By comparing the inter-hotspot distances between three Pacific hotspots, we show that Hawaii is unique in its strong, rapid southward motion from 60 to 50 Myrs ago, consistent with paleomagnetic observations. Conversely, the Rurutu and Louisville chains show little motion. Current geodynamic plume motion models can reproduce the first-order motions for these plumes, but only when each plume is rooted in the lowermost mantle.Mantle plumes upwelling beneath moving tectonic plates generate age-progressive chains of volcanos (hotspot chains) used to reconstruct plate motion. However, these hotspots appear to move relative to each other, implying that plumes are not laterally fixed. The lack of age constraints on long-lived, coeval hotspot chains hinders attempts to reconstruct plate motion and quantify relative plume motions. Here we provide  40Ar/39Ar ages that the Rurutu plume is relatively stable compared to the rapidly moving Hawaiian plume, yet it has a similar deep mantle origin.Using mantle plumes to reconstruct past plate motion is complicated, because plumes may not be fixed. Here, the authors demonstrate using  However, efforts to constrain Pacific plate motion are complicated by the potential effects of independent plume motion8. Paleomagnetic paleolatitude data from four seamounts in the Emperor chain were interpreted as showing a large southward Hawaiian plume drift of ~15° relative to Earth’s spin axis, from 80 to 50 Myrs ago8. In contrast, similar reconstructions of the Louisville hotspot indicate little (<4°−5°) to no latitudinal movement during the same time frame and thus eliminate the idea of a large-scale constant southward mantle flow6. In fact, recent geodynamic modeling suggests that the Hawaiian plume may be unique in its behavior. Focused subduction along the Aleutian arc between 100 and 50 Ma may have caused a strong southward deep mantle flow that has affected the Pacific large low shear velocity province (LLSVP)9 and the starting locations of plumes, assuming that plumes initiate from the LLSVP edges10. Alternatively, hypotheses for Hawaiian plume motion may invoke capture and subsequent release of the Hawaiian plume by the ancient Kula-Pacific ridge during the 100–50 Ma time interval8, or asthenospheric flow channeling12 towards a spreading ridge north of Hawaii8. In an effort to better constrain the degree to which hotspots move independently of each other in the Pacific mantle domain, we compare the age and geometry of the Hawaiian and Louisville chains to the newly mapped, intermittently expressed, Rurutu chain in the Mid-Pacific  and contains a distinct isotopic character that varies from HIMU (high μ = 238U/204Pb in source component16) to C (common mantle component17) that was originally identified as the “Atiu Trend”18 in the Cook-Austral islands. Here we provide new age constraints on the Tuvalu seamount chain in the mid-west Pacific between 3° S, 175° E and 10° S, 180° .The new 40Ar/39Ar age determinations , for which a short 10−0 Ma segment is documented in the Cook-Austral islands region30. Another 72–64 Ma segment of this hotspot chain is located in the Gilbert Ridge31 and HIMU seamounts can be traced even farther into the Western Pacific Seamount Province (WPSP) back to 120 Ma33. However, a lack of sampling of the seamounts between the Cook-Austral islands and Samoa, and the lack of datable material from four seamounts that carry a Rurutu composition within the Samoan archipelago34, limit our mapping of this hotspot chain between 48 and 10 Ma. It also is important to note that a rapid increase in apparent plate motion observed along the Gilbert Ridge (66–72 Ma)31 31 Fig.  is poten40Ar/39Ar age determinations and seamount locations (see Methods). Then we calculated great circle distances and uncertainties between combinations of two best-fit chains at any given age  from 60 to 48 Ma, relatively constant inter-hotspot distances between 48 and 15 Ma, and a small decrease in distance starting around 15 Ma. The comparison of Rurutu and Hawaii  as compared to Louisville and 57 ± 27 km/Myr as compared to Rurutu during that 12 Myr interval. These rates indicate a faster rate of Hawaiian hotspot motion than previous models have suggested37, if Rurutu and Louisville were approximately stationary in this time interval. Our model-independent method, based solely on chain geometry and radiometric ages, therefore shows that significant motion of the Hawaiian plume occurred, supporting the understanding that observed changes in paleomagnetic-derived paleolatitude8 cannot be solely a result of true polar wander37. The smaller change in distance between Rurutu and Louisville within the same 12 Myr interval  we favor this latter interpretation.With the age and geometry of the three longest-lived hotspots mapped out on the Pacific plate . In these models, there are three parameters that have major time-dependent controls on large-scale mantle flow and the lateral advection of mantle plumes. These major controls include the mantle density gradients that can be inferred from seismic tomographic models, the assumed mantle viscosity structure, and the assigned relative viscosity of the plume conduits that controls plume upwelling velocities. Finally, the starting age, buoyancy flux, depth of the plume root, and whether or not that root is mobile or anchored, also will affect the predicted individual plume motions. We varied all these parameters, including a suite of tomography models and published viscosity structures, in our model runs to best reproduce the observed relative motions of the Hawaii, Louisville, and Rurutu hotspots. In our approach, we applied the plate motion model of Torsvik et al.38 that for Pacific plate motions are based on rotation poles of Steinberger and Gaina39. Even though the Steinberger geodynamic models23 are limited by using tomographic models of the modern mantle to predict past large-scale mantle flow patterns, the insertion of vertically buoyant plumes at reproducible locations in every model run allows us to compute tens of thousands of inter-hotspot motion histories. These Steinberger model outcomes are directly comparable to observed inter-hotspot distances between the three longest-lived hotspot chains in the Pacific  at the base of the so-called transition zone (TZ), the motions of the plume are significantly reduced or, more commonly, advected laterally in the opposite direction to what is required to match the observed inter-hotspot distances Fig. . On the In summary, we now have evidence for three well-defined and long-lived hotspot chains across the northern, equatorial, and southern expanses of the Pacific plate, each generated from an independently moving lower mantle plume. Their relative motions to a first order are explained by geodynamic plume motion models and this work provides further evidence that the Hawaiian plume is anomalous in its rapid motion between 80 and 50 Ma. During the same interval, it appears that the Rurutu hotspot maintained a similar distance from the Louisville hotspot, showing that these two mantle plumes have been moving in tandem since 80 Ma. Most importantly, our geodynamic modeling observations indicate that it is highly unlikely that mantle plumes originate from near the base of the upper mantle at 660 km. Instead, our results strongly favor a deeper, unanchored source for the three longest-lived Pacific mantle plumes, potentially near the CMB above the Pacific LLSVP or along its edges.Roger Revelle during expedition RR1310. The expedition took place in the summer of 2013 and dredged 43 seamounts in the Tuvalu-Samoa-Tonga region of the Pacific Ocean. This work focuses on the samples recovered from the Tuvalu seamount chain, which previously has been speculated to have been generated from the Rurutu hotspot13 that currently underlays Arago Seamount in French Polynesia15. Along with dredging, seafloor bathymetric mapping was conducted using EM122 multibeam sonar. Maps were generated using the Seamount Catalog program47. All samples recovered from this expedition are archived at the Oregon State University (OSU) Marine and Geology Repository (http://osu-mgr.org) and are available upon request.All samples reported in this study were collected onboard the RV 40Ar/39Ar analyses. When available, multiple phases from a single rock were analyzed. The sample preparation protocol is based on previously established methods for obtaining useable ages from altered submarine lithologies49. Rocks were first crushed and sieved to grain sizes of either 108–180 µm  or 212–300 µm . The grains were then sonicated and rinsed in deionized H2O prior to drying in a 50 °C oven overnight. The dried grains were magnetically separated in an effort to concentrate individual phases of plagioclase, hornblende, and/or groundmass. Subsequently, the separates were leached in acid for 1 or 2 h each in 3 N HCl, 6 N HCl, 1 N HNO3 and 3 N HNO3 with rinsing in between with ultrapure H2O. Plagioclase separates were additionally sonicated in 5% HF for up to 30 min in order to further remove alteration along the edges of those grains. All samples were then sonicated in ultrapure milliQ H2O for 1 h prior to drying in an oven at 50 °C. The cleaned separates were then handpicked under a binocular microscope in an effort to create a homogenous separate free of alteration.Petrographic analyses were conducted on all potential candidates for age determinations. Rocks that contained fresh phenocrystic phases  were preferentially selected and dated. In absence of useable phenocrysts, the freshest holocrystalline groundmasses were selected for 48.Between 3 and 50 mg of each sample was loaded into aluminum packets for irradiation. Flux monitors (FCT-2 sanidine) were loaded at the base of the quartz vial and between every three samples. Secondary age standards for addressing long-term internal reproducibility (AC-2 sanidine) were also loaded with the samples. Samples were irradiated using the Oregon State University TRIGA reactor for 6 h. Incremental heating experiments were conducted for each sample with typically 9−32 steps for phenocrysts and 35−43 steps for groundmass separates. Blanks were measured at the beginning, end, and at every two to three incremental heating steps of the experiment. For groundmass separates, an increased amount of low temperature steps were undertaken in an effort to drive off alteration-derived atmospheric Ar and reduce recoil effects. Most groundmass plateaus were selected in the intermediate heating range and likely represent the degassing of the interstitial K-rich mesostasis within the groundmass2 laser. Extracted gas was exposed to a series of 400 °C, 200 °C and room temperature getters prior to being inlet into an ARGUS VI mass spectrometer. Five masses were analyzed simultaneously with 40, 39, 38, and 37 measured on 1012 Ω Faraday cups while 36 was measured using an ion-counting CuBe electron multiplier. All ages are normalized (including previously published ages) to a Fish Canyon Tuff (FCT) sanidine age of 28.201 ± 0.046 Ma50 using the decay constant of 5.530 ± 0.097×10−10 yr−1 (2σ)51. Tuvalu ages were calculated using ArArCALC v2.7.052 with errors including uncertainties on the blank corrections, irradiation constants, J-curve, collector calibrations, mass fractionation, and the decay of 37Ar and 39Ar. The consistency of the secondary AC-2 sanidine age standard analyzed during the time interval when this research was undertaken is excellent with an age of 1185.2 ± 3.8 ka  and within error with other 40Ar/39Ar laboratories53.Samples were loaded into a copper tray, which was brought under an ultra-high vacuum. Separates were precleaned using a low energy beam of a CO40Ar/36Ar isochron intercept being statistically higher than the 295.5 atmospheric value) deemed “correctable”, we set the conservative conditions that the isochron must produce an MSWD < 2, contain at least 15 consecutive heating steps on the plateau, and contain an intercept uncertainty of <10% (1σ). For all four excess Ar samples reported herein (see Supplementary Information) the excess came at the low temperature heating steps while analyzing a groundmass separate. This is hypothesized to be a function of mantle-derived Ar being retained in the glassy  portions of the groundmass upon cooling due to the hydrostatic pressure imposed on the lava flow during cooling at depths54. Thus, increased hydrostatic pressure on the Rurutu submarine lava flows may amplify the likelihood of not completely equilibrating with atmospheric argon upon eruption.In order for a sample with excess argon (40Ar/39Ar or K/Ar (for young Rurutu seamounts and some Hawaiian seamounts) with preference always given to 40Ar/39Ar ages for a given seamount. Supplementary Fig. 40Ar/39Ar age determinations