Phocoena phocoena), one of the smallest cetaceans in the Northern Hemisphere, is a primary example. These elusive, highly mobile small animals confound traditional approaches of collecting tissue samples for genetic analyses, yet their nearshore habitat makes them highly vulnerable to fisheries by-catch and the effects of habitat degradation. By exploiting the naturally shed cellular material in seawater and the power of next-generation sequencing, we develop a novel approach for generating population-specific mitochondrial sequence data from environmental DNA (eDNA) using surface seawater samples. Indications of significant genetic differentiation within a currently recognized management stock highlights the need for dedicated eDNA sampling throughout the population's range in southeast Alaska. This indirect sampling tactic for characterizing stock structure of small and endangered marine mammals has the potential to revolutionize population assessment for otherwise inaccessible marine taxa.Determining management units for natural populations is critical for effective conservation and management. However, collecting the requisite tissue samples for population genetic analyses remains the primary limiting factor for a number of marine species. The harbour porpoise ( This Accurate characterization and quantification of the genetic diversity within and among populations hinges upon the collection of an adequate number of samples representing the full genetic diversity across the geographical range of a population. While remote collection of tissue samples using biopsy darts has become a routine and highly successful method for many large cetacean species, efforts to collect significant numbers of genetic samples from some of the smaller, more enigmatic dolphin and porpoise species are still often restricted to beach-cast carcasses and fisheries by-catch. Such opportunistic samples provide insight into population structure and geographical breaks in genetic diversity, but lack of certainty about sample provenance and the infrequency of sample discovery present challenges for addressing questions pertaining to human-mediated environmental impacts on relatively local scales.Phocoena sinus), the harbour porpoise (Phocoena phocoena) is the smallest cetacean in the North Pacific. Harbour porpoise are broadly distributed through cold and temperate coastal waters along the continental shelf. The coastal habitat and prey preferences of this small, elusive marine mammal contribute to their vulnerability to anthropogenic impacts. In particular, their shallow coastal distribution makes them highly vulnerable to incidental capture during net-fishing operations [Oncorhynchus spp.) and Pacific herring  [With the sole exception of the vaquita  . Line-trpallasi) . Concernpallasi) .To overcome these sampling challenges, we explored the use of seawater environmental DNA (eDNA) to obtain population-level genetic data. Exploiting the cellular material that is continually shed into aquatic environments by inhabiting organisms is increasingly being used to detect rare and invasive organisms ,9 and to2.2.1.© polyethylene canteens. Before the start of the field season, all collection canteens were sterilized with a 10% bleach solution and triple rinsed in distilled water to ensure they were free of any exogenous DNA. Samples were kept cool and dark until further processing in an enclosed bow box on the skiff. During sampling, the air and sea-surface temperatures (approx. 10°C) in Alaska were sufficiently low such that external cooling of the collected samples with ice packs was deemed unnecessary. Negative eDNA controls consisted of seawater samples collected in regions where no porpoise were observed.Seawater samples for eDNA were collected in regions of historically high porpoise density  throughoSeawater eDNA samples were vacuum-filtered within 10 h of collection (mean = 3.42 h). The entire sample volume (2.3–3.1 l) was vacuum-filtered through a sterile, 0.45 µM nitrocellulose filter using a single-use filtration cup  attached to a PVC vacuum filtration manifold. Post-filtration, filters were stored in 5 ml of Longmire's lysis buffer  at ambie2.2.et al. [DNA extractions were carried out in a sterile biosafety cabinet in a laboratory dedicated to pre-PCR procedures. Rigorous controls for preventing and monitoring cross-sample contamination and contamination from exogenous DNA were employed throughout sample processing. Prior to each extraction set, the ventilation hood and all equipment were cleaned with DNA AWAY™ and irradiated with UV light for 30 min before and after each extraction. No harbour porpoise tissue extractions were performed concurrently during the period of eDNA extractions. Genomic DNA was extracted from each sample using a standard phenol–chloroform protocol . Filterset al. . After t2.3.b gene sequences for cetacean species known to co-occur with harbour porpoise in the inland waters of southeast Alaska were obtained from GenBank and aligned to identify species-specific regions for qPCR probe and primer design. A TaqMan© probe specific to harbour porpoise (Ppho_Cytb-PROBE CTATGACTATTAGTAGTAAGAGCACCC) was designed for the target region and modified with 6-FAM reporter dye at the 5′ end and a non-fluorescent quencher at the 3′ end. Primers Ppho_Cytb-F (GTTCTTCATTTGTCTTTATATCCATATTG) and Ppho_Cytb-R (GCACCTCAAAATGATATTTGTCCT) were used to amplify a 160 bp region of the cytochrome oxidase b gene. Confirmation of species specificity was tested against the two most common odontocetes known to co-occur in the survey region, killer whale (Orcinus orca) and Dall's porpoise , using DNA extracted from vouchered tissue samples .Quantitative PCR (qPCR) was used to detect and quantify harbour porpoise DNA in eDNA samples. Cytochrome −1 to 1 × 10−5 ng µl−1) of quantified target-species DNA extracted from a vouchered harbour porpoise tissue sample quantified on a Qubit™ (Invitrogen). The RSC slope and y-intercept were used to calculate the DNA concentration for the measured threshold cycle (Ct) values.Real-time PCRs were performed on an Applied Biosystems 7900 in 10 µl reaction volumes containing 0.3 µM probe, 0.8 µM each primer, 1× TaqMan Environmental Master Mix 2.0 (Applied Biosystems) and 3 µl template eDNA. Following Applied Biosystems TaqMan protocol, thermal cycling conditions were set at 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. Each set of reactions contained a minimum of two no-template controls to detect contamination with exogenous DNA. Relative standard curves (RSCs) were developed from 1 : 10 serial dilutions (1.3 ng µl2.4.in silico using the GenBank database (adjusting parameters for short input sequences), and in vitro with DNA extracted from vouchered tissues to confirm species specificity and ensure that the primers did not amplify the two commonly co-occurring, non-target odontocetes: killer whales and Dall's porpoise. PCR conditions were optimized to amplify the target fragment reliably in low copy number eDNA samples. PCR reactions were carried out in 10 µl volumes containing 2 µl of template eDNA, 1.2 mM MgCl2, 0.2 mM dNTP, 1.0 µM each forward and reverse primer . PCR cycling conditions were as follows: 94°C for 2 min, followed by 40 cycles of 94°C for 1 min, 49°C for 1 min, 72°C for 1 min and a final extension of 5 min at 72°C.Primer design for next-generation sequencing focused on the 5′ region of the mitochondrial control region. This section of the mitogenome is known to be hypervariable from ongoing genetic studies of North Pacific harbour porpoise ,19 and ce primer , 2 µg µl2.5.−1. Replicate 10 µl reactions were performed for all eDNA and control samples, using optimized conditions (as above) and modified primers containing Illumina sequencing primer and adapter sequences. Each sample was amplified in three independent 10 µl reactions to ensure sufficient PCR product for next-generation sequencing. Products from all three PCRs were pooled and the presence of the desired amplicon size was confirmed by electrophoresis of the pooled 30 µl in a 2% agarose gel stained with SyberSafe . Amplicon size was determined with reference to a 100 bp DNA ladder . Two negative control reactions were run in each set of PCR reactions, including template from one extraction blank and a no-template PCR sample. Bands from successful PCR of the eDNA samples and positive controls were gel excised using clean techniques to prevent cross-contamination between amplicons and purified using the Qiagen MinElute Gel Extraction spin-column protocol with a 30 µl elution volume . Gel electrophoresis of both negative controls confirmed the absence of detectable PCR product and these samples were not used for library generation.Amplifications for Illumina sequencing were carried out in triplicate on all eDNA and positive control samples. Positive controls were generated using genomic DNA from four Sanger sequenced, vouchered harbour porpoise samples to test for PCR or sequencing error. DNA from each control sample was quantified using a Qubit™ 3.0  and the Qubit DS Broad Range Assay  according to the manufacturer's instructions, and samples were normalized to a concentration of 5 ng µlSequencing capture adapters with unique pairs of Illumina Nextera XT index tags were added to each sample using a second reduced-cycle PCR protocol according to the Illumina 16S metagenomic sequencing library preparation protocol as follows. This second PCR step was performed in 50 µl reaction volumes containing 8 µl of gel-purified PCR product from the first PCR, 5 µl each of one Illumina Nextera forward and reverse index tag, and 1× NEB Phusion High-Fidelity PCR Master Mix . PCR cycling conditions were as follows: 95°C for 3 min, followed by 8 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension of 5 min at 72°C. Indexed amplicons were normalized across samples using the SequalPrep Normalization Plate Kit (Applied Biosystems) and eluted in a 20 µl volume. Following normalization, indexed amplicons were pooled, cleaned using Ampure XP beads  and eluted in 25 µl of 10 mM Tris (pH 8.5). The indexed and pooled amplicon library was quantified via qPCR using the KAPA Library Quantification Kit for Illumina libraries  according to the manufacturer's protocols and diluted to 4 nM. The final library was diluted to 12 pM and sequenced on an Illumina MiSeq Platform using the V3-600 paired-end sequencing chemistry  with a PhiX spike-in of 10%.2.6.The data analysis pipeline described below was first performed on sequence data for the four positive controls to calculate the expected rate of error generated during PCR and sequencing. Using data from each of the controls, we identified the most abundant sequence that had less than 100% identity to the known control region haplotype for that sample, and compared the read count to the total read count for the sample to derive an overall error rate due to non-specific priming and sequencing error . The obsfastq_mergepairs, with a minimum overlap of 50 bp. VSEARCH was used to cluster identical sequences (or de-replicate), and restrict sequences to a minimum aligned length of 379 bp. Chimeric sequences were identified and discarded using the VSEARCH command uchime denovo, and sequences with a frequency of occurrence less than the empirically determined error rate (above) were removed from the analysis. Remaining sequences were compared to a local database of 58 known harbour porpoise control region haplotypes from the eastern North Pacific using the pairwise BLAST  algorithm executed through the suite of command-line tools in the BLAST+ executables [ST and χ2 were used to generate preliminary estimates of genetic differentiation within southeast Alaska from mtDNA sequence data using the R package STRATAG [Cutadapt (v. 1.13)  was usedcutables . Sequenccutables  to conficutables . The bescutables  using 50 STRATAG .3.n = 4) failed to amplify above the amplification threshold within 45 cycles with species-specific primers and the TaqMan probe. The median concentration of porpoise DNA for the 36 eDNA samples was 0.0033 ng µl−1, with values ranging from 8.96 × 10−5 to 0.112 ng µl−1 . No cross-species amplification with harbour porpoise qPCR probes was detected