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Functionally complete communities result in better ecosystem functioning: Dung removal and secondary seed dispersal by dung beetles in the Western Palaearctic

Authors: Tanja Milotić, Christophe Baltzinger, Carsten Eichberg, Amy E. Eycott, Marco Heurich, Jörg Müller, Jorge A. Noriega, Rosa Menendez, Jutta Stadler, Réka Ádám, Tessa Bargmann, Isabelle Bilger, Jörn Buse, Joaquin Calatayud, Constantin Ciubuc, Gergely Boros, Pierre Jay-Robert, Märt Kruus, Enno Merivee, Geoffrey Miessen, Anne Must, Elham Ardali, Elena Preda, Iraj Rahimi, Dirk Rohwedder, Rob Rose, Eleanor M. Slade, László Somay, Pejman Tahmasebi, Stefano Ziani and Maurice Hoffmann

Handling editor: Christine Meynard

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Title page

Title

Functionally richer communities improve ecosystem functioning: Dung removal and secondary seed dispersal by dung beetles in the Western Palaearctic

Authors

1. Tanja Milotić 2. Christophe Baltzinger 3. Carsten Eichberg 4. Amy E. Eycott 5. Marco Heurich 6. Jörg Müller 7. Jorge A. Noriega 8. Rosa Menendez 9. Jutta Stadler 10. Réka Ádám 11. Tessa Bargmann 12. Isabelle Bilger 13. Jörn Buse 14. Joaquín Calatayud 15. Constantin Ciubuc 16. Gergely Boros 17. Pierre Jay-Robert 18. Märt Kruus 19. Enno Merivee 20. Geoffrey Miessen 21. Anne Must 22. Elham Ardali 23. Elena Preda 24. Iraj Rahimi 25. Dirk Rohwedder 26. Rob Rose 27. Eleanor M. Slade 28. László Somay 29. Pejman Tahmasebi 30. Stefano Ziani 31. Maurice Hoffmann

Postal addresses and email addresses of all authors

1. TM: Ghent University, Department of Biology, TEREC, K.L. Ledeganckstraat 35, 9000 Gent, Belgium; Research Institute for Nature and Forest, Havenlaan 88/73, 1000 Brussels, Belgium ([email protected]) 2. CB: Irstea, Domaine des Barres, 45290 Nogent-sur-Vernisson, France ([email protected]); Centre for Invasion Biology, School of Life Sciences, University of KwaZulu-Natal, Scottsville, 3209, Pietermaritzburg, South Africa

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3. CE: University of Trier, Regional and Environmental Sciences, Geobotany, Behringstr. 21, 54296 Trier, Germany ([email protected]) 4. AE: University of Bergen, Department of Biology, PO box 7803, 5020 Bergen, Norway ([email protected]) 5. MH: Bavarian Forest National Park, Freyunger Str. 2, 94481 Grafenau, Germany; University of Freiburg, Faculty of Environment and Natural Resources, Tennenbacher Str. 4, 79106 Freiburg, Germany ([email protected]) 6. JM: Bavarian Forest National Park, Freyunger Str. 2, 94481 Grafenau, Germany; University of Würzburg, Biozentrum, Glashüttenstraße 5, 96181 Rauhenebrach, Germany ([email protected]) 7. JAN: National Museum of Natural Science (CSIC), Department of Biogeography and Global Change, C/José Gutiérrez Abascal 2, 28006 Madrid, Spain ([email protected]) 8. RM: Lancaster University, Lancaster Environment Centre, Lancaster LA1 4YQ, UK ([email protected]) 9. JS: Helmholtz Centre for Environmental Research - UFZ, Dept. Community Ecology, Theodor- Lieser-Str. 4, 06120 Halle/Saale, Germany ([email protected]) 10. RA: MTA Centre for Ecological Research, Institute of Ecology and Botany, Alkotmány u. 2-4., H-2163 Vácrátót, Hungary; Eötvös Loránd University, Department of Plant Systematics, Ecology and Theoretical Botany, Pázmány Péter sétány 1/A, H-1117 Budapest, Hungary ([email protected]) 11. TB: University of Bergen, Department of Biology, PO box 7803, 5020 Bergen, Norway; University of Bergen, Department of Geography, PO box 7802, 5020 Bergen, Norway, (tessa.bargmann@ uib.no) 12. IB: Irstea, Domaine des Barres, 45290 Nogent-sur-Vernisson, France ([email protected]) 13. JB: University of Koblenz-Landau, Institute for Environmental Sciences, Ecosystem Analysis, Fortstr. 7, 76829 Landau, Germany; Black Forest National Park, Kniebisstr. 67, 72250 Freudenstadt, Germany ([email protected]) 14. JC: Integrated Science Lab, Department of Physics, Umeå University, Umeå, Sweden ([email protected]) 15. CC: University of Bucharest, Research Centre in Systems Ecology and Sustainability, Splaiul Independentei 91-95, 050095 Bucharest, Romania ([email protected]) 16. GB: MTA Centre for Ecological Research, Institute of Ecology and Botany, Alkotmány u. 2-4., H-2163 Vácrátót, Hungary; MTA Centre for Ecological Research, GINOP Sustainable Ecosystem Research Group, Klebelsberg u. 3., H-8237 Tihany, Hungary; Szent István University, Departure of Zoology and Animal Ecology, Páter Károly u. 1., H-2100 Gödöllő, Hungary ([email protected]) 17. PJR: CEFE, Univ. Paul Valéry Montpellier 3, Univ. Montpellier, EPHE, CNRS, IRD, Montpellier, France ([email protected]) 18. MK: Estonian University of Life Sciences, Institute of Agricultural and Environmental Sciences, Kreutzwaldi Street 1, 51014, Tartu, Estonia ([email protected]) 19. EM: Estonian University of Life Sciences, Institute of Agricultural and Environmental Sciences, Kreutzwaldi Street 1, 51014, Tartu, Estonia ([email protected]) 20. GM: independent researcher, Liège, Belgium, ([email protected]) 21. AM: Estonian University of Life Sciences, Institute of Agricultural and Environmental Sciences, Kreutzwaldi Street 1, 51014, Tartu, Estonia ([email protected]) 22. EA: Ghent University, Department of Biology, K.L. Ledeganckstraat 35, 9000 Gent, Belgium; Shahrekord University, Department of Rangeland and Watershed management, Pobox 115, Shahrekord, Iran ([email protected]) 23. EP: University of Bucharest, Research Centre in Systems Ecology and Sustainability, Splaiul Independentei 91-95, 050095 Bucharest, Romania ([email protected])

