Comparing Methodologies for Stomatal Analyses in the Context of Elevated Modern CO2.

Leaf stomata facilitate the exchange of water and CO2 during photosynthetic gas exchange. The shape, size, and density of leaf pores have not been constant over geologic time, and each morphological trait has potentially been impacted by changing environmental and climatic conditions, especially by changes in the concentration of atmospheric carbon dioxide. As such, stomatal parameters have been used in simple regressions to reconstruct ancient carbon dioxide, as well as incorporated into more complex gas-exchange models that also leverage plant carbon isotope ecology. Most of these proxy relationships are measured on chemically cleared leaves, although newer techniques such as creating stomatal impressions are being increasingly employed. Additionally, many of the proxy relationships use angiosperms with broad leaves, which have been increasingly abundant in the last 130 million years but are absent from the fossil record before this. We focus on the methodology to define stomatal parameters for paleo-CO2 studies using two separate methodologies (one corrosive, one non-destructive) to prepare leaves on both scale- and broad-leaves collected from herbaria with known global atmospheric CO2 levels. We find that the corrosive and non-corrosive methodologies give similar values for stomatal density, but that measurements of stomatal sizes, particularly guard cell width (GCW), for the two methodologies are not comparable. Using those measurements to reconstruct CO2 via the gas exchange model, we found that reconstructed CO2 based on stomatal impressions (due to inaccurate measurements in GCW) far exceeded measured CO2 for modern plants. This bias was observed in both coniferous (scale-shaped) and angiosperm (broad) leaves. Thus, we advise that applications of gas exchange models use cleared leaves rather than impressions.

Isotopic clumping in wood as a proxy for photorespiration in trees.

Photorespiration can limit gross primary productivity in terrestrial plants. The rate of photorespiration relative to carbon fixation increases with temperature and decreases with atmospheric [CO2]. However, the extent to which this rate varies in the environment is unclear. Here, we introduce a proxy for relative photorespiration rate based on the clumped isotopic composition of methoxyl groups (R-O-CH3) in wood. Most methoxyl C-H bonds are formed either during photorespiration or the Calvin cycle and thus their isotopic composition may be sensitive to the mixing ratio of these pathways. In water-replete growing conditions, we find that the abundance of the clumped isotopologue 13CH2D correlates with temperature (18-28 °C) and atmospheric [CO2] (280-1000 ppm), consistent with a common dependence on relative photorespiration rate. When applied to a global dataset of wood, we observe global trends of isotopic clumping with climate and water availability. Clumped isotopic compositions are similar across environments with temperatures below ~18 °C. Above ~18 °C, clumped isotopic compositions in water-limited and water-replete trees increasingly diverge. We propose that trees from hotter climates photorespire substantially more than trees from cooler climates. How increased photorespiration is managed depends on water availability: water-replete trees export more photorespiratory metabolites to lignin whereas water-limited trees either export fewer overall or direct more to other sinks that mitigate water stress. These disparate trends indicate contrasting responses of photorespiration rate (and thus gross primary productivity) to a future high-[CO2] world. This work enables reconstructing photorespiration rates in the geologic past using fossil wood.

C3 plant carbon isotope discrimination does not respond to CO2 concentration on decadal to centennial timescales.

Plant carbon isotope discrimination is complex, and could be driven by climate, evolution and/or edaphic factors. We tested the climate drivers of carbon isotope discrimination in modern and historical plant chemistry, and focus in particular on the relationship between rising [CO2 ] over Industrialization and carbon isotope discrimination. We generated temporal records of plant carbon isotopes from museum specimens collected over a climo-sequence to test plant responses to climate and atmospheric change over the past 200 yr (including Pinus strobus, Platycladus orientalis, Populus tremuloides, Thuja koraiensis, Thuja occidentalis, Thuja plicata, Thuja standishii and Thuja sutchuenensis). We aggregated our results with a meta-analysis of a wide range of C3 plants to make a comprehensive study of the distribution of carbon isotope discrimination and values among different plant types. We show that climate variables (e.g. mean annual precipitation, temperature and, key to this study, CO2 in the atmosphere) do not drive carbon isotope discrimination. Plant isotope discrimination is intrinsic to each taxon, and could link phylogenetic relationships and adaptation to climate quantitatively and over ecological to geological time scales.

Rapid response to anthropogenic climate change by Thuja occidentalis: implications for past climate reconstructions and future climate predictions.

Carbon isotope values of leaves (δ13Cleaf) from meta-analyses and growth chamber studies of C3 plants have been used to propose generalized relationships between δ13Cleaf and climate variables such as mean annual precipitation (MAP), atmospheric concentration of carbon dioxide ([CO2]), and other climate variables. These generalized relationships are frequently applied to the fossil record to create paleoclimate reconstructions. Although plant evolution influences biochemistry and response to environmental stress, few studies have assessed species-specific carbon assimilation as it relates to climate outside of a laboratory. We measured δ13Cleaf values and C:N ratios of a wide-ranging evergreen conifer with a long fossil record, Thuja occidentalis (Cupressaceae) collected 1804-2017, in order to maximize potential paleo-applications of our focal species. This high-resolution record represents a natural experiment from pre-Industrial to Industrial times, which spans a range of geologically meaningful [CO2] and δ13Catm values. Δleaf values (carbon isotope discrimination between δ13Catm and δ13Cleaf) remain constant across climate conditions, indicating limited response to environmental stress. Only δ13Cleaf and δ13Catm values showed a strong relationship (linear), thus, δ13Cleaf is an excellent record of carbon isotopic changes in the atmosphere during Industrialization. In contrast with previous free-air concentration enrichment experiments, no relationship was found between C:N ratios and increasing [CO2]. Simultaneously static C:N ratios and Δleaf in light of increasing CO2 highlights plants' inability to match rapid climate change with increased carbon assimilation as previously expected; Δleaf values are not reliable tools to reconstruct MAP and [CO2], and δ13Cleaf values only decrease with [CO2] in line with atmospheric carbon isotope changes.