in tests with genomic DNA from killer whale and Dall's porpoise.Forty surface seawater eDNA samples were collected during shipboard surveys in southeast Alaska inland waters on 10 different days between July and September 2016 . The numA total of 34 874 482 raw paired-end reads were produced from a pooled library consisting of eDNA samples and positive controls, sequenced on a single Illumina MiSeq flow cell. The average number of reads passing filter per sample was 237 434.50 (s.d. = 74 914.37). Positive controls had an average of 101 643.75 reads after quality filtering, and between 93.78% and 95.56% of these reads represented the true, known control region haplotype for each sample. The maximum error rate for controls was 1.70%  calculated based on the proportion of processed reads representing the most abundant sequence that was not identical to the known haplotype. This maximum error rate calculated from the control samples was carried forward in the analysis of eDNA samples; all de-replicated sequences representing less than 2% of processed reads were assumed to be either erroneous or non-target and eliminated from the dataset. All negative controls, including no-template PCR negatives, extraction blanks and four field negatives, failed to produce any amplification product detectable on the agarose gel.Harbour porpoise control region sequences with a minimum length of 379 bp were successfully generated from 29 of 36 (80.56%) targeted eDNA samples. Gel-purified product from the libraries of seven samples failed to generate sequences of sufficient read depth to meet our stringent error rate criterion of greater than 2% of processed reads per sample, based on the analysis of sequences from tissue controls (above). The most abundant sequence for these eDNA samples either failed to align to any GenBank sequence, or aligned to the genome of a marine plankton (DQ257435) when BLASTed against the NCBI database. The total number of de-replicated reads for one sample (2016HP46) was atypically low (510) and although the sequence showed a high degree of similarity (99.74% identity) to a known harbour porpoise haplotype, it was not included as a newly discovered haplotype because the processed read depth relative to the total reads (221 922 merged pair contigs) was extremely low  and may represent sequencing error.ST = 0.132, p = 0.022; χ2 = 16.24, p = 0.002) based on the geographical distribution of eDNA haplotypes suggest that genetic structure within southeast Alaska is likely.Of the 29 eDNA samples that produced unequivocal harbour porpoise control region sequences, 26 samples yielded a single control region haplotype and three samples contained eDNA from multiple porpoise having two different haplotypes . In all samples with multiple haplotypes, the less abundant haplotype was represented by greater than 20% post-processed reads. Eight unique haplotypes (based on a 379 bp fragment of the control region) were resolved from the eDNA samples, including two previously unreported haplotypes. Both were found in samples (2016HP08 and 2016HP78) that yielded multiple haplotypes , harbour porpoise successfully amplified above the minimum amplification threshold from all eDNA samples. eDNA concentration has been correlated with the density and biomass of targeted species ,30–32, aDesigning assays to target organellar genomes, such as mitochondria, will help maximize the detection of target DNA in environmental samples due to the high copy number per cell compared to the nuclear genome . TargetiHarbour porpoise mtDNA haplotypes were generated from 80% of seawater samples and the difference in relative success rates between the real-time PCR and next-generation sequencing may reflect differences in the relative sensitivities of the two PCR-based approaches with greater sensitivity for the TaqMan™ probe in detecting low concentration target DNA . Three oWe demonstrate here how high-throughput sequencing of seawater eDNA can generate mtDNA haplotypes for characterizing genetic diversity for marine vertebrate populations. Prior knowledge of haplotypic sequence diversity in Alaska harbour porpoise provided baseline data for comparison with eDNA haplotypes. Of the eight unique mtDNA sequences generated from eDNA samples, five were represented in the Alaska porpoise baseline dataset, with the most common eDNA haplotype also being the predominant haplotype from porpoise tissue samples (unpublished data). The two new haplotypes (CRx and CRxx) differed from two previously documented haplotypes  by only a single nucleotide, and with representation by greater than 20% of final processed reads, these haplotypes were determined to be true genetic variants that have not yet been found in tissue samples from fisheries by-catch and stranded animals. While it is difficult to determine empirically whether or not the new haplotypes are true haplotypes or artefacts generated by early PCR error, resequencing of these samples and the collection of additional samples throughout the surveyed waters will help to determine the veracity of new sequence variants. The discovery of new haplotypes was not unexpected, considering the relatively high diversity of control region haplotypes and frequency of unique haplotypes found in single samples from the opportunistic tissue samples collected from Alaska harbour porpoise . Furtheret al. [a priori strata rather than true geographical breaks in the population's distribution. Although this limits inference of intraspecific population structure, significant genetic differentiation was detected between the two geographically sampled regions, warranting dedicated sampling throughout suitable habitat to identify significant population boundaries. In southeast Alaska, concentrations of harbour porpoise were consistently found in the same patchy regions throughout inland waters with concentrations in the northern and southern reaches [Our goal was to examine whether population genetic data could be generated for small cetaceans through next-generation sequencing of eDNA samples. The high success rate and quality of mitochondrial sequences obtained by collecting surface seawater in the wake of focal animals offers a valuable alternative to traditional methods of collecting genetic samples that have proved to be largely ineffective for real-time biosampling of small, elusive cetaceans such as harbour porpoise. Amplicon sequencing using eDNA offers an approach that should allow the collection of a large number of samples and more thorough coverage of the surveyed geographical range, avoiding common complications in population genetic studies confronted by under-representation or bias in opportunistically collected samples. For example, Chivers et al.  noted th reaches . If sign reaches ,47,48, dPopulation genetics remains key for the conservation and management of natural populations, and contributes essential information needed to understand and mitigate human-mediated impacts on coastal marine species. This is particularly relevant for higher trophic, long-lived marine mammals which can serve as important barometers of the health of marine ecosystems ,49–51. T


The first 3-D direct observation of clusters of Nd oxide inside silicate glasses was achieved using atom probe tomography. Three-dimensional elemental maps of major chemical elements in glasses such as Si, Al, Zn and O showed no evidence of regions that had concentrations higher than the average values, whereas the Nd aggregated into regions of high concentration. Elemental maps of Nd and Pb recorded from the glasses containing PbS QDs showed highly-concentrated areas of both elements at the same locations; this result indicates that PbS QDs formation started in association with the Nd clusters. One of the fundamental difficulties associated with emission by RE ions is that when RE ions assemble into clusters, concentration quenching occurs, in which neighboring ions exchange energy non-radiatively9. Many spectroscopic analyses have provided indirect evidence of RE clustering11 but direct observation has not been reported.Rare-earth (RE)-doped glasses have been commercially used as signal amplifiers for optical communication and solid-state lasers that exploit the radiative f-f electronic transitions of trivalent RE ions19. The optical and electronic properties of QDs depend on their size, so many attempts to realize the precise control of QDs size26 have been reported. In particular, in glasses that include RE ions, QDs form near clusters of RE oxides that appeared to provide nucleation sites29. Distribution of elements analyzed from electron energy loss spectroscopy (EELS) suggested the presence of RE clusters. However, direct evidence of the presence of the RE-oxide clusters in conventional oxide glass is lacking, and their functions in nucleation of QDs in glasses are not known.Quantum dots (QDs) can provide emissions from mid-infrared to visible wavelength31. Local electrode atom probe (LEAP) uses an additional electrode to reduce the laser damage during analyses of insulating materials. Statistical tools including iso-surface image and maximum separation method can provide additional means to detect clusters or ordered phases with sizes smaller than a few nanometers33. We have employed APT to visualize the presence of RE clusters in silicate glasses. We also propose how RE clusters affect nucleation and growth of QDs in glasses.Atom probe tomography (APT) can provide single-atom sensitivity and fine spatial resolution (~0.1 nm in depth) of the distribution of chemical elements in three dimensions36. First, Pt coating was sputtered (CRESSINGTON SPUTTER COATER 208HR) on the surface of glass specimens to avoid surface damage by Ga+ ions  method using the standard lift-out technique designed for insulating materials Fig. 34–36. Fions Fig. . Then a ype Fig. . This spype Fig.  and furtype Fig.  with diaype Fig. .Figure 12–5Al2O3–25NaO–10BaO–8ZnO–2ZnS–0.8PbO–5Nd2O3 (in mol%). In tomographic reconstructions  that form the glass matrix were uniformly distributed . Average [Nd] within the clusters was 56.3 at.%, which is ~19 times higher than the average [Nd] = 3.3 at.% in the glass matrix.Afterwards, the Nd-Nd nearest-neighbor analysis between cluster and matrix was obtained from Fig. 3+ ions are surrounded by an average of seven oxygens both before and after the formation of PbS QDs in glasses29. Therefore, clusters of high [Nd]  showed no evidence clustering. When the glasses were heat-treated at 500 °C for 30 h to precipitate the PbS QDs, areas of high [Nd] and [Pb] formed in the glasses, and closely coincide with each other; this result indicates that formation of PbS QDs most probably started at the Nd clusters.2–5Al2O3–25NaO–10BaO–8ZnO–2ZnS–0.8PbO (in mol%) and a Nd-glass with an additional 5 mol% of Nd2O3 were prepared using a conventional melt-quenching process. Starting powders were mixed and melted in an alumina crucible at 1340 °C for 30 min. Each melt was poured onto a brass mold that had been preheated at 380 °C, then was quenched by pressing with another brass plate. Resulting glasses were annealed at 380 °C for 2 h to eliminate residual thermal stress. Specimens were polished to ~150-μm thickness, then used as a source for fabrication by FIB of specimens for APT.A base glass with a nominal composition of 50SiOAPT analysis  was performed using a pulsed UV laser (λ = 355 nm) and each atom was detached from the surface by irradiating the laser beam with 100-pJ pulse energy at a repetition rate of 200 kHz. An additional local electrode with a range of 2–15 kV was used to avoid the specimen damage by the laser beam. IVAS software (3.6.10a version) by CAMECA Instruments was used to reconstruct and visualize APT results. Cluster formation was identified from the iso-surface concentration. The maximum-separation algorithm was used to distinguish the clusters from the matrix, and the corresponding differences between the matrix and cluster compositions were calculated.Atom Probe Tomographic Imaging of PbS Quantum Dot Formation on Neodymium Clusters in Silicate Glasses