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24. IR: Shahrekord University, Department of Rangeland and Watershed management, Pobox 115, Shahrekord, Iran ([email protected]) 25. DR: Zoological research museum Alexander Koenig, Adenauerallee 160, 53113 Bonn, Germany ([email protected]) 26. RR: Centre for Ecology & Hydrology, Lancaster Environment Centre, Lancaster LA1 4AP, UK ([email protected]) 27. EMS: University of Oxford, Department of Zoology, South Parks Road, Oxford, OX1 3PS, UK ; University of Lancaster, Lancaster Environment Centre, Lancaster LA1 4YQ, UK ([email protected]) 28. LS: MTA Centre for Ecological Research, Institute of Ecology and Botany, Alkotmány u. 2-4., H-2163 Vácrátót, Hungary; MTA Centre for Ecological Research, GINOP Sustainable Ecosystem Research Group, Klebelsberg u. 3., H-8237 Tihany, Hungary; Szent István University, Doctoral School of Environmental Sciences, Páter Károly u. 1., H-2100 Gödöllő, Hungary ([email protected]) 29. PT: Shahrekord University, Department of Rangeland and Watershed management, Pobox 115, Shahrekord, Iran ([email protected]) 30. SZ: GEOLAB, Via Case di Dozza, 22 40026 Imola (BO) , Italy ([email protected]) 31. MHo: Ghent University, Department of Biology, TEREC, K.L. Ledeganckstraat 35, 9000 Gent, Belgium; Research Institute for Nature and Forest, Havenlaan 88/73, 1000 Brussels, Belgium ([email protected])

Running title

Dung removal and secondary seed dispersal by dung beetles

Corresponding author

Tanja Milotić ([email protected])

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Abstract

Aim In several ecosystems, the diversity of functional species traits has been shown to have a stronger effect on ecosystem functioning than taxonomic diversity alone. However, few studies have explored this idea at a large geographic scale. In a multi-site experiment, we unravelled the relationship between ecosystem function and functional completeness of species assemblages using dung beetles as a model group, focusing on dung removal and secondary seed dispersal.

Location 17 grassland locations across the Western Palaearctic.

Methods We used a randomized block design with different exclosure types to control the dung and seed removing activities of individual functional groups of the local dung beetle assemblage. We classified dung beetle species according to resource specialisation and into functional groups based on dung processing behaviour (dwellers, tunnellers, rollers) and body size (small, large). Additionally, we assessed the role of other soil macro-invertebrates. By sampling the dung beetle community and measuring the remaining dung and seeds after the experiment, the impact of each functional group was estimated.

Results Dung beetle assemblages differed along a north-south and east-west gradient. Dwellers dominated northernmost sites, whereas at lower latitudes we observed more tunnellers and rollers indicating a functional shift. Resource specialists were more abundant in southern and eastern areas. Overall, functional group diversity enhanced dung removal. More dung (+46.9%) and seeds (+32.1%) were removed in the southern sites and tunnellers and rollers were more effective. At the northernmost sites, where tunnellers were scarce or absent, other soil macro-invertebrates removed the majority of dung.

Main conclusions The conservation of functionally complete dung beetle assemblages is crucial to maintain the ecosystem functions provided by dung beetles. Given the latitudinal variation in functional group diversity, it is reasonable to expect compositional changes due to climate change. These changes could lead to increased dung removal and a higher secondary seed dispersal rate in northern regions.

Key words ecosystem service, ecosystem function, latitudinal gradient, longitudinal gradient, multi- site experiments, Scarabaeidae

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Introduction

The on-going loss of biodiversity highlights the importance of gaining insight into the relationship between species richness and ecosystem functioning (Larsen et al., 2005; Duffy et al., 2017). Even more disruptive than the loss of a single species is a functional shift in species assemblages. When species with unique traits disappear we would expect significant changes in ecosystem functioning and restructuring of ecological pathways (Barnes et al., 2014). Many studies highlight the importance of facilitation or niche complementarity and the value of multi-species assemblages for specific ecosystem functions (Cardinale et al., 2002). However, many of these studies have been criticized for their artificial and restricted species combinations and the strictly controlled abiotic environment (Naeem & Wright, 2003). In addition to these controlled (ex situ) lab experiments, large-scale field studies are necessary to explore the underlying mechanisms in a natural context. Field experiments are indispensable to verify the predictive ability of down-scaled lab experiments, for example as recently applied by Duffy et al. (2017) to link biodiversity and productivity using real world data.

In this study, we used dung beetles (Coleoptera: Scarabaeidae) as a focal taxon. Due to their broad geographic distribution and presence in all kinds of terrestrial habitats, dung beetles form a group well-fitted to field studies. Dung beetles use dung for feeding and breeding and it is relatively easy to measure dung removal by dung beetles in a standardized way. In ecosystem functioning research, dung removal can be applied as a direct measure for the relationship between ecosystem functioning and the functional completeness of dung beetle communities (Spector, 2006). Dung beetles can be classified into three major functional groups containing species with the same functional traits with regard to the direction of dung transport during nesting: tunnellers (paracoprids) make vertical shafts beneath dung pats, rollers (telecoprids) transport dung in a combined horizontal and vertical movement by rolling a dung ball a certain distance until burying it shallowly, and dwellers (endocoprids) reside within the original dung deposit (Doube, 1990). Another distinction can be made based on resource specialisation level, with generalists using different dung types and specialists only attracted to a specific dung type. Both the relative abundance of different functional groups and the intensity of resource specialisation are linked to geography and climate. Dwellers and resource generalists are dominant in northern communities and increasing numbers of tunnellers, rollers and resource specialists are found at lower latitudes (Hortal et al., 2011). The distribution of dung beetle species is closely linked with minimum temperature (Lobo et al., 2002; Menéndez & Gutiérrez, 2004). An increase in mean minimum temperature due to global warming might affect the species structure and functional composition of local dung beetle assemblages and cause an elevational migration of dung beetles in mountain ranges (Menéndez et al., 2014). Functional group richness and species composition of dung beetle assemblages strongly affect key ecological functions such as dung removal and decomposition (O'Hea et al., 2010), secondary dispersal of seeds already present in dung (Slade et al., 2007) and subsequent seed germination (D'hondt et al., 2008). Hence, spatial variation in these ecosystem functions as provided by dung beetles seems plausible.

