The Angiosperm Advantage: Evidence from carbon isotopes and the fossil record

Amanda Bender, Department of Geology, University of Maryland at College ParkAdvisors: Nathan A. Jud, Dr. A. Jay Kaufman, Dr. Thomas R. Holtz, Jr.

VI. Initial results

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Dv (m

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Leaf vein densi es

Platanus orientalis

Cloverly platanoid

Osmunda banksiaefolia

Osmunda claytoniana

Fig. 9: Initial results of Dv measurements, with error bars showing one sigma.

Fig. 10: Corrected δ13C values for initial data. Initial data points include angiosperms (triangles), conifers (circles), and ferns (squares). Data were collected from both modern (green) and fossil (red) specimens.

VII. Discussion of initial resultsThe Cretaceous platanoid measured has a The Cretaceous platanoid measured has a Dv much lower than modern Platanaceae, simi much lower than modern Platanaceae, simi-lar to lar to Dv observed in modern ferns (Osmundaceae). This could be due to an actual differ observed in modern ferns (Osmundaceae). This could be due to an actual differ-ence in leaf vein density among this lineage between the Early Cretceous and the present ence in leaf vein density among this lineage between the Early Cretceous and the present day, or due to error from insufficient fossil preservation.day, or due to error from insufficient fossil preservation.

The modern sycamore (Platanaceae) measured in the initial carbon isotope study has a The modern sycamore (Platanaceae) measured in the initial carbon isotope study has a δ1313C that is nearly identical to δC that is nearly identical to δ1313C of the conifer. It is important to keep in mind that C of the conifer. It is important to keep in mind that the sycamore material analyzed was merely the interior of a bud, as the plant had not fully the sycamore material analyzed was merely the interior of a bud, as the plant had not fully begun to leaf at the collection time, and that the value does not reflect δbegun to leaf at the collection time, and that the value does not reflect δ1313C of a fully C of a fully photosynthesizing leaf.photosynthesizing leaf.

Another interesting feature of the carbon isotope abundance measurements is that the Another interesting feature of the carbon isotope abundance measurements is that the δ1313C of two fossil taxa (between a fossil conifer and fossil angiosperm) are nearly identiC of two fossil taxa (between a fossil conifer and fossil angiosperm) are nearly identi-cal. This may signal that there is a similar fractionation between the fossil conifer and cal. This may signal that there is a similar fractionation between the fossil conifer and fossil angiosperm.fossil angiosperm.

III. Experimental designProblem: To determine whether Early Cretaceous angiosperms from the wellProblem: To determine whether Early Cretaceous angiosperms from the well-defined lineage Proteales had high vein density and high productivity rates compadefined lineage Proteales had high vein density and high productivity rates compa-rable to its modern relatives, or whether these characters were more comparable to rable to its modern relatives, or whether these characters were more comparable to contemporaneous ferns and conifers growing in the same habitat.contemporaneous ferns and conifers growing in the same habitat.

A. A. Compare leaf vein density between modern and Cretaceous angiosperms and ferns. Compare leaf vein density between modern and Cretaceous angiosperms and ferns. B. B. Measure average Measure average δ1313C from modern leaves from the same environment:C from modern leaves from the same environment:

(1) Trees of angiosperm (1) Trees of angiosperm Platanus occidentalis Platanus occidentalis (American sycamore)(American sycamore) (2) Ferns from Osmundaceae (2) Ferns from Osmundaceae (3) Conifers from Cupressaceae (3) Conifers from Cupressaceae C. C. Measure Measure δ1313C from carbonaceous compression fossils from the Early Cretaceous:C from carbonaceous compression fossils from the Early Cretaceous:

(1) Early angiosperm protealeans, thought to belong to Platanaceae lineage (1) Early angiosperm protealeans, thought to belong to Platanaceae lineage (2) Ferns from Osmundaceae lineage (2) Ferns from Osmundaceae lineage (3) Conifers from Cupressaceae lineage (3) Conifers from Cupressaceae lineage

The modern-fossil pairs of ferns and conifers create control groups. The difference in The modern-fossil pairs of ferns and conifers create control groups. The difference in δ1313C within these groups allows us to constrain change in δC within these groups allows us to constrain change in δ1313C due to diagenetic effects C due to diagenetic effects and changes in and changes in pCOCO2 and the abundance ratio of and the abundance ratio of 1313C/C/1212C in the atmosphere between C in the atmosphere between the Early Cretaceous and today.the Early Cretaceous and today.

The difference in δThe difference in δ1313C between the modern sycamore and the Cretaceous platanoid is C between the modern sycamore and the Cretaceous platanoid is expected to be driven by these same processes, and the difference in carbon expected to be driven by these same processes, and the difference in carbon fractionation as driven by leaf vein evolution.fractionation as driven by leaf vein evolution.