Lay summaryIt is poorly understood whether fish can acclimate to prolonged low-oxygen conditions (or hypoxia). Our study shows that prior long-term exposure to low-oxygen conditions improves tolerance to low-oxygen in a freshwater fish. The results of our study aid our understanding of long-term responses of freshwater fish to low-oxygen to hypoxic events. Maccullochella peelii) to low-oxygen conditions and investigated acclimation effects. Experiment 1 determined how responses could be modified by exposure to different temperatures  and oxygen conditions (control 6–8 mgO2 L−1 and low-oxygen 3–4 mgO2 L−1) over 30 days. Experiment 2 determined the acclimation ability of fish exposed to two temperatures (20 and 28°C) and low-oxygen conditions (3–4 mgO2 L−1) for three different acclimation periods . Responses were measured by determining critical oxygen tension (Pcrit), loss of equilibrium and aerobic capacity using resting respirometry. In experiment 1, resting oxygen requirements were negatively affected by long-term low-oxygen exposure except at the highest temperature (28°C). However, long-term acclimation in low-oxygen improved tolerance as measured by loss of equilibrium but not Pcrit. In experiment 2, fish could tolerate lower oxygen levels before reaching loss of equilibrium after 7 days acclimation, but this declined overtime. Murray cod were most tolerant to low-oxygen at the lowest temperature (20°C) and shortest exposure time (7 days). Extended low-oxygen exposure resulted in reduced aerobic capacity of fish particularly at the lowest temperature. While prior exposure to low-oxygen may allow fish to cope with hypoxic conditions better in the long-term, acclimation time was inversely related to tolerance, suggesting that resistance to hypoxia might decrease as a function of exposure time. Our study fills a much-needed gap in our understanding of how freshwater species acclimate to hypoxia, and in particular, how exposure to prolonged periods of low-oxygen and elevated temperatures affect organisms physiologically.Persistent hypoxic or low-oxygen conditions in aquatic systems are becoming more frequent worldwide, causing large-scale mortalities to aquatic fauna. It is poorly understood, however, whether species can acclimate to long-term hypoxic conditions. In two experiments, we exposed juvenile freshwater fish (Murray cod,  Hypoxia can be fatal to many organisms including mammals, birds, fish, reptiles and invertebrates . Extensi2 L−1 of oxygen to 8 mgO2 L−1  can have dire consequences on aquatic fauna as their capacity to respond to hypoxia is dictated by functioning physiological and biochemical systems in place at the time of exposure , a susceptible freshwater fish native to the Murray Darling Basin, an extensive river system that is frequently influenced by natural and anthropogenic hypoxic events (for further species information see SI). First, we tested how Murray cod responded to long-term low-oxygen exposure at different temperatures, and then we tested how low oxygen exposure for different lengths of time and different temperatures modified fish responses. We measured aerobic capacity, the critical oxygen limit of fish (Pcrit) and loss of equilibrium to determine if there was an acclimation response to low-oxygen. We predicted that (a) the combination of high temperatures and prolonged exposure to low-oxygen would exacerbate the effects of hypoxia and limit a fish’s ability to acclimate and (b) we predicted that duration of exposure to low-oxygen would alter the acclimation capacity of fish such that, longer duration of exposure would decrease acclimation capacity.We investigated whether prior exposure to hypoxia or low-oxygen could improve the tolerance of freshwater fish to hypoxic conditions. We used Murray cod (M. peelii), approximately 55 mm in length and 1.5 g average body mass, were obtained from aquaculture stock from the NSW Hatchery Quality Assurance Scheme accredited Silverwater Native Fish Hatchery, Grong Grong, NSW, in March 2015. Fish were kept in 250 L holding tanks at 20°C at the University of Adelaide before being assigned to 20 L tanks for the experimental treatments. All tanks  were filled using aged (dechlorinated) tap water and aerated and filtered for the duration of the experiments; evaporation was minimized using plexiglass lids. Fish were fed hatchery pellet food until satiation with excess food siphoned out an hour after feeding. Room temperature was maintained at 20°C and fish were exposed to a 12:12 hr light:dark cycle. Water changes of 25% were made daily and water quality was monitored every second day for temperature, oxygen levels and saturation, pH, ammonia and nitrite.Juvenile Murray cod (2 L−1 or 12–14 kPa and low-oxygen 3–4 mgO2 L−1 or 7–9 kPa) and three temperature treatments reflective of the species’ natural thermal range;  and two temperature treatments (20 and 28°C) again with duplicate tanks in an orthogonal design . However, only 8 new tanks (including duplicates) were used for Experiment 2 as fish from low-oxygen treatments in Experiment 1 at 20 and 28°C were used to measure 30 days of acclimation. Experiment 2 was run following the completion of Experiment 1. Fish were randomly assigned to tanks (20 L), with about 11 fish per tank. Fish in treatments ≥24°C were acclimated 2°C per day by adjusting the submersible aquarium heaters. To ensure survival of fish during experimentation, low-oxygen levels (3–4 mgO2 L−1 or 7–9 kPa) were higher than the globally accepted tolerance limit of 2 mgO2 L−1, which is believed to frequently result in mortality of aquatic species  while allowing physiological responses to be tested. For the low-oxygen treatments, we developed a simple method to deoxygenate the water  and L the total standard length  were recorded. Fasting fish were held in an isolated container in the larger water bath where respirometry experiments were conducted, to ensure there was no shock experienced prior to being placed in resting chambers. Twelve fish per treatment were randomly selected and subjected to respirometry experiments.Following 7, 14 or 30 days exposure, fish were fasted for 24 hrs prior to experimental trials to evacuate the digestive tract so that only oxygen consumption rates (ṀoThree fish were tested simultaneously using a 4-chamber system (each 300 mL volume), custom made to fit the fish . All chambers were submerged in a larger water bath (139 × 52 × 20 cm), where temperature and oxygen levels were controlled and set to match experimental treatments. A closed recirculation loop pumped low flowing water over the fish in individual chambers. To reduce background respiration, water was pumped through a heater/chiller unit fitted with a UV lamp to sterilize the water. Further, the whole system was rinsed every third day to ensure that background consumption of oxygen remained below 15% of the resting metabolic rate of fish. The remaining chamber was used to record background respiration each day and was randomized each day of recording.2 determination (mgO2 kg−1 h−1). A Ṁo2 determination period uses the slope of the line of oxygen consumption by fish for each 20 minute determination period before water is replenished to the chamber for 2 minutes (flushing period). Water was circulated intermittently after each Ṁo2 determination using a flushing pump connected to all chambers to completely replenish the chamber with oxygenated water from the water bath. During the 20 min determination period, oxygen was not reduced to less than 1 mgO2 L−1 and was above the background respiration rates. Maximum and standard metabolic rates (MMRs and SMRs) were determined using a modified version of the method described by 2 were calculated for each determination cycle using the equation:2/L) of the water at the conclusion of a flushing cycle and (t1) is the oxygen content measured at the end of a determination period, prior to the next flushing cycle. V is the volume of the chamber minus the volume of the experimental animal in L, t is t0−t1 and BW is the body weight of the experimental animal in kg. The lowest 10% of measurements were averaged to calculate SMR. Background rates were subtracted from Ṁo2 values upon calculation. The absolute aerobic scope of fish was calculated by subtracting SMR from MMR (MMR-SMR).Each chamber was fitted with a fibre optic oxygen probe , which recorded oxygen consumed during each Ṁo2 L−1 was recorded and fish were immediately removed from the chamber. The critical oxygen tension or Pcrit of fish was measured using data from this closed respirometry phase. Pcrit was defined as the point at which ṀO2 was reduced below SMR and fish shifted to an oxy-conforming state. Pcrit was determined for each fish by fitting a segmented regression using RStudio Version 1.1.419 , a method adapted from In order to record tolerance limits of fish amongst the different treatments, we left fish in chambers with the intermittent flushing cycle turned off with only access to the oxygen available from water in the chamber (closed respirometry). Fish were observed constantly during this period. Fish reached a low-oxygen tolerance limit when they lost equilibrium, at which point oxygen level in mgOPcrit and loss of equilibrium using the R-package lmerTest . All PERMANOVA+ analyses included Monte Carlo permutation tests to derive the probability value and ensure there were sufficient permutations to detect significant differences in all tests. All PERMANOVA+ statistical analyses were initially conducted as 3-factor permutational univariate ANOVAs with tank as the third factor treated as a random factor nested in temperature, oxygen or acclimation duration dependent on the experiment. No effects of tank were detected for rearing water or during any of the experimental responses in either experiment; therefore, data were pooled and 2-factor permutational univariate ANOVAs were conducted removing tank as a factor.Rearing water temperature, dissolved oxygen (DO) and oxygen saturation in the experimental tanks, as well as Fulton’s K factor, were analysed at all possible treatment levels  using a 2-factor permutational univariate analysis of variance (ANOVA) with unrestricted permutations using PRIMER 6 & PERMANOVA+ software  after 14 days exposure to low-oxygen and the lowest after 30 days exposure  was lowest at 28°C after exposure to low-oxygen for one month , and fish reared at 20°C fared much better during all acclimation periods than those reared at 28°C  did not improve with exposure to low-oxygen compared with fish exposed to normoxia.Pcrit) remained unaffected. However, the difference between average loss of equilibrium after low-oxygen exposure in our treatments was small compared to normoxic treatments (<1 mg L−1), suggesting that it may not equate to a significant response in the wild. Acclimation of fish to hypoxia may be a naturally selected trait for species living in areas prone to hypoxia. For example, acclimation to seasonal hypoxia and diel hypoxia exposure has improved the tolerance of a number of fish species  are associated with contrasting levels of aerobic capacity, such that there is a trade-off for locomotive performance and tolerance to low resource availability, in particular oxygen  could thus lead to erroneous interpretations about the acclimation abilities of fish when faced with environmental stressors such as hypoxia and elevated temperatures. Behavioural tests on the tolerance of fish to low-oxygen illustrated the possible acclimation ability of Murray cod, in particular how prolonged exposure to low-oxygen conditions may physiologically reduce tolerance long-term. Other species may be similarly affected by prolonged periods of low-oxygen conditions, and our results provide much needed hypoxia data on a species of conservation concern. Future research should target numerous species and their ability to acclimate to hypoxia in combination with other stressors. In particular, species recovery from hypoxia has been largely overlooked and will aid in understanding the capabilities of fish to not only withstand but also endure hypoxic events. Furthermore, development of a universal method to measure acclimation response to hypoxia exposure would allow direct comparisons amongst different species as research in this field continues.SuppInfoEdited_coz058Click here for additional data file.