Due to the use of dung during feeding and nesting, dung beetles have an impact on several ecologically and economically valuable ecosystem functions and services (Beynon et al., 2015) such as nutrient cycling (Sitters et al., 2014) and bioturbation (Brown et al., 2010). Furthermore, dung beetles are of great relevance for plant dispersal ecology as they facilitate secondary seed dispersal (Andresen, 2002). Larvae and adult individuals of dung beetles do not feed on the seeds present in dung but bury those seeds unintentionally (Nichols et al., 2008). Therefore, the magnitude of

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seedling competition is lowered since seeds are transported away from an environment which may contain high densities of endozoochorously dispersed seeds (Andresen & Levey, 2004).

Despite the global distribution of dung beetles, the majority of studies on the role of dung beetles in ecosystem functioning focus either on tropical and subtropical regions or are conducted in agricultural landscapes or microcosms (Nichols et al., 2007; Slade et al., 2017). In particular, studies in semi-natural landscapes in the Western Palaearctic region are scarce. To fill this gap, we conducted a large-scale field experiment at multiple sites in various biogeographic regions across the Western Palaearctic. Here we asked the following questions: (1) How are dung beetle functional assemblages affected by “geography” (i.e., latitudinal and longitudinal trends) and climate variables?; (2) How does the relative abundance of resource specialists and generalists relate to geography?; and (3) How does functional group diversity among dung beetles affect dung removal and secondary seed dispersal?

We estimated the contribution of each functional group to the ecosystem functions of dung removal and secondary seed dispersal. We locally manipulated the number of functional groups by using different exclosure types that inhibited the dung and seed removing activities of specific combinations of functional groups of the local dung beetle assemblage. Simultaneously, we sampled the local dung beetle assemblage using different dung types as bait.

Methods

Study sites

We carried out a multi-site experiment on 17 study sites across 10 countries in the Western Palaearctic realm (figure 1A and table S1). Each study site was assigned to a biogeographic region according to Udvardy (1975). The experiment was replicated both on a spatial scale by selecting study sites within the same biogeographic region (table S2) and a temporal scale by repeating the experiment in 2013, 2014, 2015 and/or 2016. All experiments and sampling were carried out during the main activity period of dung beetles in each region (table S3). The predominant vegetation type at the study site was (semi-)natural grassland. All study areas had been grazed by domestic and/or wild ungulates for at least two years prior to the experiment.

Dung beetle classification

Dung beetles were strictly defined as those species of the superfamily Scarabaeoidea that feed on dung in both the larval and adult phases (Hanski & Cambefort, 1991). Ecosystem functions such as dung decomposition and secondary seed dispersal are most likely affected by the amount of dung taken and the direction of dung transport. In our study, dung beetle species were assigned to one of the three main dung beetle functional groups: dwellers, tunnellers or rollers (Milotić et al., 2018). As the amount of dung transport is strongly correlated with beetle size (Horgan, 2001), beetles were further subdivided into size classes, as either small or large (body size smaller or larger than 1 cm). All

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Western Palaearctic dwellers are considered small species (personal communication D. Mann) and were therefore not classified according to size. Other soil macro-invertebrates, such as earthworms, ants, termites and isopods, are also often major dung feeders or decomposers (e.g., Gittings et al. (1994)) with a similar dung removal strategy to tunnellers. Therefore, we defined a sixth and seventh functional group composed of either small or large soil macro-invertebrates. In addition, we made a second classification of dung beetle species according to resource specialization with a distinction between resource generalists and specialists based on the number of dung types used for feeding (specialists: 1-2, and generalists: > 2 dung types; table S5).

Experimental design

We designed eleven exclosure types containing a standardized dung portion. By combining ground screens, vertical walls, and/or ceilings the dung removing activity by different combinations of functional groups was prevented (figure 1B). The components of the exclosures were made of plastic square mesh, and the mesh size determined the size class of the invertebrates that were able to remove dung from the experimental unit. We used 1 cm² mesh to exclude large individuals (while still including small individuals), and 1 mm² mesh to exclude all macroinvertebrate activity. In treatments lacking ground screens, ceilings or walls, tunnellers and rollers of all size classes were able to translocate dung. As dung beetles most often reach their feeding source by flying (Larsen & Forsyth, 2005), all treatments without ceilings allowed the entrance of any dung beetle functional group, but the removal of dung by tunnellers and rollers was prevented by the use of ground screens and walls. In study areas where no rollers occur we used a reduced set of exclosure types. The same basic set of 5 exclosure types was built at each site, while in areas where rolling species could occur 6 more exclosure types were constructed. We replicated each experimental treatment six times for each dung type and we grouped units using the same dung type in blocks. Within each block, we set up experimental units in a fully randomized design. Individual experimental units were 60 cm apart, while different blocks were at least 2 m apart (figure 1C).

Dung removal and secondary seed dispersal experiments

As the presence of anthelmintic residues in dung affects the attractiveness of dung to dung beetles (Errouissi & Lumaret, 2010), we collected dung from animals that had not been treated in the preceding six weeks. This duration is considered long enough to suppress these attractive effects (Beynon, 2012).

Standardized amounts of dung (table S3) were put in the centre of each experimental plot and left on site for one month, after which the remaining dung was collected and quantified.

At the start of the experiment, fresh subsamples of each dung batch were taken and weighed for use as a reference sample. Subsequently, reference samples were oven-dried (80 °C) and the dry weight was measured (Minitial). At the end of the experiment, the remaining dung in the experimental units was collected, oven-dried and the dry mass was measured (Mfinal).

Dung removal ratio (DRR) was calculated as:

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DRR = (1)

In a subset of study sites, secondary seed dispersal by dung beetles was simultaneously measured during the dung removal experiments. Therefore, ten seeds of each of three seed classes (small,

medium, and large) were mixed with the dung portions at the start of the experiment (Sinitial, table S4). In order to facilitate the retrieval of seeds at the end of the experiment and to prevent germination during the experiment, seeds were pre-treated using fluorescent paint and heating using the methodology of Milotić et al. (2017). At the end of the experiment, the dried dung was crumbled

and the remaining seeds were counted (Sfinal).

Seed dispersal ratio (SDR) was calculated for each seed size class as:

SDR = (2)

Dung beetle sampling

During each experimental run, the dung beetle community was sampled in pitfall traps baited with the dung types used in the experiments. In 2013 and 2014, two types of pitfall traps were used in order to achieve a complete representation of dung beetle diversity and abundance. The first trap type consisted of 5 small containers surrounding a central dung pile, while the second trap type was one large container with dung on top (figure 1D). During the experiments in 2015 and 2016, sampling effort was lowered by using only the latter trap type (Milotić et al. (2018)). Traps were set up randomly between the experimental units with six replicates per dung type (figure 1C). In order to minimize interference with the early colonization phase in the experimental plots, traps were put in operation one week after the start of the experiments. We emptied traps weekly for three weeks. For each dung beetle species sampled, we counted the total number of individuals per trap and sampling date.