were recalculated to same standard and decay constant discussed in the Methods. In an effort to best match the time when the hotspot was located most directly beneath the seamount (e.g. shield building stage), only the oldest age for each seamount was utilized. Supplementary Table This study focuses solely on the three best studied long-lived hotspots in the Pacific: Hawaii, Louisville, and Rurutu (new in this study). All ages used were either 34.Combinations of dated and undated seamounts were used to generate the best fit hotspot location at a given age. Each geographic track was reconstructed by interpolating along a great circle between individual seamounts on a hotspot track Fig. . The age55 .The distance between individual hotspots at a given time was calculated using the haversine equation for great circles55 Fig. ; circles23. Using this model, we predicted past geographic locations of the plumes underlying the Pacific lithosphere at any given time (limited in this study to between 80 Ma and the present day). We then used the same great circle equations discussed above to produce modeled inter-hotspot distances.The measured inter-hotspot distances since 72 Ma Fig.  were the56. This scaling factor is reduced to 50% in the upper 220 km of the mantle, such that density anomalies are not incompatible with observations. In addition, an assumed viscosity structure of the mantle is needed to constrain rates of horizontal and vertical motion in mantle flow fields and between the plume and ambient mantle. Starting ages for each plume are estimated as plumes become more advected in mantle flow through time before potentially stabilizing. Plume rising speeds are calculated as in the computations of Steinberger et al.57 and require an estimate for their buoyancy fluxes as these control the rate and ability for a plume to rise through the mantle. The depth at which a plume is rooted also is vital, because it controls through what mantle flow fields the plume rises and the timescales of ascension. Finally, we should consider whether or not the plume root (e.g. LLSVP) can be moved by the overlying mantle flow or not. A mobile root will increase the plume’s ability to move with the motions of mantle flow as opposed to resisting the motions. As upper boundary condition, the models use the absolute plate motions of Torsvik et al.38, who adopted the rotation poles of Steinberger and Gaina39 for the Pacific plate in the time period discussed.These Steinberger-style geodynamic models produce variable results depending on a few key parameters briefly summarized here. Present-day mantle densities are inferred from seismic tomography models and used to compute mantle flow assuming Newtonian viscous rheology. Past mantle densities and inferred mantle flow are then computed with backward-advection in the time-dependent flow field; however, in order to account for the increasing uncertainties back in time, backward-advection is limited to the past 68 Ma. For this study the conversion factor for seismic velocity to density anomalies uses model “2” of Steinberger and Calderwood58, Mitrovica and Forte59, the Roy and Peltier60 three-layer model and VM6 model, models A and B from Steinberger nd Calderwood56, Steinberger21 and Rudolph et al.61. For the Rudolph, et al.61 model we used a simplified version of the results with viscosities of 3×1022 Pa·s below 1000 km, 4×1021 Pa·s for 1000–670 km depth, 4×1020 Pa·s for 670–100 km and 1×1022 Pa·s above 100 km. In addition, we compared the tomography models of SMEAN62, GRAND10 (an update of Grand63), GYPSUMS64, S20RTSB65, S362ANI66, SAVANI67, SAW64268, TOPOS362d169, and TX200870. The combination of different plume parameters with various viscosity and seismic tomography models resulted in 1.8 million different outcomes . All metadata associated with the RR1310 cruise is available upon request from OSU’s Marine and Geology Repository (http://osu-mgr.org). Individual model results are available upon request.All data for Supplementary InformationPeer Review File


Transthoracic echocardiography-assessed coronary flow velocity reserve (CFVR) evaluates coronary microvascular arterial function. Coronary flow velocity measurements at baseline and during hyperemia are used to calculate CFVR. Adenosine infusion induces hyperemia but it is not known if it causes a maximal response. We hypothesized that pre-treatment with nitroglycerine before adenosine provocation enhances hyperemia.Twenty-three healthy study subjects  underwent CFVR measurements before and after pretreatment with sublingual nitroglycerine (0.5 mg). Hyperemia was induced by adenosine infusion (140 μg/kg/min). In addition, the effect of nitroglycerin on left main coronary artery diameter was assessed.p = 0.002. The increase was caused by a marked reduction in baseline coronary flow velocity 17 (15–24) vs 27 (19–31) cm/s, p < 0.0001) while hyperemic velocity remained unchanged (90 (68–116) vs 93 (75–105) cm/s, p = 0.48). Nitroglycerin significantly dilated the left main coronary artery (from median 3.1 (2.7–3.6) mm to 3.8 (3.1–4.3) mm, p = 0.018).Pretreatment with nitroglycerine increased median CFVR from 3.6 (range 2.8–4.3) to 5.0 (4.1–6.0), Pretreatment with nitroglycerine dilates coronary arteries and increases coronary flow velocity reserve indicating that adenosine alone causes a submaximal hyperemia. Transthoracic color Doppler echocardiography-assessed coronary flow velocity reserve (CFVR) is an emerging non-invasive method to quantify coronary microvascular function and evaluate volumetric coronary flow reserve (CFR) –4. CFVR Mean coronary flow velocity at baseline and during maximum hyperemia are used to calculate CFVR. Infusion of the vasodilator adenosine is an established method to induce hyperemia  and studIt is possible that other vasodilators alone or in combination with adenosine would induce an even higher level of hyperemia . NitroglWe hypothesized that pre-treatment with nitroglycerine before adenosine provocation in CFVR measurements in healthy volunteers enhances the hyperemia further in comparison to adenosine alone, due to maximum dilatation of not only the resistance vessels but also epicardial arteries. In addition, we explored the effects of nitroglycerine on basal coronary flow and coronary artery diameter and assessed the reproducibility of repeated CFVR measurements using adenosine infusion with a short wash out period.Twenty-three healthy volunteers  participated in the study. Inclusion criteria were age 18–40 years, normal BMI and no chronical or acute illness requiring medication. Exclusion criteria were history of asthma, chronic obstructive pulmonary disease, anxiety disorder, and ongoing infection. Study subjects characteristics are presented in Table All study subjects were investigated on two consecutive days. The study protocol on each study day included two sets of coronary flow velocity measurements at baseline and during adenosine infusion. On the study days all subjects were fasting for four hours and were caffeine free for at least 24 h before the investigation. On day 1 a suitable vessel segment of mid left anterior descending artery (LAD) regarding measurement quality and breathing interference was identified with transthoracic color doppler echocardiography. During the first measurement (1:1), baseline coronary flow velocity was recorded and thereafter adenosine infusion at a rate of 140 μg/kg/min was started. Hyperemic coronary flow measurements were obtained during 5 min where the highest recordings were registered. After completion of the measurements, the adenosine infusion was paused. After a ten minute washout period a new measurement period was started (1:2) when new baseline measurements were obtained before the adenosine infusion was re-started at the same rate for new hyperemic measurements. Heart rate and systolic blood pressure were recorded every minute during adenosine infusion to detect potential adverse reactions.On day 2, two new measurements were performed (2:1 and 2:2). The measurements were identical to the procedures on day 1 with the exception that after five minutes of the washout period between the two sets of measurements 0.5 mg of sublingual nitroglycerine  was administrated to the study subjects. Five minutes after nitroglycerine administration new baseline measurements and adenosine–induced hyperemic measurements were performed (period 2:2). In a subgroup of nine subjects with good visualization of the left main coronary artery, the diameter of the vessel was measured before and after nitroglycerine administration.A Siemens Acuson platform equipped with a 4V1C transducer with 3.5 MHz color Doppler frequency and 1.75 MHz spectral Doppler frequency  was used to measure coronary flow velocity (CFV) in the mid to distal left anterior descending coronary artery (LAD), in a slightly modified apical two-chamber view. The same operator performed all measurements to ensure minimum variability. CINE-loops and Doppler images were stored for offline analysis using Tomtec image analysis software . CFVR was calculated as the ratio between mean hyperemic CFV and mean baseline CFV. A typical recording of coronary flow velocity at baseline without nitroglycerine is shown in Fig. The left main coronary artery was visualized in a modified short axis view at the level of aortic root and a CINE-loop of a full cardiac circle with a frame rate up to 70 frames/s was stored for offline measurements. The diameter was measured as close as possible to the R- wave and at the same location both at baseline and after nitroglycerine administration.No data for a sample size calculation was available before study start. The number of included subjects are based on own experiences and similar studies. Data was not normally distributed. Data are thus presented as median and range. Wilcoxon matched paired test was used to compare mean CFV, CFVR and artery diameter before and after adenosine and nitroglycerine administration. Friedman’s test was used to compare the two baseline measurements on day 1 and the baseline measurement on day 2.Twenty of the 23 subjects completed all four measurements. One was excluded due to anxiety during adenosine infusion and two were excluded due to difficulties to get satisfying CFV measurements after NTG administration, due to low CFV.p = 0.82, Table p < 0.001, Table There was no significant difference in flow velocity between the two baseline measurements on day one (1:1 and 1:2) and the first baseline measurement on day two (2:1), p = 0.021), Table p = 0.20. Maximum velocity during nitroglycerine administration (measurement 2:2) did not differ significantly compared with the measurement without nitroglycerine on the same day (2:1) or to the hyperemic measurements on the first day (1:1 and 1:2), p = 0.53, Table Maximum velocity during hyperemia was higher during the second measurement period on day 1 1:2) compared to the first measurement the same day (1:1),  comparedp = 0.39). CFVR after nitroglycerine administration (measurement 2:2) was significantly higher than with only adenosine (measurement 2:1), 5.0 (2.4–9.2) vs 3.6 (2.3–6.9), (p = 0.002) and significantly higher than at the two measurements on day 1, 3.7 (2.3–7.2), (p < 0.001) and 3.9 (2.5–7.3), (p = 0.007), respectively. CFVR was significantly higher during the second measurement on day 1 (1:2) than during the first measurement the same day (1:1), (p = 0.018).CFVR during the different measurement periods are shown in Fig. p = 0.018), Fig. Left main coronary artery dilated significantly after nitroglycerine administration (from median 3.1 (2.7–3.6) mm to 3.8 (3.1–4.3) mm, The main findings of the present study were that pre-treatment with nitroglycerine before adenosine-induced hyperemia significantly increased coronary flow velocity reserve.In the present study we hypothesized that pre-treatment with nitroglycerine before adenosine provocation in CFVR measurements would enhance the hyperemia further in comparison to adenosine alone. This hypothesis was based on the known vasodilating effects of nitroglycerine on epicardial arteries and clinical experiences from fractional flow reserve (FFR) measurements and myocardial scintigraphy where pretreatment with NTG may be used to enhance hyperemia –22.After administration of NTG, there was a reduction in baseline coronary flow velocity (before hyperemia) by approximately 30%. This reduction in flow velocity is more likely a result of a dilatation of the epicardial arteries, than due to a reduction of total blood flow, since the same amount of blood at a constant flow in a larger vessel leads to a reduction in velocity. The effect on vessel diameter is supported by the present study where the diameter of the left main coronary artery increased with approximately 