Circadian rhythms are fundamental to health and are particularly important for mental wellbeing. Disrupted rhythms of rest and activity are recognised as risk factors for major depressive disorder and bipolar disorder.We conducted a genome-wide association study (GWAS) of low relative amplitude (RA), an objective measure of rest-activity cycles derived from the accelerometer data of 71,500 UK Biobank participants. Polygenic risk scores (PRS) for low RA were used to investigate potential associations with psychiatric phenotypes.NFASC) and Solute Carrier Family 25 Member 17 (SLC25A17). A secondary GWAS of RA as a continuous measure identified a locus within Meis Homeobox 1 (MEIS1). There were no significant genetic correlations between low RA and any of the psychiatric phenotypes assessed. However, PRS for low RA was significantly associated with mood instability across multiple PRS thresholds , and with major depressive disorder  and neuroticism .Two independent genetic loci were associated with low RA, within genomic regions for Neurofascin . Circadian rhythms are important for the maintenance of both physical and mental wellbeing, and disrupted rest/activity rhythms are recognised as core features of both bipolar disorder and major depressive disorder. The underlying genetic architecture of disrupted circadian rhythmicity is not yet fully understood. Genetic studies to date include candidate gene analyses of core “clock” gene variants and larger-scale genome-wide studies of subjective reports of sleep duration, insomnia and chronotype. We searched PubMed for human studies of the genetics of objectively-assessed circadian rhythmicity and mood disorders published in English before November 1, 2017. We used “accelerometry” or “actigraphy”, and “bipolar” or “depression”, and “circadian” and “amplitude”, and “gene” or “gwas” as search terms. We identified only 5 studies of interest. Some compared expression of circadian clock genes – as measures of circadian rhythmicity – between healthy controls and patients with mood disorders, or in response to different therapeutic drugs. Others applied genetic linkage analysis to actigraph (activity) data from both healthy individuals and from patients with mood disorders. Additional searches of “relative amplitude” (a common derived measure of circadian rest-activity rhythmicity) and “mood disorders” found that most studies reported lower amplitude in individuals with bipolar disorder or depression versus controls. More specifically, recently, low relative amplitude was reported to be associated with several mood disorder phenotypes within UK Biobank.To our knowledge, this is the first large scale genome-wide association study of relative amplitude, an objective measure of rest-activity cycles. We identified genes of interest with putative biological links to mood disorders. We also identified associations between polygenic risk of low relative amplitude and major depression, neuroticism and mood instability.Circadian rhythm disruption is a core feature of mood disorders and the genetic variants identified in this study may be involved in the pathophysiology of mood disorders. These findings could provide new insights into the biology of both circadian function and mood disorders, and may lead to new treatment targets for mood disorders.Alt-text: Unlabelled Box1Circadian rhythms are variations in physiology and behaviour that recur approximately every 24 h . They inCircadian rhythmicity is regulated by both exogenous environmental stimuli (“zeitgebers”) and by genetic factors . SeveralPER2, PER3, RSG16, AK5, FBXL13), in addition to novel associated genetic loci [To date, the most commonly used measure of circadian phenotypes has been subjectively-reported chronotype, defined as an individual's preference for morning or evening wakefulness and activity . Eveningtic loci .However, as a subjective measure, chronotype can be vulnerable to response bias. It may also have inconsistent associations with more objective measures of circadian rhythmicity . In a re22.1Over 502,000 United Kingdom (UK) residents aged 37–73 years were recruited to the UK Biobank cohort from 2006 to 2010. At one of 22 assessment centres across the UK, participants completed a range of lifestyle, demographic, health, mood, cognitive and physical assessments and questionnaires . Here, w2.2http://biobank.ctsu.ox.ac.uk/crystal/refer.cgi?id=131600.In 2013, 240,000 UK Biobank participants were invited to wear an accelerometer for seven days as part of a physical activity monitoring investigation . Of thes2.3From the summary five second epoch data, a measure of relative amplitude (RA) was calculated using Clocklab Version 6 (Actimetrics). This accelerometer-derived activity measure has demonstrated reliability and validity . RA is uM10 is the mean activity during the continuous 10 h period containing maximum activity in each 24 h recording period (midnight to midnight). L5 is the mean activity for the corresponding 5 h period containing the minimum activity within the same recording period. For each individual, the RA data point was the mean RA value across all included 24-h periods (seven days). RA ranges from 0 to 1, with higher values indicating greater distinction between activity levels during the most and least active periods of the day.N = 3) or who did not provide data for all one-hour periods within the 24-h cycle were excluded from analyses (excluded N = 6860). Participants were also excluded if their data was identified by UK Biobank as having poor calibration; we excluded participants with data flagged by UK Biobank as unreliable ) and participants whose wear-time overlapped with a daylight savings clock change (excluded N = 4500) [N = 91,870), mean RA was 0·87 , similar to previously reported values in healthy populations [Participants who provided accelerometer data for <72 h , or assume data take sinusoidal form (cosinor amplitude) , 30, whi2.4UK Biobank released genotypic data for over 500,000 participants using two genotyping arrays specifically designed for UK Biobank with 95% shared marker content . Approxi2.5−6 resulting in 7,969,123 variants remaining.Our primary GWAS was a study of cases of low RA defined as cases with a mean RA greater than two standard deviations below the overall mean RA, with the remaining participants as controls . Before −8.The primary association analysis was conducted using logistic regression in PLINK ; an addi2.6−8. Note that due to the imbalance in sample size between cases and controls available for low RA, we were unable to use the BOLT-LMM approach for the primary GWAS [The BOLT-LMM method allows the inclusion of related individuals within GWAS. This method relaxes the assumptions of the standard GWAS, as used above, by using a mixture of two Gaussian priors and is a generalisation of a standard mixed model. This mixed model accounts for both relatedness and population stratification; this results in greater power when compared to principal component analysis . As abovary GWAS .2.7The lead SNP from each locus, identified by GWAS, was assessed for the possibility of expression quantitative trait loci (eQTLs). This genotype-specific gene expression was assessed using the GTEx portal. The portal was also used to investigate tissue-specific expression for the implicated genes .2.8−5.The summary statistics from both the primary and secondary GWAS analyses were uploaded to FUMA web application for gene-based analyses . Gene-ba2.92SNP) [Linkage Disequilibrium Score Regression (LDSR) was applied to the GWAS summary statistics to provide an estimate of SNP heritability (h2SNP) , 38. LDS2.10a)Bipolar Disorder, Major Depressive Disorder and Generalised Anxiety Disorderb)NeuroticismA mental health questionnaire (MHQ) was developed by a mental health research reference group to collect additional mental health phenotype data in UK Biobank and was administered during 2016–2017 . The MHQc)Mood instabilityTo define neuroticism a score was taken from the 12-item neuroticism scale of the Eysenck Personality Questionnaire-Revised Short Form (EPQ-R-S) , 41. Ind“Does your mood often go up and down?” and given the option to answer “yes”, “no”, “don't know” or “prefer not to answer” [A “mood instability” outcome measure was also obtained from the EPQ-R-S questionnaire: for one question participants were asked  answer” . Individ answer” .2.11−8, p < 5 × 10−5, p < 0·01, p < 0·05, p < 0·1, p < 0·5) were divided into quartiles, with the exception of p < 5 × 10−8 which was divided into tertiles as only three different PRS were generated for this threshold. The top and bottom quantiles were compared in logistic regression models that were adjusted for age, sex, Townsend deprivation index [Associations between higher PRS for low RA and psychiatric diagnoses were examined in up to 76,018 individuals who had completed the MHQ and who were not included in the primary GWAS. Similarly, associations between low RA PRS and