Data analysis

Dung beetle diversity

To measure the effects of geography and climate variables on species richness and dung beetle abundance, we used generalized linear modelling (GLM). Climate variables during the sampling periods were downloaded from the monthly summary observations map of (NOAA, 2016) by selecting the nearest climate station for each study site. Species richness was calculated as the sum of species sampled in each trap, while dung beetle density was the total number of dung beetles in each trap. Full GLMs were built using either species richness or dung beetle density as the response variable and latitude, longitude, mean temperature and total monthly precipitation as predictor variables. We used a combined forward and backward stepwise model selection approach for simplifying the model based on AIC-values on all GLM models. As we were particularly interested in

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the distribution of dung beetle functional groups, we made additional models for each dung beetle functional group using density as a response variable.

In order to create an overview of the local species richness, we calculated Shannon Weaver diversity indices and evenness for each study site (Hill, 1973). We transformed the Shannon Weaver index to the effective number of species according to the method of Jost (2009).

The relation between resource specialization and geography (research question 2) was explored using linear regression models with the percentage of resource specialists (based on the total dung beetle density) as the response variable and latitude, longitude, mean temperature and total monthly precipitation as predictor variables.

Dung removal and secondary seed dispersal

In order to get an overview of the regional differences in dung removal by the complete local fauna assemblage, we selected the experimental plots without any walls or ground screens. Using this subset of the experiment data, we built a GLM with DRR as the response variable and geography (latitude, longitude) and climate (mean temperature, total monthly precipitation) as predictor variables. In the next step, the effects of incomplete dung fauna assemblages on ecosystem functioning was explored by GLM using the complete experimental dataset. Four different models were built with either DRR (equation 1) or SDR for small, medium or large sized seeds (equation 2) as the response variable and with geography (latitude, longitude), climate (mean temperature, total monthly precipitation), dung fauna metrics (total dung beetle abundance, the proportions of each dung beetle functional group (dwellers, small tunnellers, large tunnellers, and small rollers), and the presence or absence of soil macro-invertebrates (small or large) as predictor variables.

In order to gain further insight in the relative contribution of each functional group to the ecosystem function of dung removal, we built hierarchical partitioning models for each study site. Using this multiple regression technique, we were able to estimate the independent effect of each functional group on dung removal. We obtained significance levels for the independent effect of each predictor variable after running 1000 randomization tests.

To test the validity of our methodology for studying seed dispersal and to test whether dung beetles actively select dung particles with or without seeds, we performed simple linear analyses with SDR as the response variable and DRR as the predictor variable. All analyses were performed in R version 3.4.2. (R Core Team, 2017). The ‘hier.part’ package was used to build hierarchical partitioning models (Walsh & Mac Nally, 2013).

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Results

Dung beetle assemblage

In total, we sampled 34,994 specimens belonging to 94 Scarabaeoidea species. We assigned species to four of the five predetermined functional groups: dwellers, large and small tunnellers and small rollers (52, 8, 32 and 2 species, respectively). Apart from the study sites in the Mediterranean sclerophyl region (La Fage) and the Caucaso-Iranian highlands (Shahrekord), no rollers were recorded and even in these sites their share in the overall dung beetle community was very small. In addition, the distribution of large tunneller species of the Geotrupidae family was mainly limited to the sites in the Atlantic, Central European highlands, and Pannonian region (table S5). We found the highest species diversity at Bugac in the Pannonian region, while individual sampling units contained most specimens at the La Fage site in the Mediterranean region (table S6). Overall, more species were found in the western study sites and dung beetles were more abundant in western and southern areas (figure 2). Study sites with a high mean temperature during the experiments contained more species, whereas a negative relationship was found between the total monthly precipitation and species richness or dung beetle abundance. Similar trends were found for the abundance of each of the functional groups separately, with the exception of the negative relationship between temperature and the abundance of large tunnellers. Furthermore, the few species found in northern areas are mostly dung resource generalists, whereas richer dung beetle assemblages at lower latitudes are more variable and contain a greater proportion of resource specialists. In general, the proportion of resource specialists is negatively affected by precipitation (figure 2 and table S7).

Dung removal and secondary seed dispersal

Dung removal ratios clearly differed between regions when the local dung fauna was left intact (figure 3). More dung was removed at higher latitudes, while the opposite effect was found for increasing longitudes. At higher mean temperatures, dung removal ratios were lower, whereas a positive relation was found between dung removal and total monthly precipitation.

The presence of dwellers, small tunnellers, small rollers and small and large macro-invertebrates had a clear positive effect on dung removal, whereas the presence of large tunnellers did not result in increased dung removal (table 1). At the study site level, both large and small tunnellers were significant dung removers when abundant (e.g., in the Hungarian sites and southern European sites) (figure 4). In the Mediterranean region, rollers were significant dung removers whereas dwellers had an important role in dung removal at The Zwin, Steinbühl, Tähtvere parish, Moor House National Nature Reserve, Shahrekord, Bayreuth, Bavarian Forest National Park, and Bugac. Soil macro- invertebrates were particularly important in Le Chesnoy, The Zwin, Swindon, Tange Sayad, Bayreuth, and Lygra.

Likewise, the presence of functional groups affected secondary seed dispersal. More seeds were dispersed when tunnellers were able to enter the experimental plots, whereas macro-invertebrates other than dung beetles had the opposite effect. Furthermore, dwellers generally had a negative effect on seed dispersal. Mean monthly temperature and total precipitation had a strong positive and negative effect, respectively, for most seed size classes (table 1). We found strong positive

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correlations between secondary seed dispersal and dung removal in all studied biogeographic regions (figure S1), although there were differences among seed sizes with a higher dispersal ratio for smaller seeds.