20% after NG administration. Our baseline results thus confirm the results shown by Takagi et al.  where thDuring adenosine provocation after NTG administration one would expect a reduction of coronary blood flow velocity, compared to adenosine provocation without NTG, since NTG dilates epicardial vessels. Contrary to this hypothesis, there was no significant change in hyperemic coronary blood flow velocity when compared to the measurement without nitroglycerine. This may be interpreted as an increase in total blood flow through the myocardium after the combination of NTG and adenosine administration, compared to only adenosine. Thus, our results suggest that NTG pretreatment increased adenosine-induced coronary blood flow in healthy volunteers, supporting that epicardial vessel dilation is essential to cause maximal hyperemic flow.The reduced baseline velocity resulted in a significantly increased CFVR even though nitroglycerine did not increase hyperemic blood flow velocity Table . From a The results of the present study also demonstrate that the reproducibility of flow velocity measurements is satisfactory since the baseline measurements both at the same day and between the two study days did not differ significantly. This indicates that non.invasive flow velocity measurements are stable and can be used to detect even relatively small changes in flow velocity over time. A low intraindividual variability over time is a key factor for using this technique to evaluate possible changes in coronary flow. However, there was one potentially important exception. The flow velocity during adenosine-induced hyperemia was significantly higher during the second measurement period on day 1 in comparison to the first measurement the same day. The wash-out period between the two measurements was only 10 min and it is possible that the effects had not fully ceased, even though the half life of adenosine is only a few seconds. Alternative explanations include upregulation of adenosine receptors or increased sympathicus drive. The latter explanation may be less likely since basal flow and heart rate was unaltered between the two measurements. Independently of the reason, a longer washout period than 10 min can be recommended if repeated adenosine- induced hyperemic periods are required.The present study had some limitations. The size of the study population was limited. In particular, left main diameter was measured only in eight patients. This may reduce the validity of our observation. However, the 20% difference between left main diameter before and after nitroglycerine administration is within what previously have been reported (8–25%) , 24. A fPre-treatment with nitroglycerine before adenosine infusion could be a possible way to minimize the difference between CFVR and CFR. It could also ensure maximal coronary artery dilatation resulting in a more accurate way of evaluating maximal coronary flow reserve.


The highest DICcf (∼ × 3.2 seawater) is found during summer, consistent with thermal/light enhancement of metabolically (zooxanthellae) derived carbon, while the highest pHcf (∼8.5) occurs in winter during periods of low DICcf (∼ × 2 seawater). These opposing changes in DICcf and pHcf are shown to maintain oversaturated but stable levels of carbonate saturation (Ωcf ∼ × 5 seawater), the key parameter controlling coral calcification. These findings are in marked contrast to artificial experiments and show that pHcf upregulation occurs largely independent of changes in seawater carbonate chemistry, and hence ocean acidification, but is highly vulnerable to thermally induced stress from global warming.Coral calcification is dependent on the mutualistic partnership between endosymbiotic zooxanthellae and the coral host. Here, using newly developed geochemical proxies (δ Porites corals are linked.Coral reefs are biologically diverse and productive ecosystems, yet our understanding of the impacts of ocean acidification on biocalcification is limited. Here, the authors show that pH upregulation and the biological control of dissolved inorganic carbon in calcifying fluids of  Symbiodinium (zooxanthellae), have been spectacularly successful in building the tropical coral reef edifices that dominate many shallow-water environments and harbour more than one-third of the oceans’ biodiversity. The ongoing viability of these iconic22  are produced, transported, and then inter-converted at the site of calcification. It has also long been recognized121314cf upregulation of the calcifying fluid and biological control over the concentration of DIC in the calcifying fluid (DICcf). We find that over annual timescales there is an inverse correlation between pHcf and DICcf. This acts to maintain relatively stable levels of aragonite saturation in the calcifying fluid, and hence near-optimal rates of coral calcification, despite large seasonally driven variations in metabolically supplied DIC.Scleractinian corals together with their endosymbiotic dinoflagellates, c22 ref. . Critica2 ref. 56, there a11B) as a proxy for the calcifying fluid pHcf  records are available21Porites coral are ideal for reconstructing seasonal changes in the composition of their calcifying fluid since they are long-lived and, more importantly, the architecture of their skeleton has a relatively straightforward chronology that facilitates well-constrained timing of their skeletal growth at seasonal resolution22sw  from ∼8.02 in summer to ∼8.08 in winter  are shown in cf present under natural reef conditions becomes apparent from the exceptionally strong and inverse correlations between pHcf and DICcf (r2=0.88–0.94) present at the colony level  and lowest values in winter, whereas pHcf shows the opposite pattern. This seasonal variability in DICcf is consistent with light- and temperature-driven changes in the supply of metabolic DIC provided by the endosymbionts and/or the bicarbonate anion transporters within the coral hostPorites colony forms a distinctive subparallel array characterized by a distinctive range in DICcf that is inversely correlated to pHcf. Since the concentrations of carbonate ion [CO32−] and consequently the aragonite saturation state (Ωcf=[Ca2+]cf [CO32−]cf/Karag) of the calcifying fluid increases with increasing DICcf and pHcf, the observed antithetic seasonal changes in these parameters results in a more muted seasonal variation in Ωcf  or DICcf (±12%) acting alone. While there remains a subdued positive correlation of Ωcf with temperature . Finally and most importantly, regardless of the method employed, we find that measurements conducted under controlled, static, laboratory conditionscf and DICcf upregulation described herein.While the absolute levels of enhanced cf versus DICcf, can be explained by the ability of the coral to ‘control’ what is arguably32− relative to HCO3−, the greater increase in pHcf in winter (∼8.5) compared to summer (∼8.3) increases the concentration of carbonate ions within the calcifying fluid (and therefore Ωcf) for the same DICcf. This increase in winter pHcf therefore partially counters the seasonal slowdown in host-symbiont carbon metabolism. Hence during the cooler periods, higher pHcf enhances Ωcf and hence partially mitigates the reduced temperature-dependent kinetics of calcification because rates of mineral precipitation are proportional to (Ω−1)n, where n is the temperature-dependent order of the reactionn=1.3–2.0 for most reef habitats). During summer, the opposite behaviour is observed, with higher rates of metabolic DICcf partially offset by decreases in pHcf, resulting in a concomitant decrease in the carbonate saturation state of the calcifying fluid (Ωcf) and hence moderated  rates of calcification , are thus consistent with inferences that Ca-ATPase-driven pHcf upregulation is a relatively energetically inexpensive processcf and DICcf, therefore argue against the reduction of pHcf in summer being a result of the passive feedback from higher rates of calcification producing more protons thereby lowering pHcf. Thus, while this possibility cannot yet be entirely excluded, the higher production rates of zooxanthellae-derived metabolites that are presumably available in the summer to facilitate enhanced Ca-ATPase activity, also suggest that the lower summer levels of pHcf is not due to intrinsic limitations in the Ca-ATPase H+ pumping, but rather physiological controls on growth rate. Furthermore, similar anti-correlated changes in pHcf and DICcf are present in Porites from both Davies and Ningaloo Reefs, despite large differences in growth rates.Although our findings are based only on species of sw and/or temperature, light, nutrients, and little water motion, hence conditions that are not conducive to reproducing the natural interactive effects between pHcf and DICcf that we have documented here. A characteristic common to a variety of coral species grown under these artificial conditions is the apparently constant but limited sensitivity  of pHcf relative to external changes in seawater pHsw  of the coral’s calcifying fluid, the key driver of calcification. Although the maintenance of elevated but near-constant Ωcf in mature coral colonies is not directly influenced by ocean acidification, it is however highly susceptible to thermal stress. In extreme cases of coral bleaching, the loss of endosymbionts disrupts the metabolic supply of DICcf as well as the metabolites necessary to operate the Ca-ATPase that upregulate pHcf  Davies Reef , a mid-shelf reef ∼100 km east-northeast of Townsville, Queensland, Australia in the central Great Barrier Reef, and (2) Coral Bay , part of the Ningaloo Reef coastal fringing system of Western Australia. At Davies Reef, the annual range of daily average SST is 23–28.5 °C with a diurnal range of ∼0.5 °C or lessIn situ seawater temperature data extending back to 1987 for the core site at Davies Reef  was compiled from a number of different temperature sensors deployed between a depth of ∼2 to ∼10 m maintained by the Australian Institute of Marine Science from October 1991 to December 2013 (http://data.aims.gov.au/aimsrtds/datatool.xhtml). To estimate seasonal changes in carbonate chemistry, we used the 24-h seawater carbonate chemistry data collected by Albright et al.pCO2 solubility. We therefore assumed that daily average pH at Davies Reef also followed seasonal changes in temperature according to pHsw=−0.010 × T+8.31.et al.et al.in situ observations of temperature collected from a moored depth of ∼17 m as described by Falter et al.T  being found to be strongly correlated with seasonal changes in offshore temperature . To determine seasonal changes in pH at the back-reef site where the coral cores were recovered, the offshore pH was adjusted to account for the deviation in temperature due to local heating and cooling (see above), as well as the daily average decrease in total alkalinity of ∼10 μmol kg−1 at back-reef sites observed from measurementsAt Coral Bay, SST generally ranges from 22–23 °C in winter to 27–28 °C in summer11B (∼80%) and 10B (∼20%) are expressed in delta notation  as:Changes in the isotopic ratio of 11B/10Bcarb is the isotopic ratio measured in the coral carbonate and 11B/10BNIST951 is the isotopic ratio of the NIST SRM 951 boric acid standard. In seawater, boron exists as two different species, boric acid (B(OH)3) and the borate ion (B(OH)4−), with their relative abundance being pH dependent. The sensitivity of the δ11B proxy to the calcifying fluid pHcf arises from the incorporation of only the borate ion species into the aragonite structure454611B isotopic composition reflecting the pH sensitivity of the borate versus boric acid speciation. The pH of the calcifying fluid (pHcf) can thus be calculated from the δ11B measured in the coral carbonate (δ11Bcarb). The equation used to convert the δ11Bcarb isotopic composition measured in the coral carbonate skeleton to a pH of the calcifying fluid (pHcf) is given bywhere 11Bsw represents the δ11B in seawater (δ11Bsw=39.61‰)α(B3−B4)=1.0272 4− and CO32−. Re-analysing the Holcomb et al.KD as defined in Holcomb KD from cf as given by the inorganic data of Holcomb et al.32−]cf from measurements of (B/Ca)carb and pHcf, the latter derived from the skeletal boron isotopic ratio (δ11Bcarb). We further assume that [BT]cf is equal to the total concentration of boron of ambient seawater and only a function of seawater salinity . We therefore have:Thus, using the definition of KD=0.00297exp.Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Seawater (blue) and coral calcifying fluid parameters (orange). DIC = Dissolved Inorganic Carbon. pH*cf and G* are expected calcifying fluid (cf) pH and calcification rates (G) from fixed condition experimental calibrations (pH*cf = 0.32pHsw + 5.2). See manuscript for more details.