mood instability/neuroticism were examined in between 91,248 and 140,504 individuals (depending on the dependent variable) not included in the GWAS. PRS including SNPs at 6 different significance thresholds  accounted for <1% of the population variance . The Manhattan plot for low RA GWAS is presented in NFASC) on chromosome 1 and Solute Carrier Family 25 Member 17 (SLC25A17) on chromosome 22 . As each of these SNPs is an intronic variant, the exact effect of each polymorphism is unclear.Our primary analysis was a case-control GWAS of low RA. The GWAS data showed only a slight deviation in test statistics compared to the null . Five SNPs, all localised to one locus on chromosome 2, were associated with continuous RA at genome-wide significance (described in Supplementary Table 3). These SNPs highlight the Meis Homeobox 1 (MEIS1) gene. Again, as noted above, these are intronic SNPs and their exact effects are not known.As a secondary analysis, we performed a GWAS of a continuous measure of RA using a BOLT-LMM model. The BOLT-LMM GWAS showed a slight deviation in the test statistics compared to the null . Information on the influence of G homozygotes was not available.The lead SNPs from both GWAS were assessed for potential eQTLs. Only the lead SNP found on chromosome 22 (rs9611417) was associated with the expression of a nearby gene. Being heterozygous at rs9611417 was associated with lower expression of 3.4FOXJ1) on chromosome 17, and Zinc Finger FYVE-type Containing 21 (ZFYVE21) on chromosome 14 . The gene set analysis of continuous RA identified three genes: Copine 4 (CPNE4) and Chromosome 3 open reading frame 62 (C3orf62) on chromosome 3, and Renalase (RNLS) on chromosome 10 .Gene-based analyses of both low RA and continuous RA were undertaken. The gene-based analysis of low RA identified two genes significantly associated with low RA: Forkhead Box J1 , suggesting that the biology underlying low RA may be associated with the regulation of sleep . As low RA is derived from the continuous measure of RA, a negative correlation was expected. There were no significant genetic correlations identified between low RA and ADHD, anxiety, BD, MDD, mood instability, PTSD and schizophrenia.There was preliminary evidence of genetic correlation between low RA and insomnia (rof sleep . However3.6−8). For MDD, small positive associations were found for the low RA PRS at the top three significance thresholds (OR = 1·02–1·03), which remained significant after FDR correction (p = 0·025–0·05). A positive association with neuroticism was found for the highest threshold . However, other associations between the remaining PRS thresholds and neuroticism score were not significant. These associations did not significantly differ when including individuals with more than one psychiatric phenotype (results not shown).The findings of analyses assessing the association between low RA PRS and several mood disorder-related phenotypes are presented in 42 = 0·66–1·00) in many populations [MEIS1 gene on chromosome 2. Again, the SNPs identified are in medium to high LD with each other (r2 = 0·39–1·00) in many populations [We present the first large-scale genetic study of an objective measure of rest-activity cycles in humans, as well as the first study to examine how common risk SNPs for circadian disruption might be associated with mood disorder phenotypes. The primary GWAS of low RA identified three genome-wide significant SNPs within two independent loci; two of the SNPs highlighted were found to be in relatively high LD (rulations  and are ulations .−5 to p < 1 × 10−7) (Supplementary Tables 4 and 5). There was no genetic overlap between the SNPs identified for low RA and those identified for continuous RA at these thresholds. This suggests there is a partial genetic distinction between the pathological low RA measure and the broader range of RA.On inspection of the Manhattan plots there were several peaks whose lead SNPs had p-values below the suggestive evidence for significance threshold , gliomedin and betaIV spectrin) are important for the generation of action potentials and for the maintenance of neuronal integrity [ANK3 are robustly associated with BD [NFASC SNPs identified by the GWAS were intronic variants and the NFASC transcript undergoes extensive alternative splicing with not all variants being functionally categorised [NFASC gene is currently unclear.Of considerable interest is that one of the genes highlighted by GWAS was  protein . Neurofa neurons . It is i neurons . These Antegrity . Notably with BD . The diregorised . The preSLC25A17. This gene encodes a peroxisomal solute carrier membrane protein that transports several cofactors from the cytosol to the peroxisomal matrix [SLC25A17 gene is also involved in adrenomyeloneuropathy [RANGAP1, a GTPase activator protein involved in nuclear transport [RANGAP1 is 439 kb downstream of rs9611417 and shows relatively high expression in the brain .One of the genome-wide significant SNPs from the primary GWAS is located within l matrix . Variantl matrix . The SLCuropathy , an inheuropathy . The risransport . RANGAP1MEIS1 gene. This gene encodes a homeobox transcription factor (TF) protein crucial for the normal development of several tissues, including the CNS [MEIS1 gene is also associated with myeloid leukaemia [The GWAS of RA as a continuous measure highlighted SNPs within the  the CNS , 57. Theeukaemia  and resteukaemia , 57.4.2FOXJ1 on chromosome 17, and ZFYVE21 on chromosome 14. FOXJ1 encodes a forkhead TF protein which has a role in cell differentiation in respiratory, reproductive, immune, and CNS tissues. FOXJ1 is required for the formation of cilia [FOXJ1 was reported to be important for neurogenesis within the forebrain and olfactory bulb [ZFYVE21 gene encodes the zinc-finger FYVE-type containing 21 protein, involved in cell migration and adhesion [The gene-based analysis of low RA identified two genes: of cilia . In mousory bulb . The ZFYadhesion . The potCPNE4 and C3orf62 (chromosome 3); and RNLS on (chromosome 10). The CPNE4 gene encodes a calcium-dependent phospholipid binding protein involved in membrane trafficking and may be involved in intracellular calcium-mediated processes [For the continuous measure of RA, three genes were identified in the gene-based analysis: rocesses . Deletiorocesses . CurrentRNLS encodes a flavin adenine dinucleotide-dependent amine oxidase, known as Renalase, an enzyme hormone secreted from the kidney into the bloodstream . Renalas4.3We found little evidence of genetic correlation between low RA and psychiatric phenotypes. This is perhaps surprising given the literature on circadian rhythmicity and mood disorders. Core circadian clock genes have been associated with both BD and depression, with altered circadian biology suggested to be a vulnerability marker for mood disorders . It has Further, as noted above, within the UK Biobank cohort we recently found that lower RA was associated with several adverse mental health outcomes . In the 52 > 0·30) [Several limitations are acknowledged. UK Biobank may not be fully representative of the general UK population , with a  > 0·30) . The useIn terms of phenotyping, the mood instability phenotype was a self-reported subjective measure that may be influenced by response bias. Further, we were unable to identify a direction of causality between RA and the psychiatric phenotypes: approaches to causality such as Mendelian randomisation (MR) were not possible due to the small number of genome-wide significant SNPs identified by both GWAS analyses .Circadian rhythms are subject to influence from both biological factors and environmental stimuli. It is a potential limitation of this investigation that the analyses were not adjusted for potential non-genetic confounders operating during the accelerometry data collection period, such as medical illness, medication status, chronic pain transmeridian air travel, obesity and irregular work patterns, all factors known to affect circadian rhythmicity. The accelerometers were worn for 7 days which may not accurately represent rhythmicity, particularly in working participants where weekend rhythms might differ substantially from weekday rhythms. Further studies are required to investigate the possible interaction between RA and environmental factors; future investigations may also benefit from the inclusion of other measures of RA variability to adjust for intra- and inter-individual activity levels.6Overall, our findings contribute new knowledge on the genetic architecture of rest-activity cycles and how this overlaps with some mood disorder phenotypes, such as mood instability. Several of the genetic variants identified are located within or close to genes which may have a role in the pathophysiology of mood disorders. It is hoped that these findings will act as stimulus for future work assessing the complex biological relationships between circadian function and psychiatric disorders.


