Novel Insect Leaf-Mining after the End-Cretaceous Extinction and the Demise of Cretaceous Leaf Miners, Great Plains, USA Michael P. Donovan 1 *, Peter Wilf 1 , Conrad C. Labandeira 2,3 , Kirk R. Johnson 4 , Daniel J. Peppe 5 1 Department of Geosciences, Pennsylvania State University, University Park, Pennsylvania, United States of America, 2 Department of Paleobiology, National Museum of Natural History, Smithsonian Institution, Washington, District of Columbia, United States of America, 3 Department of Entomology and BEES Program, University of Maryland, College Park, Maryland, United States of America, 4 National Museum of Natural History, Smithsonian Institution, Washington, District of Columbia, United States of America, 5 Department of Geology, Baylor University, Waco, Texas, United States of America Abstract Plant and associated insect-damage diversity in the western U.S.A. decreased significantly at the Cretaceous-Paleogene (K- Pg) boundary and remained low until the late Paleocene. However, the Mexican Hat locality (ca. 65 Ma) in southeastern Montana, with a typical, low-diversity flora, uniquely exhibits high damage diversity on nearly all its host plants, when compared to all known local and regional early Paleocene sites. The same plant species show minimal damage elsewhere during the early Paleocene. We asked whether the high insect damage diversity at Mexican Hat was more likely related to the survival of Cretaceous insects from refugia or to an influx of novel Paleocene taxa. We compared damage on 1073 leaf fossils from Mexican Hat to over 9000 terminal Cretaceous leaf fossils from the Hell Creek Formation of nearby southwestern North Dakota and to over 9000 Paleocene leaf fossils from the Fort Union Formation in North Dakota, Montana, and Wyoming. We described the entire insect-feeding ichnofauna at Mexican Hat and focused our analysis on leaf mines because they are typically host-specialized and preserve a number of diagnostic morphological characters. Nine mine damage types attributable to three of the four orders of leaf-mining insects are found at Mexican Hat, six of them so far unique to the site. We found no evidence linking any of the diverse Hell Creek mines with those found at Mexican Hat, nor for the survival of any Cretaceous leaf miners over the K-Pg boundary regionally, even on well-sampled, surviving plant families. Overall, our results strongly relate the high damage diversity on the depauperate Mexican Hat flora to an influx of novel insect herbivores during the early Paleocene, possibly caused by a transient warming event and range expansion, and indicate drastic extinction rather than survivorship of Cretaceous insect taxa from refugia. Citation: Donovan MP, Wilf P, Labandeira CC, Johnson KR, Peppe DJ (2014) Novel Insect Leaf-Mining after the End-Cretaceous Extinction and the Demise of Cretaceous Leaf Miners, Great Plains, USA. PLoS ONE 9(7): e103542. doi:10.1371/journal.pone.0103542 Editor: Andrew A. Farke, Raymond M. Alf Museum of Paleontology, United States of America Received February 28, 2014; Accepted July 3, 2014; Published July 24, 2014 Copyright: ß 2014 Donovan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a Geological Society of America Student Research Grant, a Paleontological Society of America Student Research Grant, and the P.D. Krynine Memorial Fund, Penn State Department of Geosciences (MPD). Additional funding was provided by the American Philosophical Society, a David and Lucile Packard Fellowship, and NSF Grant DEB-0919071 (PW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Co-author PW is an editor for PLOS ONE. This does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria. * Email: mpd187@psu.edu Introduction One of the largest and most sudden mass extinctions in Earth history occurred at the end of the Cretaceous period (66.0 Ma), triggered by an asteroid impact at Chicxulub, Mexico [1–5]. The paleobotanical record from the Western Interior United States shows a ca. 30% (pollen) to ca. 57% (southwestern North Dakota macrofossils) reduction in diversity at the Cretaceous-Paleogene (K-Pg) boundary [6–9]. The richness of insect-feeding damage on fossil leaves also decreased ca. 42% across the K-Pg boundary in North Dakota [10,11]. Mines and galls, typically representing host-specific interactions, were disproportionally affected in the extinction [10,11]. Paleocene floras of the Western Interior U.S.A. typically had low diversity and were dominated by widespread species [12–14]. For ca. 10 million years following the K-Pg extinction, floral diversity was depressed compared to the latest Cretaceous and finally began to increase during the latest Paleocene and early Eocene [15–17]. Insect damage diversity was also low during most of the Paleocene, but it increased to levels similar to the latest Cretaceous during late Paleocene warming, preceding the increase in plant diversity [10,11,16,18–20]. Insect diversity generally correlates with plant diversity today [21]. Comparably, low diversity paleofloras with low diversity insect damage have been observed at all regional early Paleocene localities previously examined for insect damage, with the exception of two unusual localities: Mexican Hat (southeastern Montana) and Castle Rock (north-central Colorado) [10,11,19]. Castle Rock has a rainforest-like flora with high plant diversity and low insect damage diversity [19,22,23]. Mexican Hat has a typical, low diversity flora, but uniquely, this is paired with highly diverse insect damage across host plants [19]. Wilf et al. [19] hypothesized that these two anomalous localities represent decoupled plant and insect diversity resulting from broken food webs in the wake of a mass extinction. PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e103542