Path-dependence of the Plio-Pleistocene glacial/interglacial cycles.

We find strong path dependence in the evolution of the Plio-Pleistocene glaciations using CLIMBER-2 Earth System Model simulations from the mid-Pliocene to modern preindustrial (3 My-0 My BP) driven by a gradual decrease in volcanic carbon dioxide outgassing and regolith removal from basal ice interaction. Path dependence and hysteresis are investigated by alternatively driving the model forward and backward in time. Initiating the model with preindustrial conditions and driving the model backward using time-reversed forcings, the increase in volcanic outgassing back-in-time (BIT) does not generate the high CO2 levels and relatively ice-free conditions of the late Pliocene seen in forward-in-time (FIT) simulations of the same model. This behavior appears to originate from nonlinearities and initial state dependence in the carbon cycle. A transition from low-amplitude sinusoidal obliquity (~41 ky) and precession (~23 ky) driven glacial/interglacial cycles to high-amplitude ~100 ky likely eccentricity-related sawtooth cycles seen between -1.25 My and -0.75 My BP (the Mid-Pleistocene transition or "MPT") in FIT simulations disappears in BIT integrations depending on the details of how the regolith removal process is treated. A transition toward depleted regolith and lowered atmospheric CO2 levels are both required to reproduce the MPT.

Wildfire-induced increases in photosynthesis in boreal forest ecosystems of North America.

Observations of the annual cycle of atmospheric CO2 in high northern latitudes provide evidence for an increase in terrestrial metabolism in Arctic tundra and boreal forest ecosystems. However, the mechanisms driving these changes are not yet fully understood. One proposed hypothesis is that ecological change from disturbance, such as wildfire, could increase the magnitude and change the phase of net ecosystem exchange through shifts in plant community composition. Yet, little quantitative work has evaluated this potential mechanism at a regional scale. Here we investigate how fire disturbance influences landscape-level patterns of photosynthesis across western boreal North America. We use Alaska and Canadian large fire databases to identify the perimeters of wildfires, a Landsat-derived land cover time series to characterize plant functional types (PFTs), and solar-induced fluorescence (SIF) from the Orbiting Carbon Observatory-2 (OCO-2) as a proxy for photosynthesis. We analyze these datasets to characterize post-fire changes in plant succession and photosynthetic activity using a space-for-time approach. We find that increases in herbaceous and sparse vegetation, shrub, and deciduous broadleaf forest PFTs during mid-succession yield enhancements in SIF by 8-40% during June and July for 2- to 59-year stands relative to pre-fire controls. From the analysis of post-fire land cover changes within individual ecoregions and modeling, we identify two mechanisms by which fires contribute to long-term trends in SIF. First, increases in annual burning are shifting the stand age distribution, leading to increases in the abundance of shrubs and deciduous broadleaf forests that have considerably higher SIF during early- and mid-summer. Second, fire appears to facilitate a long-term shift from evergreen conifer to broadleaf deciduous forest in the Boreal Plain ecoregion. These findings suggest that increasing fire can contribute substantially to positive trends in seasonal CO2 exchange without a close coupling to long-term increases in carbon storage.

Growth, physiology, and stomatal parameters of plant polyploids grown under ice age, present-day, and future CO2 concentrations.

Polyploidy plays an important role in plant evolution, but knowledge of its eco-physiological consequences, such as of the putatively enlarged stomata of polyploid plants, remains limited. Enlarged stomata should disadvantage polyploids at low CO2 concentrations (namely during the Quaternary glacial periods) because larger stomata are viewed as less effective at CO2 uptake. We observed the growth, physiology, and epidermal cell features of 15 diploids and their polyploid relatives cultivated under glacial, present-day, and potential future atmospheric CO2 concentrations (200, 400, and 800 ppm respectively). We demonstrated some well-known polyploidy effects, such as faster growth and larger leaves, seeds, stomata, and other epidermal cells. The stomata of polyploids, however, tended to be more elongated than those of diploids, and contrary to common belief, they had no negative effect on the CO2 uptake capacity of polyploids. Moreover, polyploids grew comparatively better than diploids even at low, glacial CO2 concentrations. Higher polyploids with large genomes also showed increased operational stomatal conductance and consequently, a lower water-use efficiency. Our results point to a possible decrease in growth superiority of polyploids over diploids in a current and future high CO2 climatic scenarios, as well as the possible water and/or nutrient dependency of higher polyploids.