Discussion

Functional composition

Northernmost regions were dominated by dwellers, while tunnellers and rollers become more prominent with decreased latitude. This corresponds well with the known biogeographic distribution of dung beetle functional groups in the northern temperate hemisphere (Hanski & Cambefort, 1991), although roller species were scarce in our study. In general, however, Geotrupidae are known to be present in northern areas such as the British Isles and Scandinavia (Rosenlew & Roslin, 2008). Although species diversity at most sites fell within the range of expectations (Hortal et al., 2011), it is still possible that our sampled dung beetle assemblages were incomplete at the time of sampling due to stochastic variations in weather conditions, phenological population peaks, or the abundance of resources in the vicinity. Another explanation for rather low species diversity and the limited number of functional groups in some regions might be the global decline in dung beetle abundance and diversity. Roller species in particular have declined in southern Europe since the 1950s, in relation to land use changes, the use of anthelmintics and increasing urban development (Carpaneto et al., 2007). The trend of increasing complexity in dung beetle assemblage composition with decreasing latitude, and to a minor extent with increasing longitude, could be the result of the location of the 0°C isotherm both currently and during the last-glacial period (Hortal et al., 2011). Hence, the current distribution of dung beetle species in the Western Palaearctic is most likely defined by the location of refuge areas during the last glacial period, resulting in high levels of endemism and specialism in southern Europe (Lumaret & Lobo, 1996). Due to the close link between functional groups’ spatial distribution and temperature, a global increase in temperature might result in more diverse dung beetle assemblages in northern regions, while a lowered diversity is expected in southern regions of the Western Palaearctic (Dortel et al., 2013).

Ecosystem functions of dung removal and secondary seed dispersal

The presence of dung beetles results in improved ecosystem functioning in terms of dung removal and secondary seed dispersal. Dwellers, being the most abundant functional group in the northern regions, did not contribute very much to dung removal in these regions. Dwellers in the Iberian highlands on the other hand, removed a significant amount of dung although the functional group was underrepresented in the sampled dung beetle assemblage. Possibly, larval development and dung consumption by dweller larvae are faster at these sites compared with the northern sites, and could be related to higher summer temperatures (Stevenson & Dindal, 1985). Furthermore, the presence of tunnellers in southern regions might change the physical properties of dung into a more suitable habitat for dwellers, for example by increasing the dung surface. However, our method to measure dung removal by dwellers did not exclude other airborne coprophilous fauna that colonised

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the dung. With the exception of the covered treatment plots, none of the other treatments prevented oviposition by dung flies. As the larvae of dung-breeding flies play an important role in the dung ecosystem and are highly abundant and globally distributed (Hanski & Cambefort, 1991), the measured dung removal might be partially attributed to dung flies. Furthermore, we should note that tunnellers and rollers were also able to feed on the dung in the roofless treatment plots designed to measure the impact of dweller species, but were not able to move dung by tunnelling or rolling.

Many other soil macro-invertebrate groups are often found in dung, but are rarely considered as true members of the dung community (Floate, 2011). Such species are most often found in the later stages of dung degradation and are often casual visitors from adjacent habitats (e.g., woodlice (Isopoda), springtails (Collembola), and earthworms (Oligochaeta)). Especially in the cool and wet climate of northern Europe, earthworms can fulfil an important role in dung decomposition (Gittings et al., 1994) although their overall contribution is much lower compared with large tunnelling species (Kaartinen et al., 2013). According to Rosenlew and Roslin (2008), more dung is removed by large tunnellers compared with earthworms when both groups were present. However, despite the fact that earthworms and tunnellers perform similar roles in ecosystem functioning by vertically transporting dung (increasing soil respiration, and water and carbon content (Hendriksen, 1997)), these groups are not equivalent as earthworms tend to remove less dung but bury deeper (Holter, 1979). In the Mediterranean region, small rollers removed a disproportionately high amount of dung, which contrasts with some research results from (sub)tropical regions where tunnellers are considered the most important dung removers (e.g., Davis (1996), Slade et al. (2007)) and where larger beetles account for more dung removal (Braga et al., 2013). The high contribution of rolling species in southern Europe also highlights the potential negative consequences of the on-going decline in the abundance and diversity of roller species in this region for the ecosystem functions measured here (Carpaneto et al., 2007).

The strong positive relationship between secondary seed dispersal and dung removal suggests that dung beetles did not distinguish between seed-containing dung and seed-free dung. This result has been found previously for relatively small seeds (e.g., 4 mm seeds in Andresen (2002), 3.5 mm in Slade et al. (2007)), although the number of buried seeds increased with dung pat size and dung beetle size (Andresen & Feer, 2005). Moreover, the relationship might switch from positive to negative, if the proportion of seeds present in the dung is higher (Shepherd & Chapman, 1998). In contrast to most earlier studies of secondary seed dispersal by dung beetles, we used real seeds instead of plastic proxies. Although this approach is ecologically more realistic, dung beetles might handle real seeds differentially not only with respect to size but also with respect to morphology (shape, seed appendages) and smell. As a consequence of the applied experimental setting, a portion of missing seeds could have been removed (dispersed) and/or destroyed (predated) by individuals of other animal groups than the studied focal groups (e.g., ants, rodents, birds) (Andresen & Levey, 2004; Hulme & Kollmann, 2004).

Secondary seed dispersal by dung beetles may have a direct impact on the reproductive success of plants (Shepherd & Chapman, 1998; Nichols et al., 2008). The distribution of seeds away from their original dropping site can help lower the level of competition between seedlings (Andresen & Levey, 2004) and the impact of above-ground seed predators (Manzano et al., 2010). However, one of the main determinants of whether seeds germinate after secondary dispersal is the depth at which they are buried (Andresen & Feer, 2005), which differs between dung beetle species (D'hondt et al., 2008)

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and the specific germination requirements of the plant species (Limón & Peco, 2016). Rollers might provide optimal conditions for successful germination as they move seed-containing dung away in a horizontal direction and bury dung shallowly. Similarly, earthworms deposit consumed dung as casts in the soil in the upper 2 cm, where most plant species should be able to germinate (Hendriksen, 1997). By contrast, many tunnellers make deep vertical shafts below the dung pat (up to 150 cm depth; e.g., Typhaeus typhoeus (L.); Brussaard (1985)) probably suppressing germination (Fenner & Thompson, 2005). Dwellers might release seeds from the dung substrate, thus improving light availability but in many cases they will not provide as much seed-soil contact as predominantly burying species, leading to lower water availability for germination (Fenner & Thompson, 2005).

The conservation of functionally complete species assemblages is essential in order to maintain ecosystem functioning (Manning et al., 2016). Given the clear north-south shift in functional group composition and the generally high dispersal ability of dung beetles, we can assume significant changes in functional group composition due to climate change. This could lead to an increased dung removal and secondary seed dispersal rate in the northern regions, while in southern regions the combination of more frequent and longer heat waves and a drier climate might result in the disappearance of vulnerable functional groups (e.g., rollers which typically make nests close to the surface) and associated ecosystem functions (Menéndez et al., 2014; Slade & Roslin, 2016). Experimental manipulations of local dung beetle assemblages (e.g., through the introduction of new species in a mesocosm experiment) could provide further knowledge of how change in dung beetle assemblages affect ecosystem functioning.

