bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.139584; this version posted June 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. TITLE eDNA metabarcoding reveals a core and secondary diets of the greater horseshoe bat with strong spatio-temporal plasticity RUNNING TITLE Metabarcoding of the greater horseshoe bat diet AUTHORS Orianne TOURNAYRE1†, Maxime LEUCHTMANN2, Maxime GALAN1, Marine TRILLAT1, Sylvain PIRY1, David Pinaud3, Ondine FILIPPI-CODACCIONI4,5, Dominique PONTIER4,5* Nathalie CHARBONNEL1* 1CBGP, INRAE, CIRAD, IRD, Institut Agro, Université de Montpellier, Montpellier, France 2Nature Environnement 17, Surgères, France 3Centre d’Etudes Biologiques de Chizé, UM7372 CNRS, Université La Rochelle, Villiers-en- Bois, France 4LabEx ECOFECT « Ecoevolutionary Dynamics of Infectious Diseases », Université de Lyon, Lyon, France 5CNRS, Laboratoire de Biométrie et Biologie Évolutive, UMR5558, Université de Lyon, Université Lyon 1, Villeurbanne, France *equal contribution † corresponding author : [email protected] Abstract Dietary plasticity is an important issue for conservation biology as it may be essential for species to cope with environmental changes. However, this process still remains scarcely bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.139584; this version posted June 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. addressed in the literature, potentially because diet studies have long been constrained by methodological limits. The advent of molecular approaches now makes it possible to get a precise picture of diet and its plasticity, even for endangered and elusive species. Here we focused on the greater horseshoe bat (Rhinolophus ferrumequinum) in Western France, where this insectivorous species has been classified as ‘Vulnerable’ on the Regional Red List in 2016. We applied an eDNA metabarcoding approach on 1986 fecal samples collected in six maternity colonies at three sampling dates. We described its diet and investigated whether the landscape surrounding colonies and the different phases of the maternity cycle influenced the diversity and the composition of this diet. We showed that R. ferrumequinum feed on a highly more diverse spectrum of prey than expected from previous studies, therefore highlighting how eDNA metabarcoding can help improving diet knowledge of a flying elusive endangered species. Our approach also revealed that R. ferrumequinum diet is composed of two distinct features: the core diet consisting in a few preferred taxa shared by all the colonies (25% of the occurrences) and the secondary diet consisting in numerous rare prey that were highly different between colonies and sampling dates (75% of the occurrences). Energetic needs and constraints associated with the greater horseshoe bat life-cycle, as well as insect phenology and landscape features, strongly influenced the diversity and composition of both the core and whole diets. Further research should now explore the relationships between R. ferrumequinum dietary plasticity and fitness, to better assess the impact of core prey decline on R. ferrumequinum populations viability. Key-words insectivorous diet, bat, environmental DNA (eDNA), spatio-temporal variation 1 INTRODUCTION Food resources constitute a maJor environmental factor for animal populations (Hutchinson 1957; Schoener 1974). Both the quantity and quality of food resources are known to strongly impact the fitness of individuals (Eeva, Lehikoinen, et Pohjalainen 1997; Sorensen et al. 2009; Serrano-Davies et Sanz 2017) and in turn, the dynamics and viability of populations (Taylor et Schultz 2008; Vickery et al. 2001; Johnsen et al. 2017). Understanding species dietary requirements is therefore of main importance for designing conservation strategies bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.139584; this version posted June 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. (Cravens et al. 2018; Clare 2014; Brown et al. 2014; Groom et al. 2017). Several studies have even shown that some dietary characteristics could be related to elevated risk of species extinction. In particular, species at higher trophic levels in the food chain are more exposed to accumulative effects of pollutants along the food chain. This makes them more vulnerable to the deleterious effects of pollutants than species at lower trophic levels (Purvis et al. 2000; Careddu et al. 2015; Mann 2011). Besides, a narrow and specialized trophic niche (i.e. the range of possible prey) can also increase the vulnerability of species : specialists can face more constraints to respond to environmental changes in resource availability than generalist species (Boyles et Storm 2007; Pratchett, Wilson, et Baird 2006; Twining et al. 2019; Owens et Dittman 2003; Clavel, Julliard, et Devictor 2011). However, foraging can be a flexible activity. According to the optimal foraging theory, predators exploit resources that maximize the net energy intake whilst minimizing energetic costs through a trade-off between food profitability and searching time (Emlen 1966; MacArthur et Pianka 1966). As such, predators should be more selective on profitable resources when these latter are common, and more generalist when profitable resources are scarce. Such dietary plasticity is crucial to cope with environmental changes, including seasonal fluctuations (Bergmann et al. 2015; Kartzinel et Pringle 2015), climate change (de Oliveira et Val 2017; Durant et al. 2007) or anthropogenic pressure (Hempson et al. 2017; Quéméré et al. 2013; Smith et al. 2018). However, suboptimal diet can have negative impact on individual fitness (Sasakawa 2009). Dietary plasticity, despite its potential ecological and evolutionary importance, still remains scarcely addressed in the literature (Sousa, Silva, et Xavier 2019; but see Shutt et al. 2020), potentially because diet studies have long been constrained by methodological limits. Traditionally, diet has been described through direct observations of predatory events and/or through visual morphological analyses of prey remains in predator stomach, gut or scat (Nielsen et al. 2018; Sousa, Silva, et Xavier 2019). However, as these approaches depend on the presence of indigestible prey remains (e.g. exoskeletons), (i) prey are usually identified at a low taxonomic level (i.e. order or family ranks), (ii) soft-bodied prey are undetectable, and (iii) these approaches are very time consuming and are therefore usually limited in number of processed samples (Nielsen et al. 2018). The development of molecular approaches for identifying prey DNA contained in feces, in particular environmental DNA (eDNA) metabarcoding, has set aside most of the traditional methodologies limitations (Khanam et al. 2016; Clare 2014). Metabarcoding of fecal samples has been mainly applied bioRxiv preprint doi: https://doi.org/10.1101/2020.06.08.139584; this version posted June 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. for terrestrial predators, and more specifically for bats (Sousa, Silva, et Xavier 2019). Indeed these later are nocturnal, elusive, highly mobile and most of them are threatened, making it difficult to apply direct observations of their prey (Kunz et al. 2011; Kunz, Whitaker, and Wadanoli 1995; IUCN 2019). Insectivorous bats show a large range of foraging strategies, from specialists (e.g. mountain long-eared bat, Plecotus macrobullaris; Alberdi et al. 2012) to generalists (e.g. big brown bat Eptesicus fuscus; Clare, Symondson, et Fenton 2014). Bat species qualified as generalists can show preference towards certain prey (e.g. Myotis daubentonii; Vesterinen et al. 2016) and be more selective when their favorite prey are available in the environment (e.g. Eptesicus fuscus; Agosta, Morton, et Kuhn 2003). The greater horseshoe bat (Rhinolophus ferrumequinum) is an insectivorous bat species whose diet and foraging behavior have been previously explored, especially in Northern Europe where it experienced severe declines this last century (Kervyn et al. 2009; Mathews et al. 2018; Pir 2009). These declines are likely due to anthropogenic factors (i.e. agricultural intensification or landscape urbanization) that might have resulted in losses of roosting habitats, habitat fragmentation and reduction in prey availability (Froidevaux et al. 2017; Mathews et al. 2018). R. ferrumequinum populations might therefore be affected by the global decrease of arthropods (Hallmann et al. 2017; Wickramasinghe et al. 2004; Strong et al. 1996). They might also be impacted by the accumulation of pesticides in body tissues which can decrease bat fitness (Bayat et al. 2014; Clarke-Wood et al. 2016; Stahlschmidt et Brühl 2012). R. ferrumequinum is even more vulnerable that it is a long-lived species (up to 30 years) with a low reproductive rate (maximum of one pup per year) and a late sexual maturity (two to five years) (Caubère, Gaucher, et Julien 1984; Ransome 1995; Wilkinson et South 2002). Previous studies based on microscopy analyses have shown that R. ferrumequinum feeds mainly on three orders of arthropods : Lepidoptera, Coleoptera