Natural ocean iron fertilization and climate variability over geological periods.

Marine primary producers are largely dependent on and shape the Earth's climate, although their relationship with climate varies over space and time. The growth of phytoplankton and associated marine primary productivity in most of the modern global ocean is limited by the supply of nutrients, including the micronutrient iron. The addition of iron via episodic and frequent events drives the biological carbon pump and promotes the sequestration of atmospheric carbon dioxide (CO2 ) into the ocean. However, the dependence between iron and marine primary producers adaptively changes over different geological periods due to the variation in global climate and environment. In this review, we examined the role and importance of iron in modulating marine primary production during some specific geological periods, that is, the Great Oxidation Event (GOE) during the Huronian glaciation, the Snowball Earth Event during the Cryogenian, the glacial-interglacial cycles during the Pleistocene, and the period from the last glacial maximum to the late Holocene. Only the change trend of iron bioavailability and climate in the glacial-interglacial cycles is consistent with the Iron Hypothesis. During the GOE and the Snowball Earth periods, although the bioavailability of iron in the ocean and the climate changed dramatically, the changing trend of many factors contradicted the Iron Hypothesis. By detangling the relationship among marine primary productivity, iron availability and oceanic environments in different geological periods, this review can offer some new insights for evaluating the impact of ocean iron fertilization on removing CO2 from the atmosphere and regulating the climate.

The Impact of Elevated Atmospheric Carbon Dioxide Exposure on Magic Tomatoes' Nutrition-Health Properties.

The release of carbon dioxide (CO2) into the atmosphere has accelerated during the last two decades. Elevated atmospheric CO2 concentration (eCO2) is known as an agent that improves plant photosynthesis. However, eCO2 was also correlated with alterations in the macronutrient and micronutrient compositions of various dietary crops. In order to explore the effect of eCO2 on the nutritional and health properties of tomatoes, three parental lines of the Magic population, which includes a large part of the genetic diversity present in large fruit varieties, were used as models. The plants were grown in growth chambers under ambient (400 ppm) or eCO2 (900 ppm) conditions. The macronutrient and micronutrient contents were measured. The anti-oxidant and anti-inflammatory bioactivities were assessed in vitro on activated macrophages. These analyses highlighted that the carbohydrate content was not affected by the eCO2, whereas the protein, carotenoid, lycopene, and mineral contents decreased. Regarding the anti-oxidant properties, no influence of eCO2 exposure was observed. Similarly, the anti-inflammatory properties were not affected by the eCO2. These data are in contrast with previous studies conducted on different plant species or accessions, indicating that the effect of eCO2 on crops' nutrition and health properties is based on complex mechanisms in which growth conditions and genetic backgrounds play a central role.

The Effects of High CO2 and Strigolactones on Shoot Branching and Aphid-Plant Compatibility Control in Pea.

Elevated atmospheric CO2 concentrations (eCO2) regulate plant architecture and susceptibility to insects. We explored the mechanisms underpinning these responses in wild type (WT) peas and mutants defective in either strigolactone (SL) synthesis or signaling. All genotypes had increased shoot height and branching, dry weights and carbohydrate levels under eCO2, demonstrating that SLs are not required for shoot acclimation to eCO2. Since shoot levels of jasmonic acid (JA) and salicylic acid (SA) tended to be lower in SL signaling mutants than the WT under ambient conditions, we compared pea aphid performance on these lines under both CO2 conditions. Aphid fecundity was increased in the SL mutants compared to the WT under both ambient and eCO2 conditions. Aphid infestation significantly decreased levels of JA, isopentenyladenine, trans-zeatin and gibberellin A4 and increased ethylene precursor ACC, gibberellin A1, gibberellic acid (GA3) and SA accumulation in all lines. However, GA3 levels were increased less in the SL signaling mutants than the WT. These studies provide new insights into phytohormone responses in this specific aphid/host interaction and suggest that SLs and gibberellins are part of the network of phytohormones that participate in host susceptibility.

Effect of Elevated Carbon Dioxide Exposure on Nutrition-Health Properties of Micro-Tom Tomatoes.

