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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.

Premise Mechanistic models using stomatal traits and leaf carbon isotope ratios to reconstruct atmospheric carbon dioxide (CO2) concentrations (ca) are important to understand the Phanerozoic paleoclimate. However, methods for preparing leaf cuticles to measure stomatal traits have not been standardized. Methods Three people measured the stomatal density and index, guard cell length, guard cell pair width, and pore length of leaves from the same Ginkgo biloba, Quercus alba, and Zingiber mioga leaves growing at known CO2 levels using four preparation methods: fluorescence on cleared leaves, nail polish, dental putty on fresh leaves, and dental putty on dried leaves. Results There are significant differences between trait measurements from each method. Modeled ca calculations are less sensitive to method than individual traits; however, the choice of assumed carbon isotope fractionation also impacted the accuracy of the results. Discussion We show that there is not a significant difference between ca estimates made using any of the four methods. Further study is needed on the fractionation due to carboxylation of ribulose bisphosphate (RuBP) in individual plant species before use as a paleo‐CO2 barometer and to refine estimates based upon widely applied taxa (e.g., Ginkgo). Finally, we recommend that morphological measurements be made by multiple observers to reduce the effect of individual observational biases.

The Zingiberales are an iconic order of monocotyledonous plants comprising eight families with distinctive and diverse floral morphologies and representing an important ecological element of tropical and subtropical forests. While the eight families are demonstrated to be monophyletic, phylogenetic relationships among these families remain unresolved. Neither combined morphological and molecular studies nor recent attempts to resolve family relationships using sequence data from whole plastomes has resulted in a well-supported, family-level phylogenetic hypothesis of relationships. Here we approach this challenge by leveraging the complete genome of one member of the order, Musa acuminata , together with transcriptome information from each of the other seven families to design a set of nuclear loci that can be enriched from highly divergent taxa with a single array-based capture of indexed genomic DNA. A total of 494 exons from 418 nuclear genes were captured for 53 ingroup taxa. The entire plastid genome was also captured for the same 53 taxa. Of the total genes captured, 308 nuclear and 68 plastid genes were used for phylogenetic estimation. The concatenated plastid and nuclear dataset supports the position of Musaceae as sister to the remaining seven families. Moreover, the combined dataset recovers known intra- and inter-family phylogenetic relationships with generally high bootstrap support. This is a flexible and cost effective method that gives the broader plant biology community a tool for generating phylogenomic scale sequence data in non-model systems at varying evolutionary depths.

• 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.

While more commonly applied in zoology, synchrotron radiation X-ray tomographic microscopy (SRXTM) is well-suited to nondestructive study of the morphology and anatomy of both fossil and modern plants. SRXTM uses hard X-rays and a monochromatic light source to provide high-resolution data with little beam-hardening, resulting in slice data with clear boundaries between materials. Anatomy is readily visualized, including various planes of section from a single specimen, as clear as in traditional histological sectioning at low magnifications. Thus, digital sectioning of rare or difficult material is possible. Differential X-ray attenuation allows visualization of different layers or chemistries to enable virtual 3-dimensional (3D) dissections of material. Virtual potential fossils can be visualized and digital tissue removal reveals cryptic underlying morphology. This is essential for fossil identification and for comparisons between assemblages where fossils are preserved by different means. SRXTM is a powerful approach for botanical studies using morphology and anatomy. The ability to gain search images in both 2D and 3D for potential fossils gives paleobotanists a tool—virtual taphonomy—to improve our understanding of plant evolution and paleobiogeography.