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Acknowledgments

This study is the result of a pan-European multi-site project, financially supported by the ALTER-Net consortium, Europe’s Ecosystem Research Network. The project was co-financed within their multi-site research programme. Yves Israel, Robbe De Beelde, Viki Vandomme, Angelica Alcantara, Linda Stevens, Tessa Van Santen and Martine Van Hove provided practical and logistical assistance in cutting gauze and sending the experimental material to the participants. We thank Stefan Van Damme for testing the experimental materials and fine-tuning the experimental protocol in a pilot study. JAN was funded by COLCIENCIAS (Colombia). We thank Eva Cuesta, Maria Leo Montes, Ana M.C. Santos, Indradatta de Castro, Joaquin Hortal, Andrew Smithson, Matthew Rowlinson, Sally-Ann Spence, Berrycroft Farm, Capucine Tourret, Yoann Despres, Hélène Benoît-Valiergue, Claire Debulois, Rachel Barrier, Catherine Menuet, Mélanie Picard, Yves Boscardin, Marie Hauso, Joanne Inchbald, Sayuri Kiyota Beltran, Wilhelm Karch, Anne Drozynski, Anna Paquet, Anne Raber, Florian Samsony, Philipp Wagner, Helmut Hackl, Josef Nußhard, Dennis Müller, Ferenc Sipos, Zoltán Vajda, Márk Lucza, Irja Kivimägi, Marius Bujor, Mirona Zovot, Tudor Racoviceanu and Mr. Darakhshon for their assistance during the field work.

We thank the Community of Madrid, the Natural Protect Area of Soto de Viñuelas, the Regional Park of Cuenca Alta del Manzanares, Environmental Change Network, LEGTA “Le Chesnoy - Les Barres”, Sara Parisot, the Agency for Nature and Forest (ANB), the Heathland Centre (Lyngheisenteret), NABU Kirchheimbolanden, LBV Lindenhof, Bayreuth, and the Braila Research Station for providing access and logistical help at the study sites. Local permits were obtained for entering the study areas and for sampling insects.

We thank two anonymous reviewers and the handling editor for their useful suggestion on an earlier version of this manuscript.

Data Accessibility statement

All data are available as open data. Dung beetle sampling data have been published as a sampling event dataset on GBIF (https://www.gbif.org/dataset/bcbfd319-8813-4b6d-b529-07dc5a6ccf56). Results of the dung removal and secondary seed dispersal experiments can be accessed at Zenodo (https://zenodo.org/record/1115523#.WjEpHlXiapo).

Bio sketch

Tanja Milotić designed and completed this study during her PhD at the Terrestrial Ecology Unit (TEREC) of Ghent University, Belgium. Her research focuses on the dispersal of plant seeds through endozoochory and the effects of dung beetles on this process.

Author contributions

TM and MHo designed the study and applied for financing in the multi-site research program of the ALTER-Net consortium. TM, CB, CE, AE, MH, JM, JAN, RM, JS, RA, TB, IB, JB, JC, GB, PJR, MK, EM, AM, EA, EP, IR, RR, EMS and LS installed the experiment and collected field data. TM, MH, JM, JAN, RM, JB, CC, PJR, MK, EM, GM, AM, DR, EMS, LS and SZ identified dung beetles. TM did all analyses and led the writing in close cooperation with CB, CE, AE, MH, JM, JAN, RM, JS, JC and MHo. All authors discussed the analysis results and their interpretations in the manuscript.

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Tables

Table 1 – Model results of the generalized linear models for dung removal and seed dispersal by dung beetles in the Western Palaearctic. Dung removal and the dispersal of small, medium or large-sized seeds were used as response variables and geography (latitude and longitude), climate (mean temperature and total monthly precipitation, total dung beetle abundance, the proportion of each dung beetle functional group (dwellers, small tunnellers, large tunnellers, and small rollers), and the presence or absence of soil macro-invertebrates (small and large) as predictor variables. Only those variables that were retained after a combined forward and backward model selection procedure based on AIC are shown.

Response variable Predictor variable t-value p-value dung removal ratio intercept 12.723 <0.001 latitude -6.158 <0.001 longitude -10.702 <0.001 mean temperature -6.823 <0.001 total monthly precipitation 3.525 <0.001 dung beetle abundance 7.225 0.073 dwellers 2.440 <0.001 small tunnellers 4.049 0.015 small rollers 4.754 <0.001 small soil macro-invertebrates 8.914 <0.001 large soil macro-invertebrates 1.793 <0.001 small seed removal intercept 3.880 <0.001 longitude -9.661 <0.001 mean temperature 19.141 <0.001 total monthly precipitation -11.086 <0.001 dung beetle abundance 4.366 <0.001 dwellers -3.537 <0.001 small tunnellers 4.095 <0.001 large tunnellers 5.649 <0.001 small soil macro-invertebrates -3.830 <0.001 medium seed removal intercept 3.895 <0.001 latitude -7.431 <0.001 longitude -8.312 <0.001 mean temperature 21.373 <0.001 dung beetle abundance 7.546 <0.001 dwellers -3.462 <0.001 small tunnellers 2.723 <0.001 large tunnellers 7.351 0.007 small soil macro-invertebrates -8.724 <0.001 large seed removal intercept -4.443 <0.001 latitude 9.997 <0.001 longitude -6.235 <0.001 mean temperature 2.102 0.036 total monthly precipitation -8.648 <0.001 dwellers 2.737 0.006 small tunnellers -1.513 0.130 small rollers 4.694 <0.001 small soil macro-invertebrates 1.896 0.058

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Figures

Figure 1 – Set-up of the multi-site experiment estimating dung removal and secondary seed dispersal by dung beetles in the Western Palaearctic. A. Position of the study sites shown within biogeographic provinces as defined by Udvardy (1975); the digital base map was adapted from FAO Geonetwork (2015). See table S1 for site names and coordinates. At study sites marked with an asterisk, a reduced set of experimental treatments was installed as no rollers were expected to be present at these sites. B. Experimental design with a randomized distribution of 11 types of experimental units and 2 types of dung beetle sampling units. In the second year of the experiment sampling efforts were reduced with 1 sampling unit type in a similar set-up. Experimental and sampling units were grouped by dung types in a fenced experimental area. Cattle dung was used as a standard dung type. At most study sites, the experiment was replicated with up to two additional dung types. All experimental and sampling unit types were replicated six times per experimental run. C. Treatments used in the dung removal and seed dispersal experiments. In each treatment unit, a standard amount of dung was put in the center of the plot, and functional groups were excluded using ground screens, walls and/or roofs made of fine (1 mm²) or coarse mesh (1 cm²). For each treatment, functional groups unable to move dung are crossed out. The colors of the crosses refer to the colors of the building materials (see legend). Treatments excluding roller activity were not installed in regions with known roller absence and are indicated with an asterisk (*). D. Sampling strategy with three different trap types consisting of: 5 small pitfalls surrounding a central dung pile (left), and 1 large pitfall covered with a dung ball in a nylon bag (middle) used in the first year of the experiment, and 1 large pitfall covered with a large unpacked dung pile (right) used in the second year.