N2 reactions, including methyl transfer. Notably, the angles between the tetrel bond donor and acceptor atoms coincide with the prerequisite geometry for the SN2 reaction. Prompted by these findings, we surveyed crystal structures of methyltransferases in the Protein Data Bank and discovered multiple instances of carbon tetrel bonding between the methyl group of the substrate S-adenosylmethionine (AdoMet) and electronegative atoms of small molecule inhibitors, ions, and solvent molecules. The majority of these interactions involve oxygen atoms as the Lewis base, with the exception of one structure in which a chlorine atom of an inhibitor functions as the electron donor. Quantum mechanical analyses of a representative subset of the methyltransferase structures from the survey revealed that the calculated interaction energies and spectral properties are consistent with the values for bona fide carbon tetrel bonds. The discovery of methyl tetrel bonding offers new insights into the mechanism underlying the SN2 reaction catalyzed by AdoMet-dependent methyltransferases. These findings highlight the potential of exploiting these interactions in developing new methyltransferase inhibitors.Tetrel bonds represent a category of non-bonding interaction wherein an electronegative atom donates a lone pair of electrons into the sigma antibonding orbital of an atom in the carbon group of the periodic table. Prior computational studies have implicated tetrel bonding in the stabilization of a preliminary state that precedes the transition state in S N2 reaction wherein a nucleophilic atom, such as oxygen, nitrogen, or sulfur, attacks the electrophilic methyl carbon atom of AdoMet, with the sulfur atom displaced as the leaving group [N2 reaction [Methyltransferases represent a ubiquitous class of enzymes that methylate a vast array of small molecules and macromolecules and participate in numerous biological processes, including metabolism, signal transduction, and gene expression ,2,3. Theng group . The reang group  and has ng group ,7,8,9,  identified unconventional carbon–oxygen (CH∙∙∙O) hydrogen bonds between the AdoMet methyl group and oxygen atoms within the active sites of different classes of these enzymes . QuantumIn addition to unconventional hydrogen bonding, chalcogen bonding between the AdoMet sulfur cation and the active sites of methyltransferases has also been observed . A chalc3) bond.Beyond hydrogen bonding and chalcogen bonding, there is a third unconventional interaction that can occur with sulfonium cations involving a σ* orbital of a carbon atom . This in3 bond is approximately linear, precludes strong methyl CH∙∙∙X hydrogen bonding due to acute hydrogen bond angles [N2 reactions, including methyl transfer [N2 catalysis.Although aliphatic carbon atoms typically form weak tetrel bonds compared to other Group IV elements, QM calculations have demonstrated that a methyl carbon atom bonded to a sulfonium ion can form relatively strong tetrel interactions due to polarization by the adjacent cation . Notablyd angles ,30. Exped angles . In addid angles . Pertinetransfer . CollectO-methyltransferase (accession code 3OU6.pdb), the AdoMet molecules were remodeled in the electron density maps using the real space refinement and geometry tools in Coot. The remodeled AdoMet coordinates were then used to measure the tetrel bond geometries  of a small molecule inhibitor, solvent molecule, or ion were defined as exhibiting: (1) an θ(S–C∙∙∙X) interaction angle between 160° and 180°  and (2) a C∙∙∙X interaction distance less than or equal to sum of the van der Waals radii of the carbon and electronegative atoms, specifically R(C∙∙∙O) ≤3.25 Å and R(C∙∙∙Cl) ≤3.5 Å  . These gometries . FinallyQuantum calculations were carried out within the framework of the Gaussian-09 program. Active site models for SMYD2 (5ARG.pdb), SMYD3 (5CCL.pdb), COMT (5LSA.pdb), and G9A (5VSC.pdb) were generated from their respective crystallographic coordinates, with the heavy atom (non-hydrogen) positions fixed. Hydrogen atom positions were not derived from the enzymes’ X-ray structures but were added to the models followed by optimization of their positions at the M06-2X/6-31 + G** level. Energetics and NBO analyses ,39 were N2 reaction [A comprehensive survey of methyltransferase crystal structures (≤2.5 Å resolution) in the Protein Data Bank (PDB) was conducted to determine whether these enzymes exhibit evidence of methyl tetrel bonding. The survey comprised 269 structures and identified 20 nonredundant structures that display interaction geometries consistent with tetrel bonding between the AdoMet methyl group and ligands, ions, or solvent molecules within the active site . Notablyreaction ,43.The majority of the interactions observed in the PDB survey represent methyl C∙∙∙O tetrel bonds, with the exception of a single structure displaying a C∙∙∙Cl tetrel interaction between AdoMet and a small molecule inhibitor. The enzymes exhibiting tetrel bonding belong to either the (1) canonical class I methyltransferases  or 2) the Suppressor of variegation, Enhancer of Zeste, and Trithorax (SET) domain class of KMTs. This finding is not unexpected, given that these two classes are among the most abundant methyltransferases  the Supp. FurtherO-methyltransferase (COMT) display interactions indicative of C∙∙∙O tetrel bonding  Domain-containing (SMYD) methyltransferases . The humO-methyltransferase DhpI. The sulfate anion bound in the enzyme’s active site has been proposed to mimic the phosphonate group of the methyl acceptor substrate [In addition to interactions with small molecule inhibitors, the PDB survey also uncovered evidence of tetrel bonding between the AdoMet methyl group and solvent molecules as well as ions bound within methyltransferase active sites . There aubstrate , suggest2+, SMYD3/AdoMet/oxindole, and SMYD2/AdoMet/SGC BAY-598 complexes, as well as the G9A structure exhibiting a C∙∙∙O tetrel bond between AdoMet and a water molecule (+(Et)2, as previously reported [3 bond, corresponding to the σ* orbital that is also referred to as a σ-hole. A maximum occurs on the isodensity surface (0.05 au) with a value of +120 kcal/mol. It is in this region of the surface that Coulombic attraction with a Lewis base may occur.After completing the PBD survey, we selected four methyltransferase structures for computational analysis to investigate the theoretical energies and spectroscopic properties of the observed tetrel bonds with the AdoMet methyl group. These structures include the COMT/AdoMet/DNC/Mgmolecule . For thereported ,25,61. T+(Et)2 cation with a neutral partner, the COMT model contains a formal negative charge from the phenoxide anion of DNC.The geometries of the pair of relevant interacting groups in the structures of SMYD2, SMYD3, G9A, and COMT are depicted in int, where a negative quantity indicates an attractive interaction. As a frame of reference, an O–H∙∙∙O hydrogen bond in a water dimer has an interaction energy of −5.8 kcal/mol when calculated at this level of QM theory [2O complex is associated with a substantial bond energy of −7.0 kcal/mol. A much larger increment, raising the bonding energy to −65.7 kcal/mol, occurs when the partner subunit is negatively charged.As illustrated in M theory . The SMYT represents the perturbation energy consequence of transfer from the Lewis base (X) lone pair to the σ*(C–S) antibonding orbital, indicative of tetrel bond formation. Because of the proximity of CH protons to the nucleophile, there is the alternate possibility of a CH∙∙∙X hydrogen bond, which would manifest itself by a transfer into the σ*(H–C) antibonding orbital. Such a possibility is measured by EH, which is reported in the last column of T and tetrel bonding. Most importantly, this tetrel bonding is quite strong, as much as −9 kcal/mol for the neutral nucleophile, rising to more than −60 kcal/mol when the latter is an anion.In order to probe the nature of the interaction, the wave function was analyzed by the NBO procedure which considers charge transfers from one molecular orbital to another. EAn additional means to establish the presence of a tetrel bond, which can also distinguish this sort of interaction from a CH∙∙∙X hydrogen bond is by means of NMR and IR spectral data. A recent study  computedWith these patterns in mind, A glance at 2) moiety by a simple methyl group deletes any possible hydrogen bond, while leaving the tetrel bond intact. The interaction energy of the resulting derivative dimer in +(Et)2, leaving behind a neutral S(Et)2 (representing the product AdoHcy) in These two seemingly similar attractive forces need to be disentangled so as to better estimate the interaction energy of the tetrel bond itself. One way to evaluate the latter quantity is to remove the hydrogen bond entirely. Replacement of the nitro 2 cation, such that the interaction of these two species, without the DNC, amounts to 111.2 kcal/mol. The interaction energy of the MeS+(Et)2 with the DNC∙∙∙Mg2+ pair cannot overcome this strong repulsive force, so is +48.2 kcal/mol. When the pure MeS+(Et)2∙∙∙Mg2+ repulsion is subtracted from this quantity, one finds that the interaction between MeS+(Et)2 and DNC is attractive, in the amount of −63.0 kcal/mol. This quantity differs from the −65.7 kcal/mol pure MeS+(Et)2∙∙∙DNC interaction, in the complete absence of Mg2+ 2 with the new system elements, i.e., all exclusive of the DNC, is +47.4 kcal/mol. The total interaction when the DNC is added to the entire system is −16.3 kcal/mol. After subtraction of the repulsion between the Lewis acid and the extraneous elements, the interaction energy of the MeS+(Et)2 with DNC is −63.7 kcal/mol. This quantity represents a very small decrease relative to the naked MeS+(Et)2/DNC dimer, in the amount of 3%. In summary, the interaction energy computed for a pair of species, interacting directly via a combined tetrel and hydrogen bond, is scarcely affected by the addition of surrounding groups, provided one takes proper account of the newly introduced pairwise interactions.As the Mgn sphere b. In addIt is worth noting that the calculations above placed each system into an in vacuo situation, removed from the electrostatic or polarizing influence of neighboring groups. It is anticipated that these external effects will ameliorate some of the strongest Coulombic forces between the two groups, at least one of which carries a full charge. In an effort to estimate how much the surrounding protein environment might weaken these charge-assisted interactions , the var3)3+ and N-methylacetamide amounts to −13.7 kcal/mol, but to only −1.9 kcal/mol for the uncharged analogue S(CH3)2 [As in the case of halogen, chalcogen, and pnictogen bonds, tetrel bonds are much stronger for elements in lower rows of the periodic table, e.g., Si and Ge. For this reason, the majority of computational work ,75,76,77 S(CH3)2 .3 rather than pure methyl groups. Nonetheless, clear evidence was presented for the presence of tetrel bonds to this sp3-hybridized carbon atom.Our finding here that the interaction can be better described as a tetrel bond than as a trifurcated CH∙∙∙O hydrogen bond to a methyl group is consistent with earlier calculations . This grN2 reactions [N2 catalysis in methyltransferases is derived from decades of paradigmatic studies of COMT and has resulted in several models for the reaction mechanism of these enzymes. Serendipitously, crystal structures of COMT bound to AdoMet and substrate analog inhibitors, such as DNC, have provided strong evidence for C∙∙∙O tetrel bonding, given the close interaction distances of the AdoMet methyl group and phenoxide anion of DNC, as well as the S–C∙∙∙O interaction angle that approaches linearity  [2+ complex  ,7. As ob complex a, the C∙ complex  and woulreaction . The prereaction ,92. ThisIK) that are in the low nanomolar to picomolar range [Knowledge of carbon tetrel bonding can also be leveraged in the design of methyltransferase inhibitors. Indeed, the PDB survey illustrates several examples of inhibitors of COMT and SET domain KMTs that form tetrel bonds with the AdoMet methyl group . In the ar range ,94. The In addition to COMT, inhibitors of the SMYD KMTs display methyl tetrel bonding with AdoMet . With re3 bond of AdoMet, with θ(S–C∙∙∙O) angles within 20° of linearity. QM analysis of a selected set of these interactions revealed unequivocal evidence of methyl tetrel bonds, rather than what might naively be considered a trifurcated CH∙∙∙O hydrogen bond. The interaction energies of these selected tetrel bonds varied between −5 and −9 kcal/mol, comparable to or stronger than the paradigmatic hydrogen bond between a pair of water molecules . In the case of a Lewis base bearing a full negative charge, the tetrel bond energy was amplified to more than −60 kcal/mol. It is thus clear that our understanding of the forces present within biological systems must include tetrel bonding on the same footing as the venerable hydrogen bond. Finally, the discovery of AdoMet methyl tetrel bonding in methyltransferases illustrates that this interaction may have a fundamental role in the catalytic mechanism of these enzymes and thus merits further investigation. An understanding of this novel interaction can be applied in structure-guided design of potent inhibitors of methyltransferases implicated in disease.The survey of AdoMet-bound methyltransferase structures in the PDB uncovered a number of geometries that strongly indicate the presence of methyl tetrel bonding to electronegative atoms in small molecule inhibitors, solvent molecules, and ions. The C∙∙∙X distances between the AdoMet methyl carbon atom and the Lewis base vary over a wide range, but all of the C∙∙∙O interactions reported from this survey are less than 3.25 Å, with some distances as short as 2.5 Å. Further, the Lewis base is located close to the extension of the S–CH


To the editor,“PSA is a useful biomarker for predicting prognosis at levels between 20 and 70 ng / mL (1)” and “When the PSA level reaches approximately 70 ng / mL, prognosis might bottom and reach a plateau (1)”. We would like to share ideas on this report. We agree that there might be a plateau phenomenon in case of high PSA level. Nevertheless, we should also be aware on the possible false negative result in patients with low PSA level (We read the publication on “Prognosis of patients with prostate cancer and middle range prostate - specific antigen (PSA) levels of 20 – 100 ng / mL” with a great interest (SA level . In that


Reduced high latitude upwelling, a process that retains CO2 within the deep ocean over glacials, could have aided drawdown but has so far not been constrained in either hemisphere over the MPT. Here, we find that reduced nutrient upwelling in the Bering Sea, and North Pacific Intermediate Water expansion, coincided with the MPT and became more persistent at ~900 ka. We propose reduced upwelling was controlled by expanding sea ice and North Pacific Intermediate Water formation, which may have been enhanced by closure of the Bering Strait. The regional extent of North Pacific Intermediate Water across the subarctic northwest Pacific would have contributed to lower atmospheric CO2 and global cooling during the MPT.The Mid-Pleistocene Transition (MPT) is characterised by cooling and lengthening glacial cycles from 600–1200 ka, thought to be driven by reductions in glacial CO 2 from the atmosphere and caused global cooling.The causes of Mid-Pleistocene Transition global cooling 1 million years ago are still unknown. Here, the authors find the subarctic North Pacific became stratified during these glaciations due to closure of the Bering Strait, which would have removed CO Secondly, there was abrupt ice sheet growth3, and further SST reductions1 from Marine Isotope Stage (MIS) 22–23 (at ~900 ka). The absence of noteworthy secular changes in Milankovitch forcing, and the weakness of the 100 ka eccentricity insolation cycle, indicates internal feedbacks within the Earth system rather than orbital forcing alone were the cause of these two phases4. There is currently no consensus on what internal forcing shifts led to the MPT. Hypotheses have pointed to Southern Ocean dust supply increases from ~1300 ka5, the unusual insolation low over Antarctica at ~900 ka2, changing North American ice sheet dynamics6 and sea ice changes8. A secular decline in atmospheric CO2 concentrations has been suggested as a central cause11, and a slowdown in Atlantic meridional overturning circulation and North Atlantic Deep Water (NADW) production has led to the idea that there was enhanced CO2 transfer from the surface ocean to the deep at ~900 ka12. These processes are not mutually exclusive, and could each be associated with a build-up of ice on North America for which there is ample evidence4. To sequester CO2 in the deep ocean also likely requires stifled nutrient and CO2-rich deep water upwelling at high latitudes to reduce outgassing, as proposed for the last glacial;14 however, although recent records of Southern Ocean biogenic opal suggest glacial reductions in productivity occurred throughout the Pleistocene15, there are so far no direct records of high latitude upwelling from either hemisphere with which to evaluate this potentially important mechanism for the MPT glacial CO2 reduction.The Mid-Pleistocene Transition (MPT) is the last major transition in Earth’s climate, and appears as global cooling and glacial lengthening in two phases. Firstly, as gradual sea surface temperature (SST) reduction2 today is vented to the atmosphere16. The subarctic North Pacific is particularly important, as ventilation of this region may have accounted for 30% of the last deglacial rise in atmospheric CO218. High-nutrient deep water in the modern subarctic Pacific is separated from the surface by a shallow seasonal halocline. While the stratification is strong enough to prevent major intermediate or deep water formation, nutrient- and CO2-rich water sourced from North Pacific Deep Water (NPDW) upwells within eddies along the modern Bering Sea slope (termed the high productivity Green Belt) due to wind forcing, tidal mixing, instability and topographic interference20. Incomplete nutrient utilization occurs, likely due to iron and/or light limitation21, leaving surface waters oversaturated compared with atmospheric CO2 in the winter22. The modern Bering Sea intermediate water mass of low nitrate occurs above the halocline at ~300 m water depth23, but eddies along the slope can reach down 400–1000 m20.The subarctic North Pacific Fig. , includi25, and to glacial reductions in productivity in the Indian Ocean sector of the Southern Ocean (ODP Site 745)15, although the causes of reduced productivity are yet to be identified. For example, reductions in Pacific glacial productivity may be caused by light limitation due to a deepening of the mixed layer during summer24, hypothetically linked to changing atmospheric weather systems and windiness. Alternatively, changes could be related to reduced glacial upwelling of nutrients24 caused by enhanced halocline stratification21 or the glacial expansion of the nutrient-poor North Pacific Intermediate Water (NPIW) which would have resulted in a less nutrient-rich source of upwelling water27. Additional possible causes in the Bering Sea are sea ice expansion28, light limitation25, ice-rafted debris29, and reduced Fe-fertilization from riverine input and/or remobilization from shelf sediments19. A recent MPT productivity record from the Gulf of Alaska does not follow glacial cycles, and has demonstrated fertilization by glacigenic debris as an important process in that region29.Glacial reductions in Bering Sea Green Belt primary productivity are similar both to changes in subarctic Pacific primary productivity (ODP Site 882) over at least the last 800 ka15N and δ13C of organic carbon (δ13Corg) compared with organic carbon and previously published opal25 accumulation rates to decipher changes in deep ocean nutrient supply to the surface. Benthic foraminiferal δ18O is used to provide evidence of NPIW expansion. We compare our new records of surface and deep ocean changes with global sea level estimates and published data from other subarctic sites to assess the potential links between cooling, sea ice expansion, closure of the Bering Strait, NPIW production, reduced high latitude CO2 and nutrient upwelling, and development of the MPT. We find the increased presence of NPIW, and reduced nutrient upwelling, during glacial periods from the MPT onwards, and propose that coincident Bering Strait closure may have caused NPIW formation by retaining sea ice brines within the Bering Sea.In this study we produce the first high latitude nutrient upwelling record over the MPT and determine the causes of reduced glacial productivity in the Bering Sea over MIS 21–29 (840–1020 ka) by presenting new geochemical proxy records from Integrated Ocean Drilling Program (IODP) Site U1343 , located on a topographic high near the northern Bering Sea slope . The majority of our benthic δ18O record follows long-term trends in LR04 31, deep South Pacific (Site 1123)32 and North Atlantic (Site U1308) , which occurs within the MPT when sea levels fell, glacials began to lengthen, and productivity across the Bering Sea and North Pacific fell Fig. ; Fig. 3.343 Fig.  at high R04 Fig. , providimum Fig. , and posmum Fig. . These o342 Fig. . They ar18O offset seen in the Bering Sea records occurred at the glacial maximum  for three reasons. First, to shift the benthic δ18O record to better match the global composite deglacial timing would require rapid and drastic changes in sedimentation rates, which are not seen elsewhere at Site U134333. Second, manipulating the age model in this way would create an unusual three-step shape to the deglaciation, not seen in other records including the Pacific31. Third, our dinoflagellate cyst assemblages indicate that the relatively low δ18O values are accompanied by a probable cooling and/or expansion of sea ice  at ~5–30%25. Because factors such as diagenesis and low opal export may compromise the preservation of opal28, and any shifts in the balance of phytoplankton towards calcifiers