Foss. Rec., 23, 15–32, 2020 https://doi.org/10.5194/fr-23-15-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. Sampling fossil floras for the study of insect herbivory: how many leaves is enough? Sandra R. Schachat1,2, S. Augusta Maccracken1,3, and Conrad C. Labandeira1,3,4 1Department of Paleobiology, Smithsonian Institution, Washington, D.C. 20013, USA 2Department of Geological Sciences, Stanford University, Stanford, CA 94305, USA 3Department of Entomology, University of Maryland, College Park, MD 20742, USA 4College of Life Sciences, Capital Normal University, Beijing 100048, China Correspondence: Sandra R. Schachat ([email protected]) Received: 21 September 2019 – Revised: 11 January 2020 – Accepted: 17 January 2020 – Published: 21 February 2020 Abstract. Despite the great importance of plant–insect in- 1 Introduction teractions to the functioning of terrestrial ecosystems, many temporal gaps exist in our knowledge of insect herbivory in deep time. Subsampling of fossil leaves, and subsequent Plants serve as the foundation of many terrestrial ecosys- extrapolation of results to the entire flora from which they tems, and insects have herbivorized plants for hundreds of came, is practiced inconsistently and according to incon- millions of years (Labandeira et al., 2013). The fossil record sistent, often arbitrary criteria. Here we compare herbivory provides data on plant–insect interactions that encompass far data from three exhaustively sampled fossil floras to estab- longer time spans than can be examined in laboratory studies, lish guidelines for subsampling in future studies. The im- yielding insight into such timely issues as the response of in- pact of various subsampling routines is evaluated for three of sect herbivores to climate change (Currano et al., 2010). Fos- the most common metrics of insect herbivory: damage type sil evidence of insect herbivory includes coprolites (Slater diversity, nonmetric multidimensional scaling, and the her- et al., 2012) and traces of insect feeding on roots (Strullu- bivory index. The findings presented here suggest that a min- Derrien et al., 2012), wood (Pires and Sommer, 2009), seeds imum fragment size threshold of 1 cm2 always yields accu- (Schachat et al., 2014, 2015), fruit (Meng et al., 2017), and rate results and that a higher threshold of 2 cm2 should yield leaves (Pinheiro et al., 2016), with leaves being the most in- accurate results for plant hosts that are not polyphyletic form tensely studied. Insect herbivory on leaves and other plant taxa. Due to the structural variability of the plant hosts ex- organs has been categorized qualitatively using the “damage amined here, no other a priori subsampling strategy yields type” (DT) system (Labandeira et al., 2007). consistently accurate results. The best approach may be a se- Studies of insect herbivory in the fossil record vary quential sampling routine in which sampling continues un- tremendously in their intensity of coverage. At one extreme til the 100 most recently sampled leaves have caused no is the description of a single, notable DT on a single plant change to the mean value or confidence interval for damage taxon (Béthoux et al., 2004; Iannuzzi and Labandeira, 2008), type diversity and have caused minimal or no change to the which may occur on a single specimen (Jud and Sohn, 2016); herbivory index. For nonmetric multidimensional scaling, at at the other extreme is the documentation of all DTs on all least 1000 cm2 of leaf surface area should be examined and taxa in an entire fossil flora, which can contain thousands prediction intervals should be generated to verify the rela- or even tens of thousands of specimens (Labandeira et al., tive positions of all points. Future studies should evaluate 2018). Many authors have taken intermediate approaches, for the impact of subsampling routines on floras that are col- example, by documenting all DTs on a subset of leaves from lected based on different criteria, such as angiosperm floras various taxa (Filho et al., 2019), by documenting DTs for a for which the only specimens collected are those that are at specific behavior such as galling (Knor et al., 2013), or by least 50 % complete. categorizing feeding damage at a coarser scale than the DT system, such as the level of functional feeding group (Smith, Published by Copernicus Publications on behalf of the Museum für Naturkunde Berlin. 