(1) Background: The anthropogenically induced rise in atmospheric carbon dioxide (CO2) and associated climate change are considered a potential threat to human nutrition. Indeed, an elevated CO2 concentration was associated with significant alterations in macronutrient and micronutrient content in various dietary crops. (2) Method: In order to explore the impact of elevated CO2 on the nutritional-health properties of tomato, we used the dwarf tomato variety Micro-Tom plant model. Micro-Toms were grown in culture chambers under 400 ppm (ambient) or 900 ppm (elevated) carbon dioxide. Macronutrients, carotenoids, and mineral contents were analyzed. Biological anti-oxidant and anti-inflammatory bioactivities were assessed in vitro on activated macrophages. (3) Results: Micro-Tom exposure to 900 ppm carbon dioxide was associated with an increased carbohydrate content whereas protein, minerals, and total carotenoids content were decreased. These modifications of composition were associated with an altered bioactivity profile. Indeed, antioxidant anti-inflammatory potential were altered by 900 ppm CO2 exposure. (4) Conclusions: Taken together, our results suggest that (i) the Micro-Tom is a laboratory model of interest to study elevated CO2 effects on crops and (ii) exposure to 900 ppm CO2 led to the decrease of nutritional potential and an increase of health beneficial properties of tomatoes for human health.

CO2 studies remain key to understanding a future world.

Contents 34 I. 34 II. 36 III. 37 IV. 37 V. 38 38 References 38 SUMMARY: Characterizing plant responses to past, present and future changes in atmospheric carbon dioxide concentration ([CO2 ]) is critical for understanding and predicting the consequences of global change over evolutionary and ecological timescales. Previous CO2 studies have provided great insights into the effects of rising [CO2 ] on leaf-level gas exchange, carbohydrate dynamics and plant growth. However, scaling CO2 effects across biological levels, especially in field settings, has proved challenging. Moreover, many questions remain about the fundamental molecular mechanisms driving plant responses to [CO2 ] and other global change factors. Here we discuss three examples of topics in which significant questions in CO2 research remain unresolved: (1) mechanisms of CO2 effects on plant developmental transitions; (2) implications of rising [CO2 ] for integrated plant-water dynamics and drought tolerance; and (3) CO2 effects on symbiotic interactions and eco-evolutionary feedbacks. Addressing these and other key questions in CO2 research will require collaborations across scientific disciplines and new approaches that link molecular mechanisms to complex physiological and ecological interactions across spatiotemporal scales.

Climate trends account for stalled wheat yields in Australia since 1990.

Global food security requires that grain yields continue to increase to 2050, yet yields have stalled in many developed countries. This disturbing trend has so far been only partially explained. Here, we show that wheat yields in Australia have stalled since 1990 and investigate the extent to which climate trends account for this observation. Based on simulation of 50 sites with quality weather data, that are representative of the agro-ecological zones and of soil types in the grain zone, we show that water-limited yield potential declined by 27% over a 26 year period from 1990 to 2015. We attribute this decline to reduced rainfall and to rising temperatures while the positive effect of elevated atmospheric CO2 concentrations prevented a further 4% loss relative to 1990 yields. Closer investigation of three sites revealed the nature of the simulated response of water-limited yield to water availability, water stress and maximum temperatures. At all three sites, maximum temperature hastened time from sowing to flowering and to maturity and reduced grain number per m2 and average weight per grain. This 27% climate-driven decline in water-limited yield is not fully expressed in actual national yields. This is due to an unprecedented rate of technology-driven gains closing the gap between actual and water-limited potential yields by 25 kg ha-1  yr-1 enabling relative yields to increase from 39% in 1990 to 55% in 2015. It remains to be seen whether technology can continue to maintain current yields, let alone increase them to those required by 2050.

Inter-annual changes in detritus-based food chains can enhance plant growth response to elevated atmospheric CO2.