Following detailed rock magnetic, petrographical, and palaeomagnetic experiments, characteristic remanent magnetisation directions were successfully isolated from most samples using principal component analysis. The tilt-corrected direction groups yielded a palaeopole at 69.1°N, 319.8°E with A95 = 4.8° (N = 19). A primary origin for the magnetisation is consistent with positive fold tests. Our results from the Early Cretaceous units, combined with published palaeomagnetic data obtained from Cretaceous strata from the Lhasa and western Qiangtang terranes, show that these two terranes had already collided by the Early Cretaceous, the Lhasa terrane had a relatively east-west alignment, and it remained at a relatively stable palaeolatitude during the entire Cretaceous. Comparing the Cretaceous palaeolatitude calculated for the western Lhasa terrane with those from Eurasia and Mongolia suggests a latitudinal convergence of ~1400 ± 290 km and ~1800 ± 300 km, respectively, since the Early Cretaceous.To better constrain the Lhasa Those processes have a tremendous effect on global climate and the evolution of life4 and understanding the formation and evolution of the Tibetan Plateau would increase our knowledge of plate tectonic kinematics and climate change.The Tibetan Plateau, known as the Earth’s third pole, is a complex amalgamation of several continental fragments that include, from south to north, the Himalaya, Lhasa, Qiangtang, Songpan-Ganzi, and Kunlun-Qaidam blocks Fig. . These c12. The Lhasa terrane is located between the Bangong-Nujiang suture zone to the north and the Indus-Yarlung-Zangbo suture zone to the south. Its collision with the Qiangtang terrane to the north during the Mesozoic marked the closure of the Meso-Tethyan Ocean, and subsequent collision with the Tethyan Himalaya to the south during the early Cenozoic marked the closure of the Neo-Tethyan Ocean. Therefore, the Mesozoic history of the Lhasa terrane provides key context for the Lhasa-Qiangtang collisional process, the amount of intracontinental shortening within Asia, as well as the shape of the southern margin of Asia before the India-Asia collision. However, previous palaeomagnetic results obtained from both volcanic and sedimentary rocks of Cretaceous age yielded discrepant palaeolatitudes ranging from 9°N to 26°N25. Thus, the palaeomagnetically constrained Lhasa-Qiangtang collisional ages range from the Middle Jurassic (~162 Ma)26 to the Late Cretaceous27, with the consequence that the estimated amount of post-collisional crustal shortening within Asia varies from >1800 km19 to only a few hundred kilometres20. Noticeably, the large palaeolatitude discrepancy may be attributed to several factors: (1) lower palaeolatitudes, observed from sedimentary rocks, may partly result from compaction-induced inclination shallowing28; (2) some volcanic palaeomagnetic datasets originated from analyses of too few lava flows to average palaeosecular variation16, or from stacks of lava flows whose attitudes were not accurately measured; (3) some palaeomagnetic datasets were from only a few sites that may be unreliable. The Lhasa terrane is long and narrow, with a length >2000 km from west to east and a width <300 km from north to south , 2004). The earliest folding of the Early Cretaceous strata in the study area likely occurred at the end (65–75 Ma) of the Late Cretaceous2.The Early Cretaceous Zenong Group volcano-sedimentary sequences cover an area of ~2.0 × 10sed Fig. . The Zen30. We did not find any other Early Cretaceous volcanics with unambiguous, but distinctly different attitudes in the field area. We also collected 62 cores from six sites (ZZ1–ZZ6) in the Early Cretaceous Jiega Fm limestone in section B , located ~40 km southeast of the town of Zuozuo, with the goal of performing a regional fold test  and top (ZN21) samples of section A yielded weighted mean 206Pb/238U ages of 115.8 ± 0.6 Ma and 113.7 ± 0.5 Ma, respectively  region  acquisition curves displayed a rapid increase below 200 mT, and complete saturation is often reached at less than 1500 mT  and energy dispersive spectrometry (EDS) showed that Ti-Fe oxides are abundant in the studied Duoai Fm lava samples  of the 23 Duoai Fm lava sites corrected by the individual attitude, further suggesting that the k-value decrease of the Duoai Fm lava flows after unfolding is likely an artefact of field measurements and not partial pre-folding remagnetisation.One hundred and seventy-seven samples underwent stepwise thermal demagnetisation, and 36 samples were treated with stepwise alternating field demagnetisation. Representative Zijderveld diagrams are shown in Fig. 1° Table . The pre1° Table , the dec36 and excluded site and group mean directions with precision parameters  < 50 from further analysis. Nineteen of 23 palaeomagnetic sites were binned into 13 independent direction groups  of 13 independent direction groups is 2.7°, consistent with the reliability envelope of A95 min/A95 max at 1.6°/3.3° (N = 164)39. This, together with information from 13 independent direction groups from different lava flows interbedded with multiple layers of sedimentary rocks  well-determined rock age; (2) sufficient sample number ; (3) proper demagnetization techniques; (4) structural control and tectonic coherence with the relevant craton or block involved; (5) reliable field tests; (6) the presence of reversals, and (7) no resemblance to paleopoles of younger ages (by more than a period). Because there exists a long normal polarity superchron (~126–83.6 Ma) during the Cretaceous35, we considered reliable Cretaceous palaeomagnetic data from the Lhasa and Qiangtang terranes those that satisfy all of the quality criteria (the 1–5 and 7) except for the criterion 6 mentioned above. Noticeably, the potential for inclination shallowing due to sediment deposition and compaction is still a critical and often unresolved problem for the accuracy of palaeomagnetic data from red beds25. However, we note that (1) available Cretaceous palaeomagnetic data of basalt flows intercalated with red beds indicated that the two site-mean ChRM directions for the interbedded red beds and basalt flows are consistent46; (2) the inclination-only mean of site-mean ChRM directions and its corresponding palaeolatitude for red beds compared with those for volcanic rocks showed that no significant compaction-induced inclination shallowing occurred in the Cretaceous red beds of the Lhasa terrane9; (3) only four Cretaceous palaeomagnetic datasets are available from the western Qiangtang terrane, and three of them were obtained from red beds; (4) although the elongation/inclination (E/I) method, which is based on a statistical model for palaeosecular variation of the geomagnetic field that satisfies a specific latitudinal dependence on a circular distribution of virtual geomagnetic poles47, has been widely used to check and correct sedimentary palaeomagnetic datasets with inclination shallowing, the foundation of the E/I method is still questionable because the latitudinal dependency of geomagnetic palaeosecular variation may be a mathematical artefact of the conversion from directions to poles48. In this study, we used sedimentary palaeomagnetic datasets without the E/I correction to calculate the palaeomagnetic poles  and lacked a robust field test. We thus excluded this pole from further analysis. The other nine palaeopoles, which include four Early Cretaceous  and five Late Cretaceous  poles, fulfilled our quality criteria 22. These four palaeolatitudes are consistent within 95% confidence limits, and their palaeopoles generally fall along a small circle centred at the reference location 21, and 14.6° ± 2.7°N (Yare area)23. Considering that these five Late Cretaceous palaeopoles are statistically indistinguishable within 95% confidence limits and lie along a small circle centred on the reference location , a total of 10 Cretaceous palaeopoles have been published Table ; their pee sites 2 samplesion Fig. . This suion Fig. , we calcion Fig. . This in18 and two Late Cretaceous palaeopoles (NQ and QL)16 did not meet our selection criteria due to a lack of robust field tests and/or absence of a structural control; we thus excluded these three poles from further analysis. We calculated an inclination-only mean of 30.0° ± 2.2° and a corresponding palaeolatitude of 16.1° ± 1.4°N for 166 reliable Cretaceous palaeomagnetic sites from the central Lhasa terrane . We note that no reliable Late Jurassic palaeomagnetic poles are available in the western Lhasa and Qiangtang terranes, and that the Qiangtang terrane has been located along the southern margin of the Palaeo-Asian continent since the Late Triassic closure of the Palaeo-Tethys Ocean52. Therefore, we first tested whether the Lhasa terrane overlapped with the Qiangtang terrane during the Early Cretaceous. All four of the Cretaceous palaeomagnetic poles obtained from the western Qiangtang terrane satisfied the selection criteria 17, and 20.8° ± 3.1°N and 29.7° ± 5.7°N (Gaize area)27. Considering that these four poles have a wide palaeolatitude range and fall along a small circle centred in the reference location  of the western Qiangtang terrane as the southern margin of Palaeo-Asia before the Lhasa-Qiangtang collision and assume that the Lhasa terrane had an average northward velocity of 5 cm/yr between ~220 and ~130 Ma11, then the Lhasa terrane would intersect with the Qiangtang terrane at ~142 Ma, consistent with the geological interpretation that the Lhasa and Qiangtang terranes had collided by the Early Cretaceous. Geological evidence suggesting collision includes: (1) remnants of a Late Jurassic–Early Cretaceous subduction-accretion complex and forearc basin consisting of ophiolitic melange structurally overlain by Jurassic flysch near the Shiquanhe area2; (2) structural relationships and geochronological studies in the Nima basin along the Bangong-Nujiang suture zone showed that this basin underwent major structural deformation and erosion from a switch from marine to non-marine conditions between ~125 Ma and ~118 Ma, which are related to the Lhasa-Qiangtang terranes colliding during the Early Cretaceous53; (3) structural mapping and detrital zircon U-Pb dating in Domar from the western Qiangtang terrane that indicate significant shortening during Late Jurassic–Early Cretaceous, rather than the Cenozoic, in response to the India-Asia collision54; (4) synthesis of lithostratigraphical, magmatic, and metamorphic results from the Lhasa-Qiangtang collision zone that support a Bangong-Nujiang Ocean closure at ~130–140 Ma55; and (5) U-Pb and Hf isotopic analysis of detrital zircons from sedimentary rocks in the Gaize area that suggest the 140–130 Ma magmatic gap may represent the Lhasa-Qiangtang collision56.Convergence between Lhasa and Qiangtang was essentially north to south, and thus the age of their collision can be determined by the palaeolatitude overlap of these two terranes at the same reference point. Because the Lhasaion Fig. , we alsoous Fig. , suggest57. Second, we compared our results directly to a large dataset obtained from coeval lavas in Mongolia58, similar to previous approaches36. Reference poles for Mongolia and Eurasia are shown in Table The intracontinental shortening between the Lhasa terrane and stable Asia can be estimated by comparing palaeolatitudes from the same reference point with palaeolatitudes