CuticleTrace: A toolkit for capturing cell outlines from leaf cuticle with implications for paleoecology and paleoclimatology.

Premise: Leaf epidermal cell morphology is closely tied to the evolutionary history of plants and their growth environments and is therefore of interest to many plant biologists. However, cell measurement can be time consuming and restrictive with current methods. CuticleTrace is a suite of Fiji and R-based functions that streamlines and automates the segmentation and measurement of epidermal pavement cells across a wide range of cell morphologies and image qualities. Methods and Results: We evaluated CuticleTrace-generated measurements against those from alternate automated methods and expert and undergraduate hand tracings across a taxonomically diverse 50-image data set of variable image qualities. We observed ~93% statistical agreement between CuticleTrace and expert hand-traced measurements, outperforming alternate methods. Conclusions: CuticleTrace is a broadly applicable, modular, and customizable tool that integrates data visualization and cell shape measurement with image segmentation, lowering the barrier to high-throughput studies of epidermal morphology by vastly decreasing the labor investment required to generate high-quality cell shape data sets.

Toward a Cenozoic history of atmospheric CO2.

The geological record encodes the relationship between climate and atmospheric carbon dioxide (CO2) over long and short timescales, as well as potential drivers of evolutionary transitions. However, reconstructing CO2 beyond direct measurements requires the use of paleoproxies and herein lies the challenge, as proxies differ in their assumptions, degree of understanding, and even reconstructed values. In this study, we critically evaluated, categorized, and integrated available proxies to create a high-fidelity and transparently constructed atmospheric CO2 record spanning the past 66 million years. This newly constructed record provides clearer evidence for higher Earth system sensitivity in the past and for the role of CO2 thresholds in biological and cryosphere evolution.

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.

No Consistent Shift in Leaf Dry Mass per Area Across the Cretaceous-Paleogene Boundary.

The Chicxulub bolide impact has been linked to a mass extinction of plants at the Cretaceous-Paleogene boundary (KPB; ∼66 Ma), but how this extinction affected plant ecological strategies remains understudied. Previous work in the Williston Basin, North Dakota, indicates that plants pursuing strategies with a slow return-on-investment of nutrients abruptly vanished after the KPB, consistent with a hypothesis of selection against evergreen species during the globally cold and dark impact winter that followed the bolide impact. To test whether this was a widespread pattern we studied 1,303 fossil leaves from KPB-spanning sediments in the Denver Basin, Colorado. We used the relationship between petiole width and leaf mass to estimate leaf dry mass per area (LMA), a leaf functional trait negatively correlated with rate of return-on-investment. We found no evidence for a shift in this leaf-economic trait across the KPB: LMA remained consistent in both its median and overall distribution from approximately 67 to 65 Ma. However, we did find spatio-temporal patterns in LMA, where fossil localities with low LMA occurred more frequently near the western margin of the basin. These western margin localities are proximal to the Colorado Front Range of the Rocky Mountains, where an orographically driven high precipitation regime is thought to have developed during the early Paleocene. Among these western Denver Basin localities, LMA and estimated mean annual precipitation were inversely correlated, a pattern consistent with observations of both fossil and extant plants. In the Denver Basin, local environmental conditions over time appeared to play a larger role in determining viable leaf-economic strategies than any potential global signal associated with the Chicxulub bolide impact.

Quantifying the effect of shade on cuticle morphology and carbon isotopes of sycamores: present and past.

PREMISE: Reconstructing the light environment and architecture of the plant canopy from the fossil record requires the use of proxies, such as those derived from cell wall undulation, cell size, and carbon isotopes. All approaches assume that plant taxa will respond predictably to changes in light environments. However, most species-level studies looking at cell wall undulation only consider "sun" or "shade" leaves; therefore, we need a fully quantitative taxon-specific method. METHODS: We quantified the response of cell wall undulation, cell size, and carbon isotopes of Platanus occidentalis using two experimental setups: (1) two growth chambers at low and high light and (2) a series of outdoor growth experiments using green and black shade cloth at different densities. We then developed and applied a proxy for daily light integral (DLI) to fossil Platanites leaves from two early Paleocene floras from the San Juan Basin in New Mexico. RESULTS: All traits responded to light environment. Cell wall undulation was the most useful trait for reconstructing DLI in the geological record. Median reconstructed DLI from early Paleocene leaves was ~44 mol m-2 d-1 , with values from 28 to 54 mol m-2 d-1 . CONCLUSIONS: Cell wall undulation of P. occidentalis is a robust, quantifiable measurement of light environment that can be used to reconstruct the paleo-light environment from fossil leaves. The distribution of high DLI values from fossil leaves may provide information on canopy architecture; indicating that either (1) most of the canopy mass is within the upper portion of the crown or (2) leaves exposed to more sunlight are preferentially preserved.