Elevated atmospheric CO2 generally enhances plant growth, but the magnitude of the effects depend, in part, on nutrient availability and plant photosynthetic pathway. Due to their pivotal role in nutrient cycling, changes in abundance of detritivores could influence the effects of elevated atmospheric CO2 on essential ecosystem processes, such as decomposition and primary production. We conducted a field survey and a microcosm experiment to test the influence of changes in detritus-based food chains on litter mass loss and plant growth response to elevated atmospheric CO2 using two wetland plants: a C3 sedge (Scirpus olneyi) and a C4 grass (Spartina patens). Our field study revealed that organism's sensitivity to climate increased with trophic level resulting in strong inter-annual variation in detritus-based food chain length. Our microcosm experiment demonstrated that increased detritivore abundance could not only enhance decomposition rates, but also enhance plant growth of S. olneyi in elevated atmospheric CO2 conditions. In contrast, we found no evidence that changes in the detritus-based food chains influenced the growth of S. patens. Considered together, these results emphasize the importance of approaches that unite traditionally subdivided food web compartments and plant physiological processes to understand inter-annual variation in plant production response to elevated atmospheric CO2.

Mathematical modeling of vehicle carbon dioxide emissions.

The demand for transportation, driven by an increasing global population, is continuously rising. This has led to a higher number of vehicles on the road and an increased reliance on fossil fuels. Consequently, the rise in atmospheric carbon dioxide (CO2) levels has contributed to global warming. Therefore, it is important to consider sustainable transportation practices to meet climate change mitigation targets. In this research paper, a non-linear mathematical model is developed to study the dynamics of atmospheric CO2 concentration in relation to human population, economic activities, forest biomass, and vehicle population. The developed model is analyzed qualitatively to understand the long-term behavior of the system's dynamics. Model parameters are fitted to actual data of world population, human economic activities, atmospheric CO2, forest biomass, and vehicle population. It is shown that increased vehicular CO2 emissions have a potential contribution to the increase in atmospheric CO2 and the decline of human population. Numerical simulations are carried out to verify the analytical findings and we performed global sensitivity analysis to explore the impacts of different sensitive parameters on the CO2 dynamics.

Atmospheric CO2 and 14CO2 observations at the northern foot of the Qinling Mountains in China: Temporal characteristics and source quantification.

A two-year (March 2021 to February 2023) continuous atmospheric CO2 and a one-year regular atmospheric 14CO2 measurement records were measured at the northern foot of the Qinling Mountains in Xi'an, China, aiming to study the temporal characteristics of atmospheric CO2 and the contributions from the sources of fossil fuel CO2 (CO2ff) and biological CO2 (CO2bio) fluxes. The two-year mean CO2 mole fraction was 442.2 ± 16.3 ppm, with a yearly increase of 4.7 ppm (i.e., 1.1 %) during the two-year observations. Seasonal CO2 mole fractions were the highest in winter (452.1 ± 17.7 ppm) and the lowest in summer (433.5 ± 13.3 ppm), with the monthly CO2 levels peaking in January and troughing in June. Diurnal CO2 levels peaked at dawn (05:00-07:00) in spring, summer and autumn, and at 10:00 in winter. 14C analysis revealed that the excess CO2 (CO2ex, atmospheric CO2 minus background CO2) at this site was mainly from CO2ff emissions (67.0 ± 26.8 %), and CO2ff mole fractions were the highest in winter (20.6 ± 17.7 ppm). Local CO enhancement above the background mole fraction (ΔCO) was significantly (r = 0.74, p < 0.05) positively correlated with CO2ff in a one-year measurement, and ΔCO:CO2ff showed a ratio of 23 ± 6 ppb/ppm during summer and winter sampling days, much lower than previous measurements and suggesting an improvement in combustion efficiency over the last decade. CO2bio mole fractions also peaked in winter (14.2 ± 9.6 ppm), apparently due to biomass combustion and the lower and more stable wintertime atmospheric boundary layer. The negative CO2bio values in summer indicated that terrestrial vegetation of the Qinling Mountains had the potential to uptake atmospheric CO2 during the corresponding sampling days. This site is most sensitive to local emissions from Xi'an and to short distance transportation from the southern Qinling Mountains through the valleys.

Century-long stomatal density record of the nitrophyte, Rubus spectabilis L., from the Pacific Northwest indicates no effect of changing atmospheric carbon dioxide but a strong response to nutrient subsidy.