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Figure 2 – GLM model results showing relationships between species richness, dung beetle abundance or the proportion of resource specialist (y-axes) and latitude (a, e, i), longitude (b, f, j), mean temperature (c, g, k), and total monthly precipitation (d, h, l). Model-predicted means (solid lines) and standard errors are plotted for significantly correlated variables. Full model results are provided in table S7.

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Figure 3 – Dung removal in complete dung fauna assemblages in the Western Palaearctic. Graphs show the relationship between the percentage of dung removed after one month and geographic (latitude (a), longitude (b)) and climatic variables (mean temperature (c), total monthly precipitation (d)). Significantly correlated variables after GLM analysis are plotted as solid lines with standard errors (grey). Full model results are provided in table S8.

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Figure 4 – Hierarchical partitioning results for dung removal by dwellers, tunnellers, rollers and soil macro-invertebrates in the Western Palaearctic. Displayed is the percentage of independent effect contributed by each functional group in each study site. Study sites are coded according to the codes in figure 1 and table S1 and are ordered by longitude. Asterisks indicate whether the contribution of a functional group is significant (p<0.050) in a particular study site. The level of significance of the independent contribution of each functional group was estimated using randomization tests based on 1000 permutations. On top of each bar R² values of the total model is shown for each study site.

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References

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SUPPORTING INFORMATION

Functionally richer communities improve ecosystem functioning: Dung removal and secondary seed dispersal by dung beetles in the Western Palaearctic

Tanja Milotić, Christophe Baltzinger, Carsten Eichberg, Amy E. Eycott, Marco Heurich, Jörg Müller, Jorge A. Noriega, Rosa Menendez, Jutta Stadler, Réka Ádám, Tessa Bargmann, Isabelle Bilger, Jörn Buse, Joaquín Calatayud, Constantin Ciubuc, Gergely Boros, Pierre Jay-Robert, Märt Kruus, Enno Merivee, Geoffrey Miessen, Anne Must, Elham Ardali, Elena Preda, Iraj Rahimi, Dirk Rohwedder, Rob Rose, Eleanor M. Slade, László Somay, Pejman Tahmasebi, Stefano Ziani and Maurice Hoffmann

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Table S1 – Name, country, biogeographic region and coordinates (longitude, latitude and elevation) of the study sites in the multi-site experiment estimating dung removal and secondary seed dispersal by dung beetles in the Western Palaearctic. Mapping codes refer to the numbers on the map in Error! Reference source not found.. Study sites are also described by habitat, elevation and soil type.

Mapping Biogeographic region Name Country EUNIS habitat type1 Soil type2 Latitude Longitude Elevation (m) code (Udvardy 1975) 1 Castillo de Viñuelas Spain Iberian highlands Mediterranean montane grassland vertic luvisol 40° 36' 49" N 03° 39' 50" W 680 2 Moor House National United Kingdom British islands Montane grassland humic gleysol 54° 39' 28'' N 02° 37' 29'' W 446 Nature Reserve 3 Swindon United Kingdom British islands Semi-improved eutric cambisol 51° 33' 54'' N 01° 19' 14'' W 80 grassland/agricultural land 4 Le Chesnoy France Atlantic grasslands gleyic luvisol 47° 47' 07'' N 02° 44' 55'' E 90 5 INRA, La Fage France Mediterranean sclerophyl Mediterranean montane grassland eutric cambisol 43° 55' 31'' N 03° 06' 34'' E 780 6 The Zwin Belgium Atlantic Permanent mesotrophic pastures calcaric fluvisol 51° 21' 45'' N 03° 22' 02'' E 3 and aftermath-grazed meadows 7 Kalmthout Belgium Atlantic temperate shrub heathland humic podzol 51° 23' 32'' N 04° 26' 05'' E 18 8 Lyngheisenteret, Lygra Norway West Eurasian taiga Grasslands orthic podzol 60° 41' 14'' N 05° 07' 44'' E 20 9 Steinbühl Germany Atlantic Permanent mesotrophic pastures dystric cambisol 49° 40' 54'' N 08° 00' 02'' E 320 on former arable land 10 Bayreuth Germany Central European highlands Permanent mesotrophic pastures stagnic gleysol 49° 55' 02'' N 11° 35' 17'' E 355 and aftermath-grazed meadows 11 Bavarian Forest Germany Central European highlands Permanent mesotrophic pastures dystric cambisol 48° 49' 58'' N 13° 23' 53'' E 1150 National Park and aftermath-grazed meadows 12 Vácrátót Hungary Middle European forest Grasslands chromic 47° 42' 21'' N 19° 13' 47'' E 176 cambisol 13 Bugac Hungary Pannonian Grasslands calcaric regosol 46° 39' 23'' N 19° 37' 10'' E 106 14 Tähtvere parish Estonia Boreonemoral Permanent mesotrophic pastures eutric 58° 22' 20'' N 26° 35' 01'' E 67 and aftermath-grazed meadows podzoluvisol 15 Braila Islands Romania Pontian steppe grasslands calcaric fluvisol 45° 25' 08'' N 28° 02' 47'' E 2 16 Shahrekord Iran Caucaso-Iranian highlands Perennial calcareous grassland and calcic xerosol 32° 21' 43'' N 50° 49' 52'' E 2055 basic steppes* 17 Tange Sayad Iran Caucaso-Iranian highlands Perennial calcareous grassland and calcic xerosol 32° 16' 42'' N 51° 01' 17'' E 2113 basic steppes* 1 as defined in the European nature information system (EUNIS) by the European Environmental Agency (EEA, http://eunis.eea.europa.eu/). 2 as defined in the World Reference Base for Soil Resources (WRB) (IUSS Working Group, 2006). * EUNIS habitat type was identified using the definitions of the types by the EEA in case study areas fall outside the borders of the pan-European classification.

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Table S2 – Timing of the experiments, dung types used in the experiments and grazer species found in the vicinity of the study areas.