16 S. R. Schachat et al.: Subsampling fossil leaves for the study of insect herbivory 2008; McLoughlin et al., 2015). Some studies are based on 1.1 Subsampling of ecological data exhaustive examinations of a single plant lineage at multi- ple fossil assemblages (Ding et al., 2015; Glasspool et al., Subsampling can be conducted in one of two ways. Sub- 2003; Kodrul et al., 2018) on the premise that controlling for sampling can occur after data collection with the aim of plant-host affinity will produce more robust conclusions re- standardizing sampling procedures (Droissert et al., 2012) garding the evolution of plant–insect interactions. However, or can occur before data collection, with the aims of stan- because extant species, genera, and families do not have fos- dardizing sampling procedures and reducing collection ef- sil records that extend back to the Paleozoic, data from entire fort (Bowen and Freeman, 1998). Subsampling is standard assemblages must be used to compare herbivory on longer practice among neontologists; for example, transects and timescales. quadrats are very commonly used to subsample extant popu- Such data remain scant. A recent meta-analysis included lations and communities (Pilliod and Arkle, 2013). DT data from 50 exhaustively sampled floras (Pinheiro et al., When fossil floras are studied for insect herbivory, they are 2016). These 50 floras amount to an average of 1 flora per typically examined exhaustively: all leaves above a certain 7.7 million years for all habitats across the planet. Many size threshold, or all leaves that are at least 50 % complete, of these floras are separated by long temporal gaps: one of are sampled. Subsampling strategies, analogous to the tran- these gaps in the above study approaches 100 million years in sects and quadrats used by neontologists, could be applied to length and another exceeds 165 million years. The Carbonif- insect herbivory in the fossil record. Such an approach would erous and Jurassic periods are not represented by any such result in a reduction in the effort required to examine a single floras, and the Triassic and Cretaceous periods are repre- flora and would increase the rate at which such floras could sented by only two floras each. The meta-analysis examined be studied. However, at present, no guidelines are available DT diversity only and included many studies for which quan- to ensure consistent subsampling of fossil leaves or to ensure titative data (measurements of herbivorized leaf area) are not that subsampling routines adequately capture the trends that available. This study raises two main issues regarding current emerge from complete datasets. The aims of the present con- knowledge about the documentation of arthropod herbivory tribution are to test the effects of various subsampling rou- across time. First, there is an absence of available quantitative tines on herbivorized fossil leaves and to establish guidelines data that severely limits the conclusions that can be drawn for subsampling in future studies. from existing studies. Second, far more studies are needed across all intervals to document patterns of insect herbivory, 1.2 Metrics of insect herbivory as discussed by the authors (Pinheiro et al., 2016). The rate at which fossil floras are examined for insect herbivory appears In previous contributions, four metrics have typically been to be increasing, as demonstrated by the 15 recent studies used to compare insect herbivory across fossil host plants and that were not included in the above meta-analysis (see Sup- assemblages. These metrics address three aspects of plant– plement), presumably because they were published after the insect interactions. analysis was conducted. Damage type diversity (DT diversity) addresses the di- The field of ancient plant–insect associations is rife with versity of damage types for an individual plant host or as- possibilities for understanding the fossil record of arthro- semblage. DT diversity is typically reported either unstan- pod herbivory because of ample paleobotanical collections of dardized (Pinheiro et al., 2016) or standardized with sample- museums, universities, and other research institutions across based rarefaction in which each plant specimen is treated as a the world. Although fossil plant collections of such institu- sample (Currano et al., 2011; Wappler and Denk, 2011). We tions vary immensely in size, collection techniques, scope, recommend standardization with rarefaction curves that are level of plant identification, and preservation, these collec- scaled by the amount of leaf surface area examined (Schachat tions permit analyses for insect damage. However, the major et al., 2018). impediments to the study of large paleobotanical collections Nonmetric multidimensional scaling (NMDS) is an un- are time and research funding. Visits to collections that are constrained ordination method (Kruskal and Wish, 1978) of sufficient length to allow collection of quantitative data applied to address differences in herbivory across different for thousands of leaves are often cost-prohibitive. It is, there- plant hosts and assemblages. Plant hosts with identical lev- fore, imperative to discern whether and how the data from els of DT diversity (e.g., 5 DTs each) could have an identi- a subset of specimens could be used to extrapolate patterns cal suite of DTs (e.g., DTs 1, 2, 3, 4, and 5) or completely of insect herbivory for all specimens pertaining to a given different suites of DTs (e.g., DTs 1, 2, 3, 4, and 5 at one plant host or assemblage. Furthermore, it would be useful to site and DTs 6, 7, 8, 9, and 10 at the other site).