II. Hypotheses Rates of photosynthetic productivity increased during the evolution of modern Rates of photosynthetic productivity increased during the evolution of modern angiosperms. angiosperms.

A. A. Angiosperm leaf vein density increased during angiosperm evolution, Angiosperm leaf vein density increased during angiosperm evolution, from the Early Cretaceous to modern time. from the Early Cretaceous to modern time.

B. B. Increased rates of productivity in modern angiosperms results in more Increased rates of productivity in modern angiosperms results in more negative negative δ1313C values in comparison to both extinct angiosperms and C values in comparison to both extinct angiosperms and living and extinct non-angiosperm plants. living and extinct non-angiosperm plants.

C. C. δ δ1313C can be reliably measured from fossil plants, and can be constrained C can be reliably measured from fossil plants, and can be constrained to reflect on isotope fractionation during photosynthesis. to reflect on isotope fractionation during photosynthesis.

IV. Geologic settingFig. 4: Stratigraphic column of Cloverly Formation, WY, USA (Jud)

Fossilized leaves are to be collected from the Himes Fossilized leaves are to be collected from the Himes Member of the Cloverly Formation in the Bighorn Basin, Member of the Cloverly Formation in the Bighorn Basin, WY, USA. Fossil angiosperms have been collected and WY, USA. Fossil angiosperms have been collected and identified from this site (Jud 2010), marked in Figure 4 as identified from this site (Jud 2010), marked in Figure 4 as red leaves. Dated at 110-115 Ma, these represent the earlired leaves. Dated at 110-115 Ma, these represent the earli-est angiosperm macrofossils known from the fossil est angiosperm macrofossils known from the fossil record of the western UnitedStates.record of the western UnitedStates.

The sediments in the Himes Member are generally identiThe sediments in the Himes Member are generally identi-fied as fluvial depositional environments, indicating that fied as fluvial depositional environments, indicating that the leaves deposited would have had sufficient water the leaves deposited would have had sufficient water supply.supply.

I. IntroductionI. Introduction

Fig. 1: Typical angiosperm leaf and cross-section of a leaf, showing the spatial relationship of leaf veins that bring water to the stomatal pores, which lose water (gH2O)while they take in CO2 from the atmosphere to photosynthesize. [Brodribb & Feild 2010]

Fig. 2: Graph showing increase in leaf vein density of angiosperm fossils (open circles) from the Cretaceous through the Tertiary, and the steady vein density of non-angiosperm (closed circles) (Feild et al. 2011).

Fig. 3: Carbon isotope ratios are commonly expressedin δ-notation, compared to the standard Vee Pee DeeBelemnite.

Angiosperms (flowering plants) are the Angiosperms (flowering plants) are the dominant terrestrial plant group today, due dominant terrestrial plant group today, due largely to their unrivalled photosynthesic rates, largely to their unrivalled photosynthesic rates, which is limited mainly by a plant’s ability to which is limited mainly by a plant’s ability to supply water to the leaf (Brodribb & Feild supply water to the leaf (Brodribb & Feild 2010). During photosynthesis, a leaf opens its 2010). During photosynthesis, a leaf opens its stomata to exchange carbon dioxide and stomata to exchange carbon dioxide and oxygen with the atmosphere and loses water oxygen with the atmosphere and loses water via transpiration. To combat the risk of via transpiration. To combat the risk of dehydration, plants have evolved many dehydration, plants have evolved many innovative features to bring water closer to innovative features to bring water closer to these sites of water loss (Sperry 2003), including the irrigation system of leaf veins these sites of water loss (Sperry 2003), including the irrigation system of leaf veins (Roth-Nebelsick et al. 2001). Leaf veins are correlated as a measure of the maximum (Roth-Nebelsick et al. 2001). Leaf veins are correlated as a measure of the maximum capacity for photosynthesis within a leaf (Sack & Frole 2006; Brodribb et al. 2007; capacity for photosynthesis within a leaf (Sack & Frole 2006; Brodribb et al. 2007; Noblin et al. 2008; Boyce et al. 2009). Noblin et al. 2008; Boyce et al. 2009).