Angiosperm-dominated floras of the Late Cretaceous are essential for understanding the evolutionary, ecological, and geographic radiation of flowering plants. The Late Cretaceous–early Paleogene Deccan Intertrappean Beds of India contain angiosperm-dominated plant fossil assemblages known from multiple localities in central India. Numerous monocots have been documented from these assemblages, providing a window into an important but poorly understood time in their diversification. One component of the Deccan monocot diversity is the genus Viracarpon , known from anatomically preserved infructescences. Viracarpon was first collected over a century ago and has been the subject of numerous studies. However, resolution of its three-dimensional (3D) morphology and anatomy, as well as its taxonomic affinities, has remained elusive. In this study we investigated the morphology and taxonomy of genus Viracarpon , combining traditional paleobotanical techniques and X-ray micro-computed tomography (μCT). Re-examination of type and figured specimens, 3D reconstructions of fruits, and characterization of structures in multiple planes of section using μCT data allowed us to resolve conflicting interpretations of fruit morphology and identify additional characters useful in refining potential taxonomic affinities. Among the four Viracarpon species previously recognized, we consider two to be valid ( Viracarpon hexaspermum and Viracarpon elongatum ), and the other two to be synonyms of these. Furthermore, we found that permineralized infructescences of Coahuilocarpon phytolaccoides from the late Campanian of Mexico correspond closely in morphology to V. hexaspermum . We argue that Viracarpon and Coahuilocarpon are congeneric and provide the new combination, Viracarpon phytolaccoides (Cevallos-Ferriz, Estrada-Ruiz & Perez-Hernandez) Matsunaga, S.Y. Smith, & Manchester comb. nov. The significant geographic disjunction between these two occurrences indicates that the genus Viracarpon was widespread and may be present in other Late Cretaceous assemblages. Viracarpon exhibits character combinations not present in any extant taxa and its affinities remain unresolved, possibly representing an extinct member of Alismatales. The character mosaic observed in Viracarpon and the broad distribution of the genus provide new data relevant to understanding early monocot evolution and suggest that the (thus far) largely invisible Late Cretaceous monocot diversification was characterized by enigmatic and/or stem taxa.

Premise of research. Reproductive structures of modern genera of heterosporous water ferns (Marsileaceae and Salviniaceae) are widespread and abundant in plant mesofossil assemblages from the Paleogene. For Salviniaceae, whole fertile fossil plants give a good understanding of morphology. These fossils can be applied in paleoenvironmental analysis and to study water fern origin, evolution, and diversification. Methodology. New specimens were examined by SEM and TEM. Synchrotron x-ray tomographic microscopy (SRXTM) is evaluated as a nondestructive tool for investigatingAzollaLam. morphology. Pivotal results. Azolla anglicaMartin andSalvinia cobhamiiMartin (earliest Eocene, United Kingdom) are fully characterized using SEM and TEM. SRXTM enables digital rendering of the float system inAzolla, but individual floats are difficult to distinguish. Modern water fern genera characterize the Paleogene, but extinct sister taxa characterize the Cretaceous. Literature review documents that water ferns are intolerant of salinity over 5 psu. Conclusions. The oldest fully documentedSalviniaSéguier sori and spores occur in earliest Eocene deposits at Cobham, United Kingdom, probably linked to warm climates. An unusual co-occurrence ofSalviniawithAzollais preserved at this site. TheAzollaspecies differs from those present in the same region during other Eocene warm-climate intervals. SRXTM offers potential to retrieve taxonomically useful information on internal structures ofAzolla. There is a major turnover in water ferns (dominantly extinct to almost entirely modern genera) across the Cretaceous-Paleogene transition. The utility of water ferns as indicator taxa is exemplified by recognition of freshwater ocean surfaces and widespread continental wetlands during the latest Early to earliest Middle Eocene in and around the Arctic and Nordic Seas.

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 [CO 2 ] 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 C 3 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, CO 2 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.

Recent advances in microscopy, imaging, and data analyses have permitted both the greater application of quantitative methods and the collection of large data sets that can be used to investigate plant morphology. This special issue, the first for Applications in Plant Sciences, presents a collection of papers highlighting recent methods in the quantitative study of plant form. These emerging biometric and bioinformatic approaches to plant sciences are critical for better understanding how morphology relates to ecology, physiology, genotype, and evolutionary and phylogenetic history. From microscopic pollen grains and charcoal particles, to macroscopic leaves and whole root systems, the methods presented include automated classification and identification, geometric morphometrics, and skeleton networks, as well as tests of the limits of human assessment. All demonstrate a clear need for these computational and morphometric approaches in order to increase the consistency, objectivity, and throughput of plant morphological studies.

Leaf stomata facilitate the exchange of water and CO[sub.2] 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-CO[sub.2] 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 CO[sub.2] 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 CO[sub.2] via the gas exchange model, we found that reconstructed CO[sub.2] based on stomatal impressions (due to inaccurate measurements in GCW) far exceeded measured CO[sub.2] 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.