calculated from various reference poles. We employed two methods for selecting reference poles to constrain the intracontinental shortening. First, we compared our results with the Cretaceous (70–130 Ma) mean pole from the Eurasian apparent polar wander paths59. We conclude that the reference poles for Mongolia are the best determined palaeopoles for stable Asia. The ~1800 km north-south shortening deduced from comparison with reliable Cretaceous palaeomagnetic data from Mongolia is compatible with previously published palaeomagnetic estimates of 1700–2000 km since the end of the Cretaceous19.The discrepancies between the Mongolian and Eurasian reference poles could from a number of factors95 = 4.8°), corresponding to a palaeolatitude of 20.1° ± 4.8°N for the Shiquanhe area; (3) consistent inclinations measured in the Early Cretaceous Duoai Fm lava flows and Jiega Fm limestone indicate that compaction-induced inclination shallowing is insignificant in the Jiega Fm limestone; (4) the shape of the southern margin of Asia had a relatively east-west alignment before the India-Asia collision; (5) the western Lhasa terrane had accreted to the stable Palaeo-Asian continent by the Early Cretaceous and maintained a relatively stable palaeolatitude during the entire Cretaceous; (6) comparison of the Cretaceous palaeolatitudes calculated from the western Lhasa terrane with a coeval reference pole for Mongolia reveals a latitudinal convergence of ~1800 ± 300 km.We have presented geochronological and palaeomagnetic results from the Early Cretaceous Duoai Fm lava flows and Jiega Fm limestone in the western Lhasa terrane. These new data, as well as previous Cretaceous palaeomagnetic data from the Lhasa and western Qiangtang terranes, Eurasia, and Mongolia, led us to reach the following conclusions: (1) the Duoai Fm lava flows of the Shiquanhe area erupted at ~113–116 Ma, consistent with the adjacent Zenong Group volcanic rocks in the Cuoqin area; (2) 19 direction groups from the Duoai Fm lava flows and Jiega Fm limestone provide a high-quality Early Cretaceous pole located at 69.1°N, 319.8°E  were cut into 2.2-cm-long samples in the laboratory. Remanent magnetisation was measured using a JR-6A spinner magnetometer or a 2G 755-4 K cryogenic magnetometer, and demagnetisation was carried out with an ASC-TD 48 furnace with internal residual field lower than 10 nT or with a D-2000 alternating field demagnetiser. In principle, demagnetisation temperature intervals are 50 °C in the low temperature range and 30–10 °C in the high temperature range. Progressive alternating field demagnetisation was performed from 2.5 mT up to 100–120 mT. The instruments of the ASC-TD 48 furnace and magnetometers are housed in a magnetically shielded room with a magnetic field less than 300 nT. All of these experiments were completed at the Paleomagnetism and Environmental Magnetism Laboratory (PEML) at the China University of Geosciences, Beijing (CUGB). ChRM directions were obtained using principal component analysis of at least five successive stepsWith the goal of identifying magnetic carriers and guaranteeing the reliability of palaeomagnetic data, the acquisition of IRM, back-field demagnetisation of saturation IRM, and thermal demagnetisation of three-axis IRM, hysteresis loops were measured for some representative samples. The acquisition of IRM, back-field demagnetisation of saturation IRM, and thermal demagnetisation of three-axis IRM were performed using an ASC IM10–30 pulse magnetiser and were measured with a JR-6A spinner magnetometer. These experiments were also completed at the PEML. The hysteresis loops were measured at room temperature using a MicroMag™ Model 3900 Vibrating Sample Magnetometer at the Paleomagnetism and Geochronology Laboratory at the Institute of Geology and Geophysics (Chinese Academy of Sciences).SEM observations were performed with an acceleration voltage of 15 kV at the State Key Laboratory of Tribology . The polished samples were coated with carbon before analysis to yield high-quality images. Energy dispersive spectrometry (EDS) was subsequently performed to acquire compositional information.29Si as an internal standard. More detailed procedures of the analytical techniques and the configuration of the LA-ICP-MS system are found in Cai et al.64.Zircons were obtained from bulk samples after they were crushed and ground, and separated by a combination of heavy liquids and magnetic techniques at the Langfang Laboratory of Geophysical Exploration . Individual zircons were hand-picked under a binocular microscope, mounted in epoxy resin and polished to clearly expose the grain structure. Cathodoluminescence images were produced to reveal internal structures and choose potential target sites for U-Pb isotopic analyses. U-Pb zircon dating was conducted by laser ablation multicollector inductively coupled plasma mass spectrometry (LA-ICP-MS) at the Key Laboratory of Continental Collision and Plateau Uplift . To eliminate contamination, each zircon surface was cleaned with ethanol before analysis. U-Th-Pb concentrations were calibrated using National Institute of Standards and Technology (NIST) SRM 612 as an external standard and Supplementary Information


Ursus arctos) in the Greater Yellowstone Ecosystem (GYE). In recent decades, whitebark pine forests have declined markedly due to pine beetle infestation, invasive blister rust, and landscape-level fires. To date, no study has reliably estimated the contribution of whitebark pine seeds to the diets of grizzlies through time. We used stable isotope ratios  measured in grizzly bear hair and their major food sources to estimate the diets of grizzlies sampled in Cooke City Basin, Montana. We found that stable isotope mixing models that included different combinations of stable isotope values for bears and their foods generated similar proportional dietary contributions. Estimates generated by our top model suggest that whitebark pine seeds (35±10%) and other plant foods (56±10%) were more important than meat (9±8%) to grizzly bears sampled in the study area. Stable isotope values measured in bear hair collected elsewhere in the GYE and North America support our conclusions about plant-based foraging. We recommend that researchers consider model selection when estimating the diets of animals using stable isotope mixing models. We also urge researchers to use the new statistical framework described here to estimate the dietary responses of grizzlies to declines in whitebark pine seeds and other important food sources through time in the GYE , as such information could be useful in predicting how the population will adapt to future environmental change.Past research indicates that whitebark pine seeds are a critical food source for Threatened grizzly bears ( Ursus arctos) forage for a wide variety of foods [Pinus albicaulis) seeds in the GYE [Oncorhynchus clarki) when they were abundant in the Yellowstone Lake watershed [Euxoa auxiliaris) in the eastern region of the GYE [In the Greater Yellowstone Ecosystem (GYE), Threatened grizzly bears , which were exacerbated partly due to climate warming [Cronartium ribicola) [Currently, whitebark pine trees are listed as Endangered by the IUCN and as a high priority Candidate Species under the U.S. Endangered Species Act. These designations resulted from massive die-offs in western North America caused by severe infestations of mountain pine beetles  . Understibicola) . The maiibicola) .δ34S values (believed to be an indicator of whitebark seed consumption) derived from grizzly tissues (hair and blood) and cone production (mean number of cones per tree); and ii) to estimate the contribution of whitebark pine seeds to the diets of grizzly bears using δ34S and δ15N values , except for one year (2000) of seven in which δ34S values were relatively high for bears and cone production was low. They explained that high cone production in 1999 likely caused bears to forage heavily on overwintered masts in the spring and summer of 2000, increasing δ34S values for bears that year. For objective 2, they estimated that 11–100% of bears’ assimilated diets were composed of whitebark pine seeds, depending on year [n+1 sources, where n is the number of isotope tracers, does not have a unique, deterministic solution) to obtain unique solutions. They also showed that some isotope values for these sources were collinear  with those for bears, leading to non-identifiable solutions. Recently, Schwartz et al. [δ34S values for grizzly hair and cone production, but reported that other bear foods may have similar δ34S signatures as whitebark pine seeds, confounding any results generated by stable isotope mixing models .Felicetti et al.  and Schw on year . They stz et al.  conductez et al.  and founEven though there have been considerable advancements in modeling the diets of omnivores using SIMMs over the past decade , no studPlotting stable isotope data corrected for isotopic discrimination. It is important to use accurate isotopic discrimination factors when estimating the assimilated diets of free-ranging animals using stable isotope analysis  [∆13C, ∆15N, ∆34S) for omnivorous rats [Rattus norvegicus) to the isotope values of each major food source  or animals  [δ13C and δ15N values for plants and animals used in this study (∆13C for rat hair (∆34S) for laboratory rats calculated from a regression equation in Florin et al. [δ34S values for all sampled foods ((1) er, SIA) . We addeous rats  (laboratd source . Correct as rats . We adde9±0.3 ‰)  to the δis study . We note9±0.9 ‰; ) was sim5±0.9 ‰; ). We alsed foods .δ13C/δ15N, δ34S/δ15N, δ13C/δ34S, and δ13C/δ15N/δ34S.We plotted isotope values for bear hair and discrimination-corrected isotope values for the three main bear foods in Cooke City using 4 multivariate plots . HereaftCorrecting the mixing space for digestible elemental concentrations and assessing collinearity between mixtures and sources. Not accounting for differences in digestible elemental concentrations among food sources can bias dietary estimates calculated by SIMMs [www.nal.usda.gov/fnic/foodcomp/search; (2) by SIMMs . We calcby SIMMs  or compaCollinearity among sources and mixtures in an isotopic mixing space  results in multiple solutions for dietary contributions that are statistically equivalent. We assessed the geometry of each mixing space and assumed that those showing signs of collinearity would not converge or yield estimates that were consistent with those generated by other combinations of stable isotope measurements.Fitting SIMMs to stable isotope data for consumers. We initially ran four models in each candidate set that estimated the diets of bears at the individual-level (as a random effect), as we assumed diet heterogeneity exists in the population. Each candidate set was designated as such based on a different 2- or 3-isotope combination of δ13C, δ15N, and δ34S values derived from bear hair (the responses). Each candidate set contained a null model, which included stable isotope values but did not include digestible elemental concentrations  for each food source (concentration dependence), and a series of models that included stable isotope values and incorporated concentration dependence. We developed a notation for SIMMs in order to facilitate the comparison of the wide array of models tested in this work. Our notation utilizes a similar structure to that used in generalized linear models in R [δ13C, δ15N, and δ34S values), and its argument can be used to note any modifications to the mixing model such as the inclusion of concentration dependence (CD). The right-hand side of the model formula is used to denote the covariates used to model diet proportions. For example, CN ~ (1|BearID) is a null model that uses δ13C and δ15N values to estimate dietary proportions for each individual bear (as a random factor).