Mapping code Name Timing experiments Used dung types Nearby grazer species 1 Castillo de Viñuelas June 2016 cattle cattle 2 Moor House National Nature Reserve mid June-mid July 2014, July 2015 cattle, sheep sheep 3 Swindon mid June-mid July 2014 cattle cattle, sheep 4 Le Chesnoy June 2014*, June 2015 cattle, sheep, red deer cattle, horse, sheep, red deer 5 INRA, La Fage May 2015 sheep sheep 6 The Zwin Aug 2014, mid Sept-mid Oct 2014 cattle, horse, sheep cattle, horse, sheep 7 Kalmthout Aug 2014, mid Sept-mid Oct 2014, mid Sept-mid Oct 2015 cattle, horse, sheep cattle, horse, sheep 8 Lyngheisenteret, Lygra Aug 2014 cattle, horse, sheep sheep 9 Steinbühl June 2014, June 2015 cattle, horse cattle, horses 10 Bayreuth June 2014, July 2015 cattle, horse, sheep cattle, horse, sheep 11 Bavarian Forest National Park Aug 2014, July 2015 cattle, horse cattle, horse, red deer 12 Vácrátót June 2015, mid Sept-mid Oct 2015 cattle, horse, sheep cattle, horse, sheep 13 Bugac June 2014, June 2015, mid Sept-mid Oct 2015 cattle, horse, sheep cattle, horse, sheep 14 Tähtvere parish June 2014, mid June-mid July 2015 cattle, sheep cattle, sheep 15 Braila Islands July 2014 cattle, horse, sheep cattle, horse, sheep 16 Shahrekord Sept 2013, Oct 2013, mid Nov -mid Dec 2014, May-June 2015 cattle, sheep, goat cattle, sheep 17 Tange Sayad Aug 2013, Nov 2013, mid Nov -mid Dec 2014, May-June 2015 cattle, sheep, goat cattle, sheep * Experimental run limited to 14 days due to adverse meteorological conditions.

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Table S3 – Dung types used in the dung and seed removal experiments and the amount used in each year. Study sites refer to the mapping codes (see table S1).

Dung type study sites year mass (g) cattle 16, 17 2013-2015 300 2, 3, 4, 6, 7, 8, 9, 10, 11, 13, 14, 15 2014 300

2, 4, 7, 10, 11, 12, 13, 14 2015 500

9 2015 600

1 2016 500 red deer 4 2014-2015 300 goat 16, 17 2013-2015 300 horse 6, 7, 8, 9, 10, 11, 13 2014 300 10, 11, 12, 13 2015 500 sheep 16, 17 2013-2015 300 6, 7, 8, 10, 13 2014 200

2, 4, 14, 15 2014 300

2, 5, 7, 12, 13, 14 2015 100

10 2015 500

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Table S4 – Plant species used in the secondary seed dispersal experiments with their diaspore morphology (with Fa= fruit with appendages, MC= mericarp), minimum and maximum diaspore mass ('m'), length ('l'), width ('w') and height ('h') as defined in the BiolFlor database (www.ufz.de/biolflor, accessed January 2017), seed shape, seed size class used in the analyses, and seed number per dung pat. Study sites refer to the mapping codes (see table S1).

species diaspore type m (mg) l (mm) w (mm) h (mm) seed shape seed size class seed number study sites Alopecurus myosuroides Fa 2.0-2.8 4.0-7.5 1.4-2.3 0.5-1.1 elongated large 10 2, 4, 6, 7, 9, 11, 14, 15 Galium aparine MC 5.7-9.9 1.9-7.0 1.9-4.0 1.5-3.5 spherical medium 10 2, 4, 6, 7, 9, 11, 14, 15 Poa pratensis Fa 0.2-0.4 2.2-4.0 0.6-1.0 0.3-0.8 elongated small 10 2, 4, 6, 7, 9, 11, 14, 15

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Table S5 – Dung beetle species abundance (with functional group assignment) by study area (numbered columns), followed by total abundance and species richness by functional group, summed for all experimental runs at each study site. Functional groups (FG) are coded as follows: D= dwellers, Rs= small rollers, Tl= large tunnellers, and Ts= small tunnellers. Habitat specialisation (HS) is coded as follows: G= generalists, S= specialists, and NA= no data available. Habitat specialisation data were adapted from Buse et al. (2015), Carpaneto, Mazziotta, Pittino, and Luiselli (2011), Frolov and Akhmetova (2013), Gharakloo and Ziani (2010), IUCN (2016), Janssens (1960) and Jessop (1986). Dung beetle nomenclature and systematics follow Löbl and Löbl (2016).

Species FG HS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Geotrupidae Anoplotrupes stercorosus (Scriba, 1791) Tl G 0 0 0 0 0 0 0 0 0 1 36 0 0 0 0 0 0 Geotrupes mutator (Marsham, 1802) Tl S 0 0 0 0 0 0 0 0 0 0 0 0 81 0 0 0 0 Geotrupes spiniger (Marsham, 1802) Tl G 0 0 0 0 0 11 118 0 0 1 0 10 2 0 0 0 0 Geotrupes stercorarius (Linnaeus, 1758) Tl G 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 Trypocopris vernalis (Linneus, 1758) Tl S 0 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 Typhaeus typhoeus (Linneus, 1758) Tl S 0 0 0 0 0 0 483 0 0 0 0 0 0 0 0 0 0 Scarabaeidae Aphodiinae Acanthobodilus immundus (Creutzer, 1799) D S 0 0 0 0 0 0 0 0 0 0 0 4 5 0 0 0 0 Acrossus depressus (Kugelann, 1792) D S 0 321 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acrossus luridus (Fabricius, 1775) D S 0 0 0 1 456 0 0 0 1 0 0 0 0 0 0 1 0 Acrossus rufipes (Linnaeus, 1758) D G 0 4 1 0 0 91 67 3 25 4 2 0 0 0 0 0 0 Agoliinus lapponum (Gyllenhal, 1808) D G 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Agrilinus ater (De Geer, 1774) D G 0 242 13 70 0 1 0 0 0 15 0 0 0 38 0 0 0 Aphodius fimetarius (Linnaeus, 1758) D G 0 0 3 36 0 2 5 0 1 48 0 73 108 4 0 0 0 Aphodius foetidus (Herbst, 1783) D S 13 0 0 1 0 0 0 0 0 0 0 0 29 0 0 0 0 Aphodius sp. D NA 0 0 0 1 0 0 0 0 0 0 1 0 0 2 4 0 0 Biralus satellitius (Herbst, 1789) D S 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 Bodiloides ictericