DNA extraction rooms maintained under positive air pressure. The purified PCR products were pooled in equal volumes to achieve an expected sequencing depth of 500,000 reads per sample before DNA library preparation.For the fish analyses, 10 libraries were prepared using a PCR-free library protocol (https://www.fasteris.com/metafast) at Fasteris, Geneva, Switzerland. Four libraries were sequenced on an Illumina HiSeq 2500 (2 × 125 bp) (Illumina, San Diego, CA, USA) with a HiSeq SBS Kit v4 (Illumina), three were sequenced on a MiSeq (2 × 125 bp) (Illumina) with a MiSeq Flow Cell Kit Version3 (Illumina), and three libraries were sequenced on a NextSeq (2 × 150 bp + 8) (Illumina) with a NextSeq Mid kit (Illumina). The libraries run on the NextSeq were equally distributed in four lanes. Sequencing was performed according to the manufacturer’s instructions at Fasteris. For the mammal analyses, eight libraries were prepared using a PCR-free library protocol (https://www.fasteris.com/metafast) at Fasteris. Two libraries were sequenced on an Illumina HiSeq 2500 (2 × 125 bp) (Illumina) using a HiSeq Rapid Flow Cell v2 and a HiSeq Rapid SBS Kit v2 (Illumina), three libraries were prepared on a MiSeq (2 × 125 bp) (Illumina) with a MiSeq Flow Cell Kit Version3 (Illumina), and three libraries were prepared using a NextSeq (2 × 150 bp + 8) (Illumina) and a NextSeq Mid kit (Illumina). The libraries run on the NextSeq were equally distributed in four lanes. As different sequencing platforms were used (MiSeq and NextSeq for the Maroni and HiSeq 2500 and MiSeq for the Oyapock; Supplementary Fig. 6 and Supplementary Data 3), the possible influences of the platforms on the sequencing results were verified. To this end, we compared the differences in species numbers between the sample replicates assigned to the same platform (accounting for replicate effect only) against those of the sample replicates assigned to different platforms (accounting for replicate and platform effects). As there were more sites with their two replicates sequenced with the same platform than sites with their replicates sequenced with different platforms (see Supplementary Fig. 6), sites with replicates on the same platform were randomly selected for the comparisons. We repeated this procedure 50 times. The number of species between replicates sequenced on the same platform and those sequenced on different platforms did not differ for >98.5% of all fish and mammal samples (Supplementary Fig. 7 and Supplementary Note 2). Similar to these results, a previous study on 16 S rRNA amplicon has shown that the samples were not influenced by the Illumina sequencing platform used62.To monitor for contaminants, 13 negative extraction controls were performed for each of the primers (“teleo” and “12S-V5”); one control was amplified twice. All of them were amplified and sequenced by the same methods as the samples and in parallel to them. Therefore, for the negative extraction controls, 168 amplifications were prepared with the “teleo” primers (13 negative controls; one amplified and sequenced twice) and 156 amplifications with the “12S-V5” primers (13 negative controls). Fourteen negative PCR controls (ultrapure water; 12 replicates) were amplified and sequenced in parallel to the samples. Eight were amplified with the “teleo” primers and six were amplified with the “12S-V05” primers. Thus, for the PCR negative controls, there were 96 amplifications with the “teleo” primers and 72 amplifications with the Vert01 primers. Sequencing information for the controls is shown in Supplementary Data 3c.An updated version of the reference database from ref. 43 was used. There were 265 Guianese species for the fish analyses (ref. 47). The GenBank nucleotide database was consulted, but it contained little information on the Guianese fish species. Most of the sequences were derived from ref. 43. For the mammal analyses, the vertebrate database was built using ecoPCR software63 from the releases 134 and 138 of the European Nucleotide Archive (ENA), for the Maroni and Oyapock river samples, respectively. The two releases were compared, and it was established that the new mammal species added to each version did not originate from French Guiana. Hence, the results were not influenced by the EMBL release number. The relevant metabarcoding fragment was extracted from this database with ecoPCR63 and OBITools64. Therefore, the reference database comprised the local database of French Guianese mammals60, which references 576 specimens from 164 species as well as all available vertebrate species in EMBL.The sequence reads were analyzed with the OBITools package according to the protocol described by Valentini et al.58. Briefly, the forward and reverse reads were assembled with the illuminapairedend programme. The ngsfilter programme was then used to assign the sequences to each sample. A separate dataset was created for each sample by splitting the original dataset into several files with obisplit. Sequences shorter than 20 bp or occurring less than 10 times per sample were discarded. The obiclean program was used to identify amplicon sequence variants (ASVs) that have likely arisen due to PCR or sequencing errors. It uses the information of sequence counts and sequence similarities to classify whether a sequence is a variant (“internal”) of a more abundant (“head”) ASV64. After this step, we matched the ASV with the reference database to obtain the taxonomic assignation for each ASV. Sequences labelled by the obiclean programme as ‘internal’’ and probably corresponding to PCR errors were discarded. The ecotag programme was then used for taxonomic assignment of molecular operational taxonomic units (MOTUs). The taxonomic assignments from ecotag were corrected to avoid overconfidence in assignments. Species-level assignments were validated only for ≥98% sequence identity with the reference database. Sequences below this threshold were discarded.Measuring disturbance intensity using GIS dataIn riverine systems, the disturbances may accumulate because of hydrologic connectivity, which is the downstream transfer of matter and pollutants4. Hence, the upstream sub-basin drainage network was considered to determine the size of the upstream sub-basin affecting local biodiversity (Fig. 1). The sub-basins were delineated by applying a flow accumulation algorithm to the SRTM global 30 m digital elevation model65. Deforestation was measured over 14 upstream spatial extents with radii of 0.5, 1.5, 3, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, and 90 km for each sampling site. Then, these 14 upstream spatial extents were intersected with the sub-basin drainage network. In addition, mammals and fish can also be affected by disturbances other than those mediated by hydrologic connectivity. Thus, deforestation was also measured upstream and downstream from the eDNA sampling sites using the same foregoing 14 radii.At each sampling site, deforestation intensity was quantified for each of the 14 spatial extents. We summed upstream (only accounting for disturbances mediated by river hydrologic connectivity) or upstream and downstream (not only considering disturbances mediated by hydrologic connectivity) deforested surfaces from Landsat satellite image datasets. Forest loss surfaces were obtained from the Global Forest Change dataset66. The Global Forest Change dataset identifies areas deforested between 2001 and 2017 on a 30 m spatial scale. To incorporate deforested areas prior to 2000, tree canopy cover data for that year were also used. Except for river courses, all pixels with More

We express our gratitude to Lukáš Falteisek, Richard Dorrell, Jan Petrášek, Stanislav Volsobě, Kateřina Schwarzerová and Jana Krtková for constructive discussions. English has been kindly corrected by William Bourland. Furthermore, we thank to Dovilė Barcytė, William Bourland, Antonio Calado, Dora Čertnerová, Yana Eglit, Ivan Fiala, Martina Hálová, Miroslav Hyliš, Dagmar Jirsová, Petr Kaštánek, Viktorie Kolátková, Alena Kubátová, Alexander Kudryavtsev, Frederik Leliaert, Julius Lukeš, Jan Mach, Joost Mansour, Jan Mourek, Yvonne Němcová, Fabrice Not, Vladimír Scholtz, Alastair Simpson, Pavel Škaloud, Jan Šťastný, Róbert Šuťák, Daria Tashyreva, Dana Savická, Jan Šobotník, Zdeněk Verner, Jan Votýpka for kindly providing cultures and taxonomic identifications. More

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Yuuka Fukui
Yuuka Fukui received Ph.D. degree from Keio University in 2012 under the supervision of Prof. Keiji Fujimoto. She was a JSPS research fellow (DC2) from 2010 to 2012. She joined the laboratory of Prof. Keiji Fujimoto at Keio university as a research associate in 2012 and was promoted to an assistant professor in 2017. Her research interests focus on the design and synthesis of polymeric materials (particles, membranes, porous structures) and organic-inorganic hybrid materials inspired from biological systems.About the award article: The authors reported a new technique to prepare nanoparticles from biomass-derived polymers, which will be utilized as an eco-friendly alternative to synthetic particulate plastics. Nanosized agarose gel particles were produced via sol-to-gel transition of agarose inside water nanodroplets prepared by W/O miniemulsion method. Subsequently, the water evaporation was carried out to generate xerogel nanoparticles (AgarX). The morphologies and crystal structure of AgarX were controlled by changing the pressure and temperature during the water evaporation. The resultant AgarX possessed high crystallinity and exhibited a water dispersibility and a water resistance.

Mikihiro Hayashi
Mikihiro Hayashi received his Ph.D. degree from Nagoya University (Prof. Yushu Matsushita group) in 2015. During his doctor course, he had been selected as a JSPS research fellow (DC2) and experienced researches in ESPCI Paris-Tech (Prof. Ludwik Leibler) and in Shanghai Jiao Tong University (Prof. Xinyuan Zhu). He then re-joined Ludwik Leibler’s group as a postdoc, and experienced another postdoc in Prof. Masatoshi Tokita in Tokyo institute of technology. In 2017, he became an assistant professor in Prof. Akinori Takasu group (Nagoya institute of technology), and currently manages his own laboratory as a PI. His research interest is the design of functional cross-linked materials.About the award article: the authors reported a preparation vitrimer-like elastomers with dynamic bond-exchangeable cross-links. A poly(ethyl acrylate)-based copolymer bearing random pyridine groups was synthesized, which was cross-linked by quaternization reaction with dibromo cross-linkers. In this system, the bond exchange was operated via trans-N-alkylation of the quaternized pyridine groups, showing useful sustainable functions, such as reprocessability, recyclability, and dissolution ability in some selective solvents.

Ryohei Ishige
Ryohei Ishige received his Ph.D. from Tokyo Institute of Technology in 2011 under the supervision of Prof. Junji Watanabe. He joined Prof. Atsushi Takahara’s laboratory at Kyushu University (2011–2013) and Prof. Yoshinobu Tsujii’s laboratory at Kyoto University (2013–2014). From 2014, he joined Prof. Shinji Ando’s laboratory at Tokyo Institute of Technology as an assistant professor and was promoted to an associate professor in 2021. His research interests are liquid-crystalline aromatic polymers and those structure-property relationships.About the award article: the authors developed a novel analytical technique integrating spectroscopies (infrared pMAIRS, and spectroscopic ellipsometry) and scattering methods (GI-WAXS), applied to the process where thin film polyimide, PI, is generated from linear poly(amic ester), PAE, precursors whose backbone consists of para-linkage. They revealed that PAE-based thin PI films form heterogeneous structure composed of non-oriented amorphous region and oriented ordered region which includes anisotropic nanopores causing structural birefringence. This method enables comprehensive evaluation of the evolution in complex hierarchical structures following chemical reactions for every noncrystalline thin film polymers.

Ryohei Kakuchi
Ryohei Kakuchi received his Ph.D. degree from the Hokkaido University in 2009 with a JSPS (Japan Society for Promotion of Science) research fellowship. After the Ph.D., he has made postdoctoral works in Germany from 2009 to 2014 and joined Kanazawa University as a research assistant professor in 2014. Based on the Leading Initiative for Excellent Young Researchers program, he was then appointed as an assistant professor (PI) at Gunma University in 2017. His research interests are the novel polymer synthesis based on unique organic transformation reactions including multicomponent reactions.About the award article: The authors proposed a new synthetic strategy to utilize wood-biomass sourced compounds in a green fashion. To achieve sustainable material chemistry, the intrinsic reactivity of lignin-derived poly(methacrylated vanillin) (PMV) was spotlighted because many multicomponent reactions employ aldehydes as a reactant. First, the Passerini three-component reaction (Passerini-3CR) of the PMV was revealed to proceed with >90% aldehyde conversions. Taking advantage of this high reactivity of the PMV, its immobilized cellulose fabric, a wood-biomass sourced organic hybrid, was revealed to accept the surface Passerini-3CR with amino acid derivatives, thereby demonstrating a fully bio-based material fabrication. More

Site descriptionThe respective sites were classified into five climatic regions in California, containing San Cruz and San Rose in region 1, Saint Helena and San Jose in region 2, Livermore and Cloverdale in region 3, Davis, Lodi and Fontana in region 4, Fresno and Bakerfield in region 5 (Fig. 1). There were differences in annual mean temperature among five climatic regions, ranging from 14.3°C to 18.6°C. In each region, the GHDs, quality of musts and wines, and wine tasting notes were recorded for 148 cultivars from 1935 to 1941. Meanwhile, in region 2, namely in Napa, the GHDs and must sugar content (in °Brix) were recorded for four representative cultivars (Cabernet Sauvignon, Chardonnay, Merlot and Sauvignon Blanc) during 1991–2018.Fig. 1The locations of five climatic regions for wine grape classed by Winkler index in California. The insert plot represents the distinct Winkler index (WI) during 1935–1941 in five climatic regions.Full size imageClimate dataThe climate data was collected from five stations for over one hundred year-period (1911–2018), including daily average, maximum and minimum temperature (Table 1). Climate data was retrieved from the National Oceanic and Atmospheric Administration (NOAA)’s National Centers for Environmental Information (NCEI). The database from which the data was retrieved was the “Global Historical Climatology Network – Daily (GHCN-Daily), Version 3” (https://www1.ncdc.noaa.gov/pub/data/ghcn/daily/by_station/)25,26. Table 1 showed the search codes and names of five stations in the website. The climate data of region 1 and region 5 were for the periods of 1911–2011 and 1938–2018, respectively.Table 1 Description of weather stations and time-span in five climatic regions.Full size tableBioclimatic indicesHere, we presented seven temperature-related indices to explore the changing climate in five climatic regions during the last 100 years. We compared the changes of these indices between the past (1935–1941) and current climate conditions (1991–2018). Thereafter, four indices were chosen to describe annual changes, including average, maximum, minimum temperature and diurnal temperature range (DTR). Furthermore, other indices were used to analyse growing season temperature (GST), Winkler index (WI) and Huglin index (HI) for the grape-growing season5,27,28. The equations used to calculate the bioclimatic indices of grape-growing season are:$$GST=frac{{sum }_{Apr1}^{Oct31}frac{{T}_{max}+{T}_{min}}{2}}{n}$$
(1)
$$WI={sum }_{Apr1}^{Oct31}left(frac{{T}_{max}+{T}_{min}}{2}-10right)$$
(2)
$$HI={sum }_{Apr1}^{Sep30}left(frac{{T}_{max}+{T}_{ave}}{2}-10right)times K$$
(3)
where Tmax, Tmin and Tave represent daily maximum, minimum and average temperatures, respectively. K is a length of day coefficient ranging from 1.02 to 1.06 between 40 and 50 of latitude in the northern hemisphere.Sample collection, harvest dates, quality of musts and wines measurementSample collection, harvest dates, quality of musts and wines measurement were detailed in the report of Amerine and Winkler24. Briefly, grape berries (22–220 kg) were picked in the morning from representative vines of variety collections or commercial vineyards by Amerine and Winkler24, as well as numerous vineyard owners. The harvest dates were recorded after picking. All grapes picked were crushed within 24 hours except for a few samples in 1935. The clear juice was taken after the coarse sediment had settled, in order to measure total soluble solids (°Brix), total acid (grams per 100 cc), and pH of must. The must was placed in an open oak fermenting tank. After fermentation, it was completed in a closed oak container. Then, the alcohol (percent by volume), extract (grams per 100 cc), tannin (grams per 100 cc), and fixed acid (grams per 100 cc) of wine were measured. The must °Brix was measured with a Brix hydrometer floating in a cylinder, must total acid was determined by titration with sodium hydroxide to a phenolphthalein end point, and must pH was measured with a quinhydrone electrode or a Beckman pH meter. In addition, wine alcohol was measured by the hydrometer and reported as percentage by volume, the extract and tannin of wine were measured by means of a special 0° to 8° Balling hydrometer and the Association of Official Agricultural Chemists method24. Note that the fixed acid of wine are equal to total acid minus volatile acid, where the total acid was measured by titration with phenolphthalein as an indicator while the volatile acid was determined also by titration with pretreated wines by method II of the Association of Official Agricultural Chemists24.Wine tasting notesThe purpose of wine tasting was to evaluate the cultivars based on the merits and defects of wine. The descriptive terms used for recording the results of the organoleptic examination contained appearance, color, odors, volatile acidity, total acidity, dryness, body, taste, smoothness and astringency, and general quality. More