Fossil Atmospheres: a case study of citizen science in question-driven palaeontological research.

Palaeontologists increasingly use large datasets of observations collected from museum specimens to address broad-scale questions about evolution and ecology on geological timescales. One such question is whether information from fossil organisms can be used as a robust proxy for atmospheric carbon dioxide through time. Here, we present the citizen science branch of 'Fossil Atmospheres', a project designed to refine stomatal index of Ginkgo leaves as a palaeo-CO2 proxy by involving citizen scientists in data collection through the Zooniverse website. Citizen science helped to overcome a barrier presented by the time taken to count cells in Ginkgo samples; however, a new set of challenges arose as a result. A beta-testing phase with Zooniverse volunteers provided an opportunity to improve instructions to ensure high fidelity data. Exploration of citizen scientists' estimates shows that volunteer counts of stomata are accurate with respect to counts made by the project's lead scientist. However, counts of epidermal cells have a wide range, and mean values tend to underestimate expert counts. We demonstrate a variety of approaches to reducing the inaccuracy in the calculated stomatal index that this variation causes. Zooniverse serves as an ideal tool for collection of palaeontological data where the distribution of fossils would be impossible, but where specimens can be easily imaged. Such an approach facilitates the collection of a large palaeontological dataset, as well as providing an opportunity for citizens to engage with climate research.This article is part of the theme issue 'Biological collections for understanding biodiversity in the Anthropocene'.

Cuticle and subsurface ornamentation of intact plant leaf epidermis under confocal and superresolution microscopy.

Plant cuticle micromorphology is an invaluable tool in modern ecology and paleoecology. It has expanded our knowledge of systematic relationships among diverse plant groups and can be used to identify fossil plants. Furthermore, fossil plant leaf micromorphology is used for reconstructing past environments, most notably for estimating atmospheric CO2 concentration. Here we outline a new protocol for imaging plant cuticle for archival and paleoecological applications. Traditionally, both modern reference and fossil samples undergo maceration with subsequent imaging via environmental SEM, widefield fluorescence, or light microscopy. In this paper, we demonstrate the capabilities of alternative preparation and imaging methods using confocal and superresolution microscopy with intact leaf samples. This method produces detailed three-dimensional images of surficial and subsurface structures of the intact leaf. Multiple layers are captured simultaneously, which previously required independent maceration and microtome steps. We compared clearing agents (chloral hydrate, KOH, and Visikol); mounting media (Eukitt and Hoyer's); fluorescent stains (periodic acid Schiff, propidium iodide); and confocal vs. superresolution microscopes. We conclude that Eukitt is the best medium for long-term preservation and imaging. Because of nontoxicity and ease of procurement, Visikol made for the best clearing agent. Staining improves contrast and under most circumstances PAS provided the clearest images. Supperresolution produced higher clarity images than traditional confocal, but the information gained was minimal. This new protocol provides the botanical and paleobotanical community an alternative to traditional techniques. Our proposed workflow has the net benefit of being more efficient than traditional methods, which only capture the surface of the plant epidermis. Microsc. Res. Tech. 81:129-140, 2018. © 2016 Wiley Periodicals, Inc.

New methods reveal oldest known fossil epiphyllous moss: Bryiidites utahensis gen. et sp. nov. (Bryidae).

PREMISE OF THE STUDY: Epiphyllous bryophytes are a highly characteristic feature of many humid tropical forest ecosystems. In contrast to the extensive fossil record for the leaves of their host plants, the record is virtually nonexistent for the epiphylls themselves, despite a fossil record for mosses that begins in the Middle Carboniferous Period, 330 million years ago. METHODS: Epifluorescence optical microscopy, scanning electron microscopy, and atomic force microscopy were employed to investigate an intimate association between a newly discovered epiphyllous moss and a Lauraceae plant host from the middle Cretaceous. KEY RESULTS: We describe the oldest fossil specimen of an epiphyllous moss, Bryiidites utahensis gen. et sp. nov., identified from an individual specimen only 450 µm long, situated on an approximately one millimeter square fossil leaf fragment. The moss epiphyll is exquisitely preserved as germinating spores and short-celled protonemata with transverse and oblique cross-walls closely matching those of extant epiphyllous mosses on the surface of the plant-leaf hosts. CONCLUSIONS: The extension of the epiphyll record back to the middle Cretaceous provides fossil evidence for the appearance of epiphyllous mosses during the diversification of flowering plants, at least 95 million years ago. It also provides substantive evidence for a tropical maritime climate in central North America during the middle Cretaceous.