Triangle Island on Canada's Pacific coast is home to a large, globally important seabird breeding colony. The shrub Salmonberry Rubus spectabilis and tussock-forming Tufted Hairgrass Deschampsia cespitosa together form ~70% of vegetation coverage and contain the vast majority (~90%) of seabird nesting burrows. Salmonberry has in recent decades greatly expanded its coverage, while that of Tufted Hairgrass has receded. Seabirds prefer not to burrow under Salmonberry, making its ongoing expansion a potential conservation issue. We investigated three hypotheses proposed to explain Salmonberry's expansion (climate change, biopedturbation, and nutrient input), using comparisons of stomatal density of Salmonberry leaves sampled from Triangle Island, other seabird colonies, other coastal locations, and from historical specimens in herbaria. Stomatal density helps regulate photosynthetic gain and control water loss, and responds to light, nutrient, carbon dioxide, and water availability. Differing patterns of stomatal density are expected among sample locations depending on which of the hypothesized factors most strongly affects Salmonberry's performance. Our data are most consistent with the nutrient input hypothesis. We discuss possible reasons why Salmonberry has expanded so recently, even though Triangle has been a large seabird colony for at least a century and likely much longer.

Will elevated atmospheric CO2 boost the growth of an invasive submerged macrophyte Cabomba caroliniana under the interference of phytoplankton?

The growth of most submerged macrophytes is likely to be limited by the availability of carbon resource, and this is especially true for the obligatory carbon dioxide (CO2) users. A mesocosm experiment was performed to investigate the physiological, photophysiological, and biochemical responses of Cabomba caroliniana, an invasive macrophyte specie in the Lake Taihu Basin, to elevated atmospheric CO2 (1000 µmol mol-1); we also examined the possible impacts of interferences derived from the phytoplankton proliferation and its concomitant disturbances on the growth of C. caroliniana. The results demonstrated that elevated atmospheric CO2 significantly enhanced the biomass, relative growth rate, and photosynthate accumulation of C. caroliniana. C. caroliniana exposed to elevated atmospheric CO2 exhibited a higher relative maximum electron transport rate and photosynthetic efficiency, compared to those exposed to ambient atmospheric CO2. However, the positive effects of elevated atmospheric CO2 on C. caroliniana were gradually compromised as time went by, and the down-regulations of the relative growth rate (RGR) and photosynthetic activity were coupled with phytoplankton proliferation under elevated atmospheric CO2. This study demonstrated that the growth of C. caroliniana under the phytoplankton interference can be greatly affected, directly and indirectly, by the increasing atmospheric CO2.

CO2 Sensing and CO2 Regulation of Stomatal Conductance: Advances and Open Questions.

Guard cells form epidermal stomatal gas-exchange valves in plants and regulate the aperture of stomatal pores in response to changes in the carbon dioxide (CO2) concentration ([CO2]) in leaves. Moreover, the development of stomata is repressed by elevated CO2 in diverse plant species. Evidence suggests that plants can sense [CO2] changes via guard cells and via mesophyll tissues in mediating stomatal movements. We review new discoveries and open questions on mechanisms mediating CO2-regulated stomatal movements and CO2 modulation of stomatal development, which together function in the CO2 regulation of stomatal conductance and gas exchange in plants. Research in this area is timely in light of the necessity of selecting and developing crop cultivars that perform better in a shifting climate.

[13 C-based Sources Partitioning of Atmospheric CO2 During Youth Olympic Games, Nanjing].

Observations of urban atmospheric CO2 molar fraction and its 13 C isotope composition (δ13 C) is of great importance to interpret the effect of anthropogenic and biologic sources on local or regional carbon cycle. High-frequency in-situ observation on atmospheric in urban airsheds was performed during Youth Olympic Games (YOG) in Nanjing. The hourly, diurnal and daily differences of CO2 concentration and its δ13 C between the period with and without temporary CO2 emission controls were compared. The results showed that short-term emission reduction measures could cause 21×10-6 decrease in atmospheric CO2 concentration in a regional and short-term scale. The reduction of coal combustion during YOG in YRD was about 5%. The overall isotopic signature of local surface sources δ13 CS in Yangtze River Delta (YRD) was determined by Miller-Tans, and the isotopic signatures of anthropogenic and natural sources in YRD were also determined based on literature investigation. According to the above results, the surface net CO2 flux, plant flux and anthropogenic flux in YRD were quantified using mass-balance equation. The CO2 emission from cement production (non-energy industrial process) was the key human factor of high atmospheric δ13 C of CO2 in YRD during summer (2.36‰). The plant effect could offset 23% to 39% anthropogenic CO2 emission in YRD during summer. In this study, we tried to provide new solution to partition carbon sources in urban areas by combining top-down atmospheric observation and traditional IPCC's emission inventory.

Gas valves, forests and global change: a commentary on Jarvis (1976) 'The interpretation of the variations in leaf water potential and stomatal conductance found in canopies in the field'.

Microscopic turgor-operated gas valves on leaf surfaces-stomata-facilitate gas exchange between the plant and the atmosphere, and respond to multiple environmental and endogenous cues. Collectively, stomatal activities affect everything from the productivity of forests, grasslands and crops to biophysical feedbacks between land surface vegetation and climate. In 1976, plant physiologist Paul Jarvis reported an empirical model describing stomatal responses to key environmental and plant conditions that predicted the flux of water vapour from leaves into the surrounding atmosphere. Subsequent theoretical advances, building on this earlier approach, established the current paradigm for capturing the physiological behaviour of stomata that became incorporated into sophisticated models of land carbon cycling. However, these models struggle to accurately predict observed trends in the physiological responses of Northern Hemisphere forests to recent atmospheric CO2 increases, highlighting the need for improved representation of the role of stomata in regulating forest-climate interactions. Bridging this gap between observations and theory as atmospheric CO2 rises and climate change accelerates creates challenging opportunities for the next generation of physiologists to advance planetary ecology and climate science. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society.

Photosynthetic responses to CO2 enrichment of four hardwood species in a forest understory.

We compared the CO2- and light-dependence of photosynthesis of four tree species (Acer rubrum, Carya glabra, Cercis canadensis, Liquidambar styraciflua) growing in the understory of a loblolly pine plantation under ambient or ambient plus 200 µl l-1 CO2. Naturally-established saplings were fumigated with a free-air CO2 enrichment system. Light-saturated photosynthetic rates were 159-190% greater for Ce. canadensis saplings grown and measured under elevated CO2. This species had the greatest CO2 stimulation of photosynthesis. Photosynthetic rates were only 59% greater for A. rubrum saplings under CO2 enrichment and Ca. glabra and L. styraciflua had intermediate responses. Elevated CO2 stimulated light-saturated photosynthesis more than the apparent quantum yield. The maximum rate of carboxylation of ribulose-1,5-bisphosphate carboxylase, estimated from gas-exchange measurements, was not consistently affected by growth in elevated CO2. However, the maximum electron transport rate estimated from gas- exchange measurements and from chlorophyll fluorescence, when averaged across species and dates, was approximately 10% higher for saplings in elevated CO2. The proportionately greater stimulation of light-saturated photosynthesis than the apparent quantum yield and elevated rates of maximum electron transport suggests that saplings growing under elevated CO2 make more efficient use of sunflecks. The stimulation of light-saturated photosynthesis by CO2 did not appear to correlate with shade-tolerance ranking of the individual species. However, the species with the greatest enhancement of photosynthesis, Ce. canadensis and L. styraciflua, also invested the greatest proportion of soluble protein in Rubisco. Environmental and endogenous factors affecting N partitioning may partially explain interspecific variation in the photosynthetic response to elevated CO2.

High-resolution alkenone palaeobarometry indicates relatively stable pCO(2) during the Pliocene (3.3-2.8 Ma).

Temperature reconstructions indicate that the Pliocene was approximately 3(°)C warmer globally than today, and several recent reconstructions of Pliocene atmospheric CO2 indicate that it was above pre-industrial levels and similar to those likely to be seen this century. However, many of these reconstructions have been of relatively low temporal resolution, meaning that these records may have failed to capture variations associated with the 41 kyr glacial-interglacial cycles thought to have operated in the Pliocene. Here we present a new, high temporal resolution alkenone carbon isotope-based record of pCO2 spanning 3.3-2.8 Ma from Ocean Drilling Program Site 999. Our record is of high enough resolution (approx. 19 kyr) to resolve glacial-interglacial changes beyond the intrinsic uncertainty of the proxy method. The record suggests that Pliocene CO2 levels were relatively stable, exhibiting variation less than 55 ppm. We perform sensitivity studies to investigate the possible effect of changing sea surface temperature (SST), which highlights the importance of accurate and precise SST reconstructions for alkenone palaeobarometry, but demonstrate that these uncertainties do not affect our conclusions of relatively stable pCO2 levels during this interval.