Modern angiosperms exhibit a Modern angiosperms exhibit a larger density of leaf veins than larger density of leaf veins than other plant groups (e.g. conifers and other plant groups (e.g. conifers and ferns) (Brodribb et al. 2005), ferns) (Brodribb et al. 2005), allowing for their increased rates of allowing for their increased rates of transpiration and higher rates of transpiration and higher rates of productivity. However, the evolution productivity. However, the evolution of this increased leaf vein density is of this increased leaf vein density is not well-constrained. Recent not well-constrained. Recent phylogenetic and fossil evidence phylogenetic and fossil evidence suggest that the key innovation(s) suggest that the key innovation(s) allowing for increased leaf vein allowing for increased leaf vein density (>5 mm mm density (>5 mm mm-2-2) first ) first appeared in leaves at ~100 Ma appeared in leaves at ~100 Ma (Late Albian) (Brodribb & Feild (Late Albian) (Brodribb & Feild 2010; Feild et al. 2011). 2010; Feild et al. 2011).

Of the two stable isotopes of Of the two stable isotopes of carbon, carbon, 1212C and C and 1313C, the lighter C, the lighter 1212C comprises significantly more CO comprises significantly more CO2 in the modern atmosphere (98.89%). in the modern atmosphere (98.89%). Carbon isotope signatures measured from modern and fossil leaves record how the leaf Carbon isotope signatures measured from modern and fossil leaves record how the leaf discriminates against the heavy discriminates against the heavy 1313C isotope during photosynthesis, which occurs in CC isotope during photosynthesis, which occurs in C3 plants mostly due to the enzyme RuBisCO. Farquhar et al. (1982) relate stomatal plants mostly due to the enzyme RuBisCO. Farquhar et al. (1982) relate stomatal conductance to isotope discrimination, or fractionation. When the stomatal conductance conductance to isotope discrimination, or fractionation. When the stomatal conductance is small (i.e. stomata are more closed), intercellular partial pressure is small, which nearly is small (i.e. stomata are more closed), intercellular partial pressure is small, which nearly disables enzymatic fractionation. Consequently, the leaf fixates more disables enzymatic fractionation. Consequently, the leaf fixates more 1313C in leaf tissueC in leaf tissue when its stomata are more closed (Farquhar & Sharkey 1982). Because leaf veins bring when its stomata are more closed (Farquhar & Sharkey 1982). Because leaf veins bring water closer to the stomatal sites of water closer to the stomatal sites of water loss, a greater density of leaf water loss, a greater density of leaf veins enable greater stomatal veins enable greater stomatal conductance, resulting in an increased conductance, resulting in an increased intercellular partial pressure, and a intercellular partial pressure, and a greater discrimination against heavy greater discrimination against heavy 1313C. This results in a more negative C. This results in a more negative δ1313C for leaves with high vein density.C for leaves with high vein density.

Step 2:Prepare specimensScrape carbon from fossilized leafWeigh ~100 μg in tin cupDry modern leavesCrush modern leavesWeigh 30-70 μg in tin cup Step 3:

Analyze for stable carbon isotopes

Gas carried to mass spectrometer,ionized, and de lected intocollector cupsIons measured by computer,outputs in Excel ile

Combust samples inelemental analyzerLoad samples into loadingcarousel

Step 1:Collect leaf specimensCarbonaceous fossil leavesfrom Cloverly Formation

Fig. 7: Osmundrastrum cinnamomeum, fern fiddlehead Fig. 8: Early Cretaceous platanoid

Modern leaves fromSeneca Creek State Park

Fig. 5 (left): Early Cretaceousplatanoid, Dv=1.71 (±0.19 SD)

Fig. 6 (right): Modern Osmundaclaytoniana from Cleared Leaf

Collection, Dv=1.80 (±0.37 SD)

A. Leaf vein density measurements A. Leaf vein density measurements Leaf vein density: Leaf vein density: length of veins (mm) divided length of veins (mm) divided by surface area of lamina (mm by surface area of lamina (mm2),), measured using ImageJ (NIH measured using ImageJ (NIH Image, Bethesda, MD, USA) Image, Bethesda, MD, USA)