(3) In addition to the null model, each candidate set was composed of a set of concentration dependence models with and without group structure . We considered sex and the year that bears were sampled as two different groups. In addition to bear ID, we treated these groups as random effects in our models. Hereafter, we refer to models that include concentration dependence with group structure (sex or year) and those without these random effects as our three “main” models. We did not include an interaction between sex and years because all male bears were sampled in one year (2009) and did not include additional levels of hierarchy (sex and years as random effects in the same model) because our sample was small. We expected that the null model would fit the data poorly compared to other models in each candidate set because this model did not take into account concentration dependence . After rδ13C±0.2 ‰, δ15N±0.3 ‰, and δ34S±0.4 ‰), as these estimates of error were higher, and therefore more conservative, than those from the Washington State lab. For all models, we applied discrimination error  for each mean marginal posterior density distribution  for each major food source. We combined chains by concatenation to calculate summary statistics. We calculated rhat (not reported) and DIC values following the calculations in R’s CODA package [We used IsotopeR  to estim factors ); used u package  and in t package ).Assessing goodness of fit and relative support for competing models. We calculated the goodness of fit of each stable isotope tracer in each SIMM model and conducted model selection within and between candidate sets. We used IsotopeR to estimate proportional dietary contributions for bears using each model. We then compared the goodness of fit of each isotope tracer in each model by calculating the normalized root-mean-squared error (NRMSE),ns values of the estimated (ysi) stable isotope ratios for tracer s and consumer i. Each NRMSEs  h NRMSEs  is a stapD). The latter term penalizes the deviance as the number of parameters increase. We also reported the penalized expected deviance (PED) for each model because this information criterion might be better suited than DIC for evaluating hierarchical mixture models [We used the deviance information criterion (DIC) to select the best model in each candidate set because it is widely used for Bayesian model selection. The DIC is composed of a goodness of fit term  for each model by rescaling the expected deviance (nsample) to a new sample size :rescaled metric to compare 2- (nsample = 34) and 3-isotope (nsample = 51) models under the hypothetical scenario that they have the same amount of data . We used this calculation to determine whether future work will benefit from collecting additional stable isotope data (3- vs. 2-isotope models).We also rescaled the deviance and recalculated DIC (Eq 2) to compare models with the same model structure  between candidate sets with different sample sizes. We recalculated DIC . We also estimated the probability of similarity , which includes the top model overall  for botSurprisingly, no effort has been dedicated to developing an approach for model selection to compare alternative SIMMs. In particular, no studies to our knowledge have tested competing models that use different isotopic mixing spaces  to estimate the dietary contributions of consumers. Such analyses are particularly important for modeling the diets of omnivores, as subtle changes to an isotopic mixing space due to the inclusion or exclusion of covariates  can have an effect on the proportional dietary contributions estimated by SIMMs.δ13C, δ15N, and δ34S values) derived from the hair of each individual bear and their foods to estimate the proportional contributions of major food sources to the diets of grizzlies sampled in Cooke City Basin in 2007–2009. We conducted our analyses using 4 sets of SIMM models. Each candidate set was defined by a different combination of stable isotope values from grizzly bear hair and their foods. We compared δ13C, δ15N, and δ34S values from bear tissues through time and tested the explanatory power of each stable isotope tracer in each model using a goodness of fit measure. We then conducted model selection within and among candidate sets to determine the best models for estimating the diets of grizzly bears. In the end, we provide a modeling strategy to estimate the grizzly bear diets through time in the GYE using stable isotopes.The main purpose of our study was to demonstrate the utility of model selection for SIMM analysis by estimating the diets of grizzly bears in Cooke City Basin, Montana . We firsCooke City Basin is located northeast of Yellowstone National Park within the Grizzly Bear Recovery Zone (U.S. Fish and Wildlife Service 1993) . Our stuPicea engelmannii), whitebark pine , lodgepole pine (Pinus contorta), and/or subalpine fir (Abies lasiocarpa). Like much of the GYE, the Cooke City Basin supports a diversity of large ungulates, including elk (Cervus elaphus), bison (Bison bison), mule deer (Odocoileus hemionus), white-tailed deer (O. virginianus), and moose .The vegetation in the study area is a mosaic of forested community types fragmented by talus, avalanche chutes, non-forested openings, and alpine meadows. Forested areas in Cooke City Basin may contain Engelmann spruce  available to grizzlies in Cooke City: plants, ungulates, and whitebark pine seeds . We usedOncorhynchus clarkii) were historically available to bears as prey in the Yellowstone Lake watershed, introductions of whirling disease  and non-native lake trout  severely reduced their numbers over the past few decades [Although spawning cutthroat trout  to identify individuals. Before sending the hair, we sub-selected 333 samples to reduce the probability of identifying the same individual multiple times each survey . WGI extWe measured the abundances of stable isotopes in the hair for each bear. Washington State University prepared and analyzed all samples as described in Felicetti et al. . We rinsWashington State University Stable Isotope Core Laboratory measured the abundances of carbon, nitrogen, and sulfur stable isotopes in bear foods using a ECS 410  elemental analyzer interfaced with a Delta Plus XP  mass spectrometer via continuous flow isotope ratio mass spectrometry. The Colorado Plateau Stable Isotope Laboratory at Northern Arizona University analyzed carbon and nitrogen stable isotopes in hair using a Carlo Erba NC2100 elemental analyzer interfaced via a CONFLO III device to an isotope ratio monitoring mass spectrometer. The same lab measured sulfur stable isotopes using a DELTA plus Advantage IRMS configured through a CONFLO III using a Costech ECS4010 Elemental Analyzer .δ) notation to report the differences of stable isotope ratios, expressed as δ13C, δ15N, and δ34S values , between samples and Vienna Peedee belemnite (VPDB), atmospheric N2 (Air), and Vienna Cañon Diablo Toilite (VCDT), respectively. The Colorado Plateau lab used the following international measurement standards: IAEA CH6 and IAEA CH7 for δ13C, IAEA N1 and IAEA N2 for δ15N, and IAEA S1-S4, IAEA SO5, and SO6 for δ34S. The Colorado Plateau lab estimated an analytical error (+1 SD) of ±0.2 ‰ for δ13C, ±0.3 ‰ for δ15N, and ±0.4 ‰ for δ34S. The Washington State lab used the following international measurement standards: USGS 40 and 41 for δ13C, USGS 32, 25, and 26 for δ15N, and IAEA S-2, S05, and S3 for δ34S. The Washington State lab estimated an analytical error (+1 SD) of ±0.1 ‰ for δ13C, ±0.2 ‰ for δ15N, and ±0.35 ‰ for δ34S.We used conventional delta  of each sex and year were normally distributed and homoscedastic following the assumptions of normal mixture models . We thenFor our main analysis, we estimated the diets of grizzlies using a four-step approach .n = 17; The genetics lab identified 18 grizzly (6M:12F) bears. Thirteen of the 18 (6M:12F) grizzlies were recaptured at least once during other years. We measured the stable isotope ratios in hair samples from 14 (4M:10F) of 18 individuals captured in 2007–2009 and considered three recaptured bears as independent n = 17; . We did δ13C and δ34S among years  sampled in Cooke City were relatively low in 2007–2009 . We als5,7.8 ‰; ); 1994–2P<0.001; ); and Yeδ13C had the lowest NRMSE values, especially when paired with δ15N, suggesting δ13C had potentially more predictive power than other tracers (δ13C/δ15N model without group structure (CN(CD) ∼ (1|Bear)) and year models (CN(CD) ∼ (1|Year) + (1|Bear)) were the top models overall   . The δ13stimates  because ne seeds . We did ne seeds . We notene seeds , they yine seeds .Rescaled values for the more complex 3-isotope models were higher than the 2-isotope models, suggesting that δ13C/δ15N/δ34S may not be the best choice for modeling the diets of bears without the inclusion of army cutworm moths or trout as a major diet source  yielded similar mean proportional dietary contributions  did not track dietary δ13C values as cleanly as δ15N and δ34S values. Therefore, they did not use the ∆13C factors estimated in their study to adjust the δ13C values of bear foods in their isotopic mixing space. No study has used this classic stable isotope tracer (δ13C) to aid in understanding the assimilated (plant-based) diets of free-ranging grizzlies in the GYE. We found that the ∆13C for rat hair was similar to those derived from the hair of other mammals [∆13C factors [δ13C values for major food sources were isotopically distinct  were relatively low in 2007–2009 compared to bears sampled throughout the GYE in 1994–2001 and 2000–2010. Isotopic data suggest that grizzlies sampled in Cooke City Basin may have consumed less whitebark, prior to sampling, than bears sampled during other time periods, or the contribution of whitebark may have declined in grizzly diets through time in the GYE. Similar to Schwartz et al. [δ34S values for bears decreased with increased whitebark pine mortality from 2007 to 2009. Although our sample sizes were small, such corroborating evidence  suggestδ13C/δ34S model, the 2-source concentration dependence models with no group structure were the most parsimonious (lowest DIC and PED) models in each candidate set , we still recommend the use of the 3-isotope models to estimate the diets of grizzlies in the GYE when trout are available. We validate this suggestion by showing that the top δ13C/δ15N model (no δ34S) and 3-isotope model (with δ34S) yielded comparable parameter estimates (∆34S) for mammal tissues because the use of δ34S to investigate animal diets is gaining popularity.Although we recognize the uncertainty associated with the digestibility of S ; and iii) generate reliable estimates of whitebark pine seed consumption by grizzly bears in the GYE using stable isotope data. We hope researchers use our new statistical framework to reconstruct the diets of other free-ranging animals, as such analyses could be useful in answering many important, and often fundamental, questions in both basic and applied ecology.SIMM analysis is gaining popularity among ecologists. To our knowledge, this is the first study to i) demonstrate that different combinations of stable isotope values for consumers and their foods can generate similar proportional dietary contributions using SIMMs; ii) use model selection to evaluate SIMMs Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


