DOI: 10.5061/dryad.g1jwstqzv
title: Data from: Estimates of late Early Cretaceous atmospheric CO2 from Mongolia based on stomatal and isotopic analysis of Pseudotorellia

description: Our dataset includes 115 leaf cuticles related to two species of
 Pseudotorellia Florin from three stratigraphically similar samples at the
 Tevshiin Govi lignite mine in central Mongolia (~119.7–100.5 Ma,
 Aptian–Albian, Cretaceous). We apply a well-vetted paleo-CO2 proxy based
 on leaf gas-exchange principles (the Franks model) to those leaves for
 paleo-CO2 reconstruction, which requires leaf stomata and carbon isotope
 analysis. All cuticle measurements are summarized in this
 dataset. 
description: The leaves studied here are from the Tevshiingovi lignite mine in
 central Mongolia, approximately 220 km south-southwest of Ulaanbaatar. We
 excavated lignite blocks from three stratigraphically similar samples from
 lignite #2 along the Tevshiin Govi lignite mine wall. The blocks were
 disaggregated with detergent, 3% hydrogen peroxide, and deionized water.
 Because chemical treatments can affect δ<sup>13</sup>C
 measurements (Barral et al., 2015), we measured one leaf (cut in half)
 both with and without peroxide treatment and found an isotopic difference
 of 0.22 ‰, which propagates to a small change in estimated
 CO<sub>2</sub> (~2.6 %). The lithological column was
 illustrated using SDAR package version 0.9-55 (Ortiz and Jaramillo,
 2020). Leaves were first cut in half, with one half set
 aside for isotope analysis. The remaining half was cleared following
 standard practices (Dilcher, 1974; Wang, 2010; Liang et al., 2022).
 Fragments were soaked in deionized H<sub>2</sub>O
 (dH<sub>2</sub>O) for 1 hour. Softened leaf fragments were cut
 at the margin and then cleaned using dilute household bleach
 (<em>c.</em> 1% sodium hypochlorite solution (NaClO)). Once
 the leaves turned transparent (or light yellow), the reaction was
 immediately terminated by neutralizing with dH<sub>2</sub>O
 before being transferred to a microscope glass slide. A fine dissecting
 needle was used to separate the leaf into the upper (adaxial) and lower
 (abaxial) sides, each containing one epidermis and part of the mesophyll
 tissues. They were then gently washed with dH<sub>2</sub>O
 twice to remove loosened mesophyll tissue.  Clean
 abaxial epidermises were observed with a Leica DMLB microscope under
 transmitted light and epifluorescence with mercury light and a 420–490 nm
 filter cube. ZStack photos of four different focal planes were taken with
 a Jenoptik Gryphax digital microscope camera and then integrated into a
 single composite image. We measured stomatal density mostly at 100× (0.80
 mm<sup>2</sup> field-of-view), but occasionally at 200× (0.20
 mm<sup>2</sup> field-of-view) when lower-quality preservation
 limited large fields-of-view.  The stomata in both species appear randomly
 distributed.  For each leaf fragment, we measured 10
 guard cells for their length and width as well as the length and width of
 the bounded stomatal pore. These images were taken at 400× and measured
 using ImageJ (Rasband 1997). Both species of
 <em>Pseudotorellia</em> are hypostomatous and have sunken
 guard cells. <em>Pseudotorellia resinosa </em>possesses
 thickened subsidiary cells that partly overlap the underlying guard cells
 (Shi et al. 2018; see their Fig. 16C-E). These thickened areas are
 prominent under both transmitted light and epifluorescence, and comprise a
 smaller footprint than the underlying guard cells (see representative
 lines for guard cell length [gcl] and single guard cell width
 [sgw].  Bulk-leaf δ<sup>13</sup>C values
 were measured at the Light Stable Isotope Mass Spec Lab at the Department
 of Geological Sciences of the University of Florida using a Thermo
 Electron DeltaV Advantage isotope ratio mass spectrometer coupled with a
 ConFlo II interface linked to a Carlo Erba NA 1500 CNHS Elemental
 Analyzer. After combustion in a quartz column at 1000 °C in an oxygen-rich
 atmosphere, the sample gas was transported in a He carrier stream and
 passed through a hot reduction column (650 °C) consisting of elemental
 copper to remove oxygen. The effluent stream then passed through a
 magnesium perchlorate trap to remove water followed by a 0.7 m GC column
 at 125 °C to separate N<sub>2</sub> from
 CO<sub>2</sub>. The sample gas next passed into a ConFlo II
 interface and into the inlet of a Thermo Electron Delta V Advantage
 isotope ratio mass spectrometer running in continuous flow mode where the
 sample gas was measured relative to laboratory reference
 CO<sub>2</sub> gas. All carbon isotopic results are expressed
 in standard delta notation relative to VPDB. Instrument error across the
 multiple runs ranged from 0.01-0.19 ‰. We analyzed 36 samples in
 duplicate, with an average difference of 0.33 ‰ (0.06–1.03 ‰
 range).
description: # Data from: Estimates of late Early Cretaceous atmospheric CO2 from
 Mongolia based on stomatal and isotopic analysis of Pseudotorellia
 [https://doi.org/10.5061/dryad.g1jwstqzv](https://doi.org/10.5061/dryad.g1jwstqzv) This dataset is the Appendix S2 for a manuscript ("AJB-S-24-00018") in the American Journal of Botany, entitled "Estimates of late Early Cretaceous atmospheric CO2 from Mongolia based on stomatal and isotopic analysis of *Pseudotorellia*". This dataset is collected from two species of *Pseudotorellia* Florin from three stratigraphically similar samples at the Tevshiin Govi lignite mine in central Mongolia (~119.7–100.5 Ma) for a paleo-CO2 proxy based on leaf gas-exchange principles (the Franks model). ## Description of the data and file structure All data, including fossil leaf stomatal measurements and carbon isotope analysis, are listed in an Excel spreadsheet. For each leaf fragment, we measured stomatal density based on 4 field-of-view, which mostly at 100× (0.80 mm2 field-of-view), but occasionally at 200× (0.20 mm2 field-of-view) when lower-quality preservation limited large fields-of-view. We also measured 10 guard cells for their length and width as well as the length and width of the bounded stomatal pore. Species: Currently valid scientific name.  Catalog number: Related accession numbers of the fossil leaf slides. Those slides have been formally reposited in the Field Museum in Chicago, IL.  Column D-S: stomatal density under 4 field-of-view.  Field-of-view: Field-of-view under microscope (10x magnification: 1.1315 x 0.707 mm, OR 20x: 0.35 x 0.56 mm) \# stomates: Number of stomates in field-of-view.  area (mm2): area of field-of-view Column U-CB: Measurements of 10 guard cells for their length and width as well as the length and width of the bounded stomatal pore. GCW pair (mm): Guard cell width for a pair of guard cell in the stomata complex.  single GCW (mm): Single guard cell width.  GCL (mm): Guard cell length.  Column CD-CG: carbon isotope analysis. δ13C (‰): a measure of the ratio of the two stable isotopes of carbon—13C and 12C—reported in parts per thousand (per mil, ‰) in a fossil leaf sample.  δ13C duplicate (‰): duplicate carbon isotope analysis for some of leaf samples. NA in this column means the fossil leaf sample amount are not enough for a duplicate.  error δ13C: error of carbon isotope result.  s.e.m. : Standard error of the mean.