could impact opal accumulation, absolute values may not constitute a quantitative proxy for export production. However, in this high flux region37 the relative changes in opal accumulation are large and likely to reflect first-order changes in productivity. Site U1343 constitutes a continuous long record that has been orbitally-tuned using δ18O stratigraphy over the past 1.2 Ma33 allowing glacial-interglacial calculation of sedimentation and biogenic opal accumulation rates. As the age model is constructed with a relatively small number of tie points (see Methods), the resulting opal and total organic carbon mass accumulation rates (MARs) are likely to approximate true flux rates only on long glacial-interglacial timescales. The adjustment of opal and total organic carbon percentages to MAR does not change the overall pattern in a significant way, although it does lower the glacial MAR to some extent  accumulation over the interval 840–1020 ka at Site U1343  record of organic carbon , which can be impacted by local or regional water column denitrification ;42 nitrate utilization during marine organic production ;42 diagenetic alteration;43 and contributions of terrestrial organic material which has lower δ15N (~3 ‰)44 and δ13Corg (–26‰)45 values than marine organics. Diagenesis is unlikely a major influence in the high sedimentation rate and productive Bering Sea47. Although it is possible that increased terrestrial material caused low δ15N and δ13Corg during low export production intervals before 930 ka, we regard this as unlikely due to the relatively stable sediment accumulation rates from 920–1020 ka, and relatively low C/N values  that do not co-vary with δ15N or δ13Corg , from our δ15N record we subtract eastern North Pacific Site 101248  is lagging productivity . The lag is more pronounced in Δδ15N than δ15N, which may be real or possibly due to an issue with age model uncertainty of the two sites. The 28 ka cyclicity disappears after 930 ka, and Δδ15N remains high and productivity  low for the entirety of MIS 22–23  both opal accumulation and δ012 Fig.  shows br–23 Fig.  signifyi15NU1343–1012 nutrient utilization proxy for U1343 is potentially impacted by the low-resolution nature of the Site 1012 record, by possible discrepancies between age models at both sites, and by possible denitrification. Although glacials have been considered times of reduced denitrification in the Bering Sea and subarctic North Pacific due to lower export production21, peaks in Bering Sea δ15N during deglacial oxygen minima have been proposed as possible denitrification signals21. To test this we compare the Δδ15NU1343–1012 to the Site U1343 δ13Corg  throughout the entirety of MIS 22–23, with highest values during MIS 22 before a rapid return to low nutrient utilization during interglacial MIS 21  as the modern Pacific halocline is relatively shallow (upper 300 m) and eddies at the slope can reach down to 1 km water depth20. These strong eddies are also likely to rule out a change in the depth of the shallower mixed layer as the primary cause of reduced productivity. We propose the cause of the reduced glacial nutrient upwelling from 930–1020 ka was due to enhanced NPIW  NPIW to at least ~0.8 km water depth during glacials over the past 1.2 Ma, evidenced by Site U1342 (Bowers Ridge) negative offsets in benthic foraminiferal δ18O from deep Pacific end members27. These records indicate that the lower boundary of NPIW deepened to >0.8 km water depth during glacials27, offsetting shallow benthic δ18O from U1343 values at ~2 km water depth 21, and the last glacial cycle from the Bering Slope  frequency in opal accumulation cycles at ~930 ka  northward flow since 3.3 Ma59. There is no direct evidence for how the water depth of the Bering Strait may have changed since, and it is possible that the strong currents did not allow significant sediment deposition and therefore any significant change from its current depth of ~50 m. We hypothesise that the failed MIS 23 deglacial may have directly coincided with the first closure of the Bering Strait  has been lowest when sea level2 was below 50 m of present (Table 18O (combined deep ocean temperature and ice volume) was above 4‰ . This provides some support for a closed Bering Strait having a stronger influence on NPIW and nutrient upwelling in the Bering Sea than either the average global surface or bottom water temperatures alone.Glacial expansions of sea ice influence may have been controlled by reduced air temperatures and/or increased windiness, and another factor promoting these conditions could have been closure of the Bering Straitnt3 Fig. . The modone Fig. . This woone Fig. 5N has be2 atmospheric exchange not only in the Bering Sea, but also across the subarctic North Pacific16, creating a wide regional barrier for the upwelling of CO2-rich deep water and associated primary productivity. Recent studies have highlighted the importance of North Pacific deep overturning60 for increased outgassing of deep ocean CO2 during the last deglacial18, and the role that expanded NPIW may have played in suppressing North Pacific CO2 ventilation to the atmosphere during the last glacial maximum18. A closed Bering Strait, therefore, could have contributed to MPT cooling by promoting local sea ice8 and regionally-extensive NPIW and reduced nutrient upwelling (this study). NPIW would have created an effective barrier to deep North Pacific overturning, and subsequent atmospheric ventilation of CO2. In this scenario, overturning of the North Pacific from the MPT onwards was only instigated at the termination of severe glacial conditions and associated global ocean circulation changes such as a shutdown of Atlantic Meridional Overturning Circulation60.The regional significance of reduced MPT nutrient upwelling along the Bering Slope, caused by NPIW expansion Fig. , is that1 and may have been associated with a reduction in atmospheric CO2 from enhanced dust fertilization in the Southern Ocean5 . In agreement with this, we find that MIS 22–24 was associated with a severe and extended period of reduced Bering Slope nutrient upwelling and NPIW expansion to >2 km water depth, similar to subsequent severe glacials , they do not show a clear increase in stratification over the MPT15 highlighting the potential importance of NPIW in increased oceanic sequestration of CO2 during the MPT. Although a further enhancement of dust deposition across the Southern Ocean may have contributed to MIS 22–24 cooling and CO2 decline , due at least in part to NPIW expansion across the subarctic North Pacific and associated reduced upwelling. Further high-resolution continuous sea ice and upwelling records over the last 1.5 Ma, from both the subarctic North Pacific and the Southern Ocean, and numerical modelling studies with integrated shelf processes, are now needed to explore the full climatic impacts of a closed Bering Strait and development of the MPT.The cause of MPT glacial lengthening is still unknown, but it may have emerged from the ‘skipping’ of insolation peaks20. Deep water that bathes Site U1343 is fed from NPDW, which enters and exits the Bering Sea through various passes in the Aleutian Island Arc (sill depths <4000 m). The clastic sediments from Site U1343 are predominantly fine clay and biogenic material64. There are no transported shelf-characteristic benthic foraminifera recorded65, which is to be expected as Site U1343 is situated on a topographic high separated from the shelf  was designed to override the major effects of bioturbation. We sampled between 219.87 and 281.74 m composite core depth (m CCSF-A). Site U1343 was double piston-cored to ~262 m CCSF, and the constructed splice leaves no apparent coring gaps in this section65.Surface water in the Bering Sea enters from the North Pacific, primarily from the relatively warm Alaskan Stream  (Supplementary Data 13Corg is expressed as per mil (‰) in conventional delta notation relative to the 13C/12C ratio calculated to the Vienna Pee Dee Belemnite (VPDB) scale, utilising within-run laboratory and international standards. Analytical reproducibility for the within-run standards was <0.1‰ for δ13C, and <0.7% for TOC.We measured ~740 samples for total organic carbon (TOC) and isotopes of Corg δ1Corg  were run on a Carlo Erba 1108 elemental analyzer, interfaced to a Thermo Finnigan Delta Plus XP IRMS at the University of California, Santa Cruz (Supplementary Data 15N is expressed as per mil (‰) in conventional delta notation relative to atmospheric nitrogen. Precision for δ15N is 0.2‰, based on replicates and in-house sediment standards.For δorg MAR) was calculated as (dry bulk density) × (sedimentation rate) × (wt %TOC). Opal MAR was calculated as (dry bulk density) × (sedimentation rate) × . Measured dry bulk density was only carried out on U1343 Hole A, and the bottom part of Hole E (ref. 65). We therefore estimated dry bulk density for the U1343 splice by converting the depths to composite m CCSF values, and modelling a 9-point loess smoothing spline for the entire record. We then estimated sample dry bulk density values to the nearest 10 m (by linear interpolation).Total organic carbon (TOC) mass accumulation rate  when our record is smoothed, with a consistent offset of ~0.5‰. This correlation suggests no significant missing intervals, as is expected with high sedimentation rates at U1343. Although our age model is markedly more precise than previously reported for U1343, when comparing our records with other locations it should be noted that up to 103 yr offsets exist between δ18O records in different ocean basins33.We constructed a composite benthic foraminiferal δ18O record, we firstly removed all δ18O data points where the values for δ13C were below –5‰ , and removed all significantly high/low δ18O data points if there was a replicate measurement for that sample. We then calculated offsets between all species compared with Elphidium . We used the previously defined offsets for Globobulimina pacifica (0.85 ± 0.22‰) and Nonionella labradorica (0.65 ± 0.27‰) from U134333, as our data were collected on a much lower number of samples and produced similar offset values . To estimate offsets for the other species, we calculated a smoothing spline because we did not have paired measurements in enough samples. The offsets for the species Islandiella norcrossi, Valvulineria aurucana, Lenticulina spp. and Cassidulina spp. were estimated by subtracting their values from the smoothing spline value at that location  and HF (49%) several times to dissolve carbonate, silicate, and aluminosilicates. The residue was finally mounted between a slide and cover slide in glycerin gel. Organic-walled dinoflagellate cysts were counted using an optical microscope at magnification of ×400 to ×1000 . At least 300 dinoflagellate cysts were counted for each sample when possible, otherwise the entire slide was scanned and the average count is 214 cysts (Supplementary Data Palynological samples were chemically treated following the procedure used at GEOTOPSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4