Using a detailed investigation of the age-related changes in cerebral tissue oxygenation in the barrel cortex of healthy, awake aged mice, we demonstrate decreased arteriolar and tissue pO2 with age. These changes are exacerbated after middle-age. We further uncovered evidence of the presence of hypoxic micro-pockets in the cortex of awake old mice. Our data suggests that from young to middle-age, a well-regulated capillary oxygen supply maintains the oxygen availability in cerebral tissue, despite decreased tissue pO2 next to arterioles. After middle-age, due to decreased hematocrit, reduced capillary density and higher capillary transit time heterogeneity, the capillary network fails to compensate for larger decreases in arterial pO2. The substantial decrease in brain tissue pO2, and the presence of hypoxic micro-pockets after middle-age are of significant importance, as these factors may be related to cognitive decline in elderly people.Despite the possible role of impaired cerebral tissue oxygenation in age-related cognition decline, much is still unknown about the changes in brain tissue pO While the exact mechanisms are not known, declined cerebrovascular function has been the subject of intense investigation due to its crucial role in oxygen supply to the neuronal units5. Clinical findings show a correlation between cognitive impairment and vascular disorders8 as well as between cognitive impairment and cerebral blood flow (CBF)9. These correlations suggest that gradual changes in the brain microvasculature and oxygen delivery which occur with aging may significantly contribute to cognition decline.It has been established that cognitive function declines even in healthy aging11. Strikingly, it has been shown that hypoxia, even in vitro, promotes the formation of amyloid β peptide, which is believed to be the primary neurotoxic element of Alzheimer’s disease (AD)11. There are several lines of evidence supporting the possible role of disrupted cerebral oxygenation in cognition decline. Clinical studies suggest that conditions which lead to restricted oxygen delivery to the brain promote the onset of cognitive disorders. In the extreme case of a stroke, the likelihood of developing dementia is several-fold higher in subjects that survive the event11. Oxygen supply to the brain is also globally decreased during high altitude excursions12 which are associated with cognitive defects in both human15 and experimental animals16. Furthermore, it has been shown in rats that intermittent hypoxia leads to increased incidence of neuronal death in the cortex17.Cellular studies suggest that restricted oxygen supply can contribute to neuronal death and increased incidence of cognitive impairment by promoting reactive oxygen species (ROS) formation and calcium dyshomeostasis2  changes with age. Previous aging studies investigating brain oxygenation were limited to near-infrared spectroscopy (NIRS) and functional magnetic resonance imaging (fMRI) measurements of attenuated oxygenated and total hemoglobin concentration response to hypercapnic challenges18 or neuronal activation23. Translation of these findings to tissue pO2 is not straightforward because cerebral tissue pO2 is affected by several other factors, including CBF, geometry and morphology of the vascular network, capillary density, and the cerebral metabolic rate of oxygen consumption (CMRO2); all of which are potentially modulated by age. Furthermore, the brain tissue pO2 distribution is highly heterogeneous24 leaving open the possibility of microscopic hypoxic domains as opposed to homogeneous pO2 decrease.Despite its critical importance, to our knowledge, there is no data available in the literature regarding brain tissue pO2, capillary flow and non-capillary blood flow parameters were measured to investigate the underlying vascular substrates for observed tissue oxygenation changes.Here we study age-related changes in brain tissue oxygenation within the barrel cortex of healthy, awake aged mice. In addition, vascular pO2 in penetrating arterioles and venules of young (6–8 month-old), middle-aged (13–15 month-old) and old (24–26 month-old) mice (n = 8 in each) using two-photon phosphorescence lifetime microscopy24 , 58.1 ± 2.7 (middle-aged) and 49.9 ± 2.1 (old) mmHg. Oxygen extraction fraction (OEF) was higher in the old mice  measured via the tail using pulse oximetry also showed a decreasing trend , 6 middle-aged (15–16 month-old) and 7 old (26–28 month-old)) up to a depth of 250 µm . As expected, the total blood flow values obtained from penetrating arterioles and venules were almost equal  was studied using Doppler Optical Coherence Tomography (OCT)ual Fig.  although34 and 0.5–2.0 ml/g/min in anesthetized young mice under varying conditions36. One study using the quantitative autoradiographic iodo[14 C]antipyrine method obtained a CBF value of ~0.7 ml/g/min for the barrel field of anesthetized young rats, but CBF was ~1.8 ml/g/min under awake conditions37, which is in agreement with our CBF estimate in the barrel cortex of young mice.CBF estimates from average arteriolar and venular flow maps were 1.7 ± 0.4, 2.3 ± 0.4 and 1.9 ± 0.4 ml/g/min for young, middle-aged and old mice, respectively Fig. . Estimatet al. also shows that in awake, non-restrained mice, HR follows an inverted U-shaped trend from 3 to18 months of age, peaking at 14 months38. A number of studies on mice39, monkeys40 and humans41 have shown that cardiac output (CO) is directly linked to HR. Therefore, the observed increase in HR at middle-age suggests increased CO, which is necessary to support a higher demand for oxygen and other nutrients in middle-aged mice having a larger body mass. Indeed, a study found lower CO in 2-month-old rats compared with 6- and 24-month-old rats, but CO normalized to total body mass was not different among age groups42. We also observed that HR normalized to the body weight did not differ in our age groups  and body weight of the animals also followed the same inverted U-shaped trend Fig. . Data fr44. Capillary flow measurements  were performed on the mice in which vascular pO2 was imaged .Red blood cell (RBC) flow in capillaries (diameter < 10 µm) was imaged using two-photon laser-scanning fluorescence microscopy  a denser capillary network at middle-age is hypothesized to be able to deliver the same amount of oxygen to the tissue by a smaller pO2 gradient (between the capillaries and tissue). This smaller gradient leads to unchanged tissue pO2 from young to middle-age despite a decrease in intravascular pO2, provided that CMRO2 does not change significantly (see below). Interestingly, regional analysis of tissue pO2 data confirmed this conclusion. We observed that tissue pO2 near arterioles declines continuously with age, but distant from the arterioles (in the capillary bed) tissue pO2 is maintained until middle-age followed by a significant decrease afterwards . No significant change in OC and Rt or their spatial heterogeneities was observed between young and middle-aged mice age Fig. . Therefo2 imaging over the same region , we could identify diving arterioles in which we had both the arterial pO2 versus depth and their blood flow . While we aimed to minimize confounds, our use of the probe PtP-C343 is also associated with known limitations when measuring absolute pO2 values: potential interaction with plasma proteins when doing vascular measures, temperature dependence, O2 consumption by the probe itself and background signal that increases with depth, more so in a thin-skull preparation which increases excitation light scattering. However, we have no reason to believe these confounds would differentially affect our group comparisons.All measurements were done in awake mice, removing the confounding effects of anesthesia. Awake imaging is particularly important in aging studies since anesthesia may affect physiological and hemodynamic parameters differently in young and old subjects. In addition, anesthetized experiments often need mechanical ventilation and/or adjustment of physiological parameters which the results because many of these parameters normally change with aging. To minimize animal stress during imaging, a custom-built treadmill wheel was used in which the animal was able to freely walk or run, while the head was fixed, similar to that of Lyons 2 in old mice, a possible shift in the oxyhemoglobin dissociation curve with age can also affect the oxygen uptake in the lungs and its delivery to tissue. In human erythrocytes, it has been shown in vivo that RBC aging shifts the oxygen dissociation curve to the left57. However, it has been reported that the RBC age distribution does not differ in male human subgroups of different ages57. Investigation of the oxygen dissociation curves of blood samples from different age groups could answer the question more clearly, but we only found one human study showing a slight rightward shift of the oxyhemoglobin dissociation curve in elderly subjects32.In addition to the role of hematocrit, capillary density and CTTH in decreasing tissue pO58 are very common in the brain samples from old individuals, particularly people suffering from mild cognitive decline or AD59. A causal role of these microinfarcts in age-related cognitive disorders has been suggested. An animal study also reported neuronal loss and cognitive impairment in a mouse model of multiple diffuse microinfarcts59. These and our findings support the hypothesis that impaired tissue oxygenation in healthy aging may be one of the mechanisms involved in cognition decline. Unfortunately, in our tissue pO2 images the small capillaries were not clearly visible in most cases because of the background fluorescence of the PtP-C343 dye. Therefore, it was not possible to study the correlation between hypoxia and local capillary loss. Although our data showed an overall reduction in mean capillary density and hematocrit in old mice, future studies need to be performed to correlate the hypoxic micropockets with local capillary density or capillary flow parameters, as well as local neuronal death.Our observation of hypoxic micropockets in old age could be linked to previous neuropathological findings which report that tiny microinfarcts (mean diameter ~200–1000 µm)60 or unchanged CBF62 with age in humans. However, there are a number of studies that report either stable CBF until middle-age with decline thereafter66, or a parabolic trend in global CBF peaking at an age of ~60 years67. In anesthetized rats, CBF was also shown to decrease with age69, but two studies on conscious rats observed a parabolic trend, peaking between 3–14 months of age71. Differences in the techniques, brain regions, the age range, the criteria for the selection of healthy old subjects and inclusion or exclusion of middle-aged subjects may account for the disparate findings. Our investigation underlines the necessity of including a middle-age group in aging studies. Our