B. Carbon isotope abundance measurements B. Carbon isotope abundance measurements

V. Methods

10 mm10 mm

IX. ReferencesBoyce, C.K., T.J. Brodribb, T.S. Feild & M.A. Zwienecki. 2009. Angiosperm leaf vein evolution was physiologically and environmentally Boyce, C.K., T.J. Brodribb, T.S. Feild & M.A. Zwienecki. 2009. Angiosperm leaf vein evolution was physiologically and environmentally transformative. transformative. Proceedings of the Royal Society BProceedings of the Royal Society B 276: 1771-1776. 276: 1771-1776.Brodribb, T.J., N.M. Holbrook, M.A. Zwienecki & B. Palma. 2005. Leaf hydraulic capacity in ferns, conifers and angiosperms: impacts of Brodribb, T.J., N.M. Holbrook, M.A. Zwienecki & B. Palma. 2005. Leaf hydraulic capacity in ferns, conifers and angiosperms: impacts of photosynthetic maxima. photosynthetic maxima. New PhytologistNew Phytologist 165: 839-846. 165: 839-846.Brodribb, T.J., T.S. Feild & G.J. Jordan. 2007. Leaf maximum photosynthetic rate and venation are linked by hydraulics. Brodribb, T.J., T.S. Feild & G.J. Jordan. 2007. Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant PhysiologyPlant Physiology 144: 1890-1898. 144: 1890-1898.Brodribb, T.J. & T.S. Feild. 2010. Leaf hydraulic evolution led a surge in leaf photosynthetic capacity during early angiosperm Brodribb, T.J. & T.S. Feild. 2010. Leaf hydraulic evolution led a surge in leaf photosynthetic capacity during early angiosperm diversification. diversification. Ecology LettersEcology Letters 13: 175-183. 13: 175-183.Farquhar, G.D., & T.D. Sharkey. 1982. Stomatal conductance and photosynthesis. Farquhar, G.D., & T.D. Sharkey. 1982. Stomatal conductance and photosynthesis. Annual Review of Plant PhysiologyAnnual Review of Plant Physiology 33: 317-345. 33: 317-345.

IX. References (continued)Feild, T.S., T.J. Brodribb, A. Iglesias, D.S. Chatelet, A. Baresch, G.R. Upchurch, Jr., B. Gomez, B.A.R. Mohr, C. Coiffard, J. Kvacek & C. Feild, T.S., T.J. Brodribb, A. Iglesias, D.S. Chatelet, A. Baresch, G.R. Upchurch, Jr., B. Gomez, B.A.R. Mohr, C. Coiffard, J. Kvacek & C. Jaramillo. 2011. Fossil evidence for Cretaceous escalation in angiosperm leaf vein evolution. Jaramillo. 2011. Fossil evidence for Cretaceous escalation in angiosperm leaf vein evolution. Proceedings of the National Academy of Proceedings of the National Academy of Sciences Sciences Early Edition. Early Edition.Jud, N.A. 2010. Angiosperms from the Early Cretaceous of Wyoming. Jud, N.A. 2010. Angiosperms from the Early Cretaceous of Wyoming. Geological Society of America Abstracts with ProgramsGeological Society of America Abstracts with Programs 42: 482. 42: 482.Noblin, X., L. Mahadevan, I.A. Coomaraswamy, D.A. Weitz, N.M. Holbrook & M.A. Zwienecki. 2008. Optimal vein density in artificial Noblin, X., L. Mahadevan, I.A. Coomaraswamy, D.A. Weitz, N.M. Holbrook & M.A. Zwienecki. 2008. Optimal vein density in artificial and real leaves. and real leaves. Proceedings of the National Academy of Sciences Proceedings of the National Academy of Sciences 104: 9140-9144.104: 9140-9144.Roth-Nebelsick, A., D. Uhl, V. Mosbrugger & H. Kerp. 2001. Evolution and function of leaf venation architecture: a review. Roth-Nebelsick, A., D. Uhl, V. Mosbrugger & H. Kerp. 2001. Evolution and function of leaf venation architecture: a review. Annals of Annals of Botany Botany 87: 553-566. 87: 553-566.Sack, L. & K. Frole. 2006. Leaf structural diversity is related to hydraulic capacity in tropical rain forest trees. Sack, L. & K. Frole. 2006. Leaf structural diversity is related to hydraulic capacity in tropical rain forest trees. EcologyEcology 87: 483-491. 87: 483-491.Sperry, J.S. 2003. Evolution of water transport and xylem structure. Sperry, J.S. 2003. Evolution of water transport and xylem structure. International Journal of Plant SciencesInternational Journal of Plant Sciences 164: S115-S127. 164: S115-S127.

VIII. Timeline of future work Summer 2011: Collection of modern and fossil specimens Fall 2011: Continue collection of modern specimens Identify appropriate fossil specimens Collect vein density and carbon isotope measurements Interpret and analyze results Speculate on further work to improve results/sampling method/etc.

X. AcknowledgementsI am immeasurably grateful for Nathan Jud’s guidance. His endless patience, infectious excitement, and passion for learning have been beyond invaluable for leading me to discover the world of paleobotany.I would like to thank Jay Kaufman for his wisdom and leadership, especially with technical laboratory work and writing suggestions.I owe many thanks to Thomas Holtz for his insight and encouragement, and especially for sparking my initial interest in geology and paleontology.Thanks to Scott Wing for providing advice and consultation.Thanks to Yongbo Peng, Natalie Sievers, and Sara Peek for assisting me in the geochemistry laboratory.Thanks to my fellow seniors for supplying their good humor and various assistance.Thanks to my family and friends for their unwavering support and encouragement.