CBF estimates were obtained assuming unchanged cortical thickness with age. As a rough analysis, we normalized the CBF to total body weight and we observed a flatter trend in age-related change  stimulation at 24 months of age compared with 3 and 12 months80, suggesting maintained capacity for cerebral angiogenesis until middle-age, but a decline thereafter.In accordance with the CBF data, we also saw a parabolic trend in capillary density. This is in line with the strong correlation between the capillary density and the regional CBF found in rats2 in conscious mice which was manifested mainly after middle-age. The findings suggest regulated capillary oxygen supply until middle-age to maintain the oxygen availability in the brain tissue. With further aging, tissue pO2 decreased significantly, hypothesized to be due to a capillary network failing to compensate larger decreases in arterial pO2. The observed substantial decrease in brain tissue pO2 and the presence of hypoxic micro-pockets after the middle-age are of significance because they could be involved in neurodegenerative diseases and cognition decline in elderly people.Overall, our study reveals an age-related decrease in resting cerebral tissue pOAlthough this study was not blinded, the analysis of microscopic data was automated and the same parameters and algorithms were used to analyze all images limiting investigator bias. In addition, no data was discarded and care was taken to treat all groups equally during surgery, handling, training and imaging.2- sensitive molecule PtP-C343 was synthetized following the general procedures reported in Finikova et al.25 and Vinogradov et al.81. Polyethylene glycol (PEG) units with the MW of ~2000Da were used for the PEGylation of the dendrimer periphery. All compounds were characterized using routine techniques, including NMR, mass spectroscopy, GPC, and UV-Vis spectroscopy. The dye was then calibrated at 37 °C and 7.2 pH as described before82.The O2. In batch 2 (8 young (average age = 6.9 ± 0.2 month-old), 8 middle-aged (average age = 14.1 ± 0.1 month-old), 8 old (average age = 24.9 ± 0.1 month-old)), vascular pO2 and capillary blood flow were measured. OCT and tail pulse oximetry were performed on both batches. 8–10 days before measurements, a thinned skull window was created over the left barrel cortex under 2.0% isoflurane anesthesia, as described in Shih et al.26. Briefly, the scalp was removed and a titanium-made head-plate was fixed on the skull using dental cement. The skull was then slowly thinned to translucency with a micro-drill . A 150 µm-thick cover glass was glued to the window using cyanoacrylate glue and the edges were sealed with dental cement to form a 3 mm diameter cranial window. For tissue pO2 measurements, a small thinned region at the edge of the cover glass (~0.5 mm) was left uncovered with dental cement to allow later injection of the PtP-C343 dye into tissue through the soft thinned membrane. During the surgery, animals were fixed on a controlled physiological monitoring system  which enabled continuous monitoring of the rectal temperature, respiration and heart rate. Ketoprofen  and buprenorphine  were injected before the surgery and baytril  was injected after the surgery. Injections were repeated 24 hours after the surgery.Animal handling and surgical procedures were approved by the ethics committee of the research center of the Montreal Heart Institute. All experiments were performed in accordance with the Canadian Council on Animal Care recommendations. Young adult (6–9 month-old), middle-aged (13–16 month-old) and old (24–28 month-old) C57BL/6 J healthy male mice were obtained from the colony of aged mice of the Quebec Network for Aging Research (RQRV) and housed in 12-hr light-dark cycle until imaging. Two batches of mice were used. The mice in batch 1 (7 young (average age = 8.8 ± 0.1 month-old), 6 middle-aged (average age = 15.3 ± 0.1 month-old), 7 old (average age = 27.0 ± 0.1 month-old)) were used to record tissue pODuring all measurements, animals were fixed on an angled treadmill wheel which allowed free movement of the limbs while the head was restrained by a titanium bar  capillary volumetric flow ; (4) RBC flux (cells/s), determined as the number of dark shadows in the space-time images divided by the acquisition time of the images ; (5) hematocrit, calculated using the obtained values for RBC flux and volumetric flow44 ; and (6) capillary resistance, estimated from diameter and hematocrit using the equations given in literature85. All measured capillaries in each age group were pooled for age comparisons. For each animal, 1–3 angiograms  were also recorded for capillary density calculations.~200 µL 2MDa dextran-FITC  was injected through the tail vein. Due to the injected fluorescent dye, the plasma appeared bright in the images while RBCs appeared as dark shadows. Capillary flow parameters were obtained from longitudinal and perpendicular line-scans over capillaries using the contrast in the images  metric with a patience of 25 epochs. The model was regularized with a weight decay of 1e−5 and a dropout rate of 0.2. We processed the output of the neural network by 3D morphological erosion and dilation operations to refine the segmentations. The vascular density was measured for each slice of the binarized (segmented) two-photon angiograms before and after applying a median filter  was slowly injected into the brain tissue ~300 µm below the surface with a glass micropipette using a microsyringe injector . ~200 µl 2MDa FITC-Dextran  was also injected through the tail vein to visualize the vasculature. For each animal, grid measurements (225–400 points) were performed over 3–4 400 µm × 400 µm adjacent planes at different depths   were graphed and labeled as arterioles or venules. The distance of each pO2 sampling point to closest arteriole and venule (in 3D) was then found. All sampling points in each age group were pooled to find the average tissue pO2, spatial heterogeneity of tissue oxygenation (defined as SD/mean) and the fraction of sampled points with hypoxic pO2 (here defined as pO2 < 5 mmHg). In addition, pO2 profiles from arterioles and venules were obtained by plotting the points versus their distance from closest arteriole or venule. For venular profiles, the points within 100 µm from an arteriole were excluded to avoid the contamination of venular profiles with the dominant effect of arterioles. For a regional study of tissue oxygenation, sampled points were divided into three categories: (1) “near arterioles”, including the points within 100 µm from an arteriole; (2) “near venules”, including the points within 100 µm from a venule, but at least 100 µm from the closest arteriole, and (3) “capillary bed” including the points which were at least 100 µm distant from the closest arteriole or venule. Average tissue pO2 was then obtained for each region.For tissue pOep) Fig. . Before 2 supply in tissue, and a modification of the Krogh model50 was used to estimate the net oxygen consumption rate in the tissue (OC), defined as CMRO2 minus capillary O2 supply per unit volume of the tissue 51, and D is oxygen diffusivity in tissue (~4000 µm2/s)51. Tissue pO2 profiles around arterioles, obtained from tissue pO2 maps, were fitted with the Krogh model to find OC and pO2,art. Rt was determined as the distance at which the curve reached a plateau.For simplicity, we assumed a uniform capillary Osue Fig. :1\docume2 was measured at 30–40 µm depth intervals, starting right after they branched from pial vessels up to the depth of ~200 µm  using the Hill’s equation (for C57BL/6 mice: n = 2.59 and P50 = 40.2)87. For each vessel, average pO2 and SO2 over the first 150 µm of depth were obtained. Average pO2 and SO2 values were then pooled in each group for age comparisons. Oxygen extraction fraction (OEF = (SaO2-SvO2)/SaO2) was also obtained from average arteriolar and venular SO2 values.The PtP-C343 dye solution  was injected through the tail vein. The fluorescent signal of the dye allowed visualization of the vasculature without the need for FITC-Dextran injection. In each mouse, a few penetrating arterioles and venules were chosen and vascular pO µm Fig. . Each vaSupplementary Fig. et al.88. Optimization was done on the first frame of each acquisition to obtain the dispersion coefficients which were then applied to the rest of the acquisition. Reconstruction of flow speed was based on a moving-scatterer-sensitive reconstruction technique90 which uses the Kasai Autocorrelator91. A digital high pass filter was used to remove the stationary scattering components from the OCT image. For angiography image reconstruction, spectral shaping and dispersion compensation was identical to above. Frame differences were then computed in pairs (for repeated frames) and their variance were averaged over the 10 volumes repetition.For each mouse, an OCT angiogram and a 3D Doppler OCT volume were acquired over a cortical surface of ~700 µm × 700 µm. First, an angiogram volume (512 × 512 pixels) was acquired Fig. . In eachen face slices of z-projection blood velocity maps at cortical depth intervals of ~3.8 µm. Pial vessels generated both positive and negative Doppler shifts because of their undulating shape, but penetrating arterioles and venules exhibited pure positive or negative Doppler shifts . Capillaries were defined as microvessels a few branches away from penetrating vessels with diameter less than 10 µm.The camera images taken during the surgery were used to identify pial arterioles and venules based on their morphology; pial arteries tend to be thinner, straighter, and gradually branching into smaller vessels, while pial veins are thicker, more curvy, and branching into vessels of all calibers2 and CBF measures over the same cortical region, we estimated the CMRO2 from CMRO2 = CBF([O2]a-[O2]v)  over the same depth using the equation [O2] = α.pO2 + 4 Hct.CHb.SO295. CHb is hemoglobin content of RBC (5.3 µmole Hb/ml RBC)95 and Hct is hematocrit, assumed to be equal to the systemic hematocrit.For the mice in which we had both vascular pO]v) Fig. . CBF was2 and Doppler OCT measures over the same region, we were able to detect arterioles for which we had both vascular pO2 versus depth and mean blood flow . Two hours after the beginning of the light cycle, blood was harvested from the saphenous vein of non-fasted mice and put in microhematocrit tubes by capillary action. Tubes were then sealed and spun in a microhematocrit centrifuge at high speed for 5 minutes. The length taken up by centrifuged red cells was measured and divided by the whole microhematocrit tube length to determine the hematocrit percentage.The results are presented as mean ± s.e.m. Statistical significance was calculated using ANOVA followed by Tukey HSD post hoc test. Statistical significance was assigned at *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and *****p < 0.00001. The sample sizes were chosen empirically based on our previous experience.Custom-written code in Matlab or Python are available from the corresponding author upon reasonable request.The authors declare that the main part of data supporting the findings of this study is available within the paper and its supplementary figures. Data not presented within the paper or supplementary figures are available from the corresponding author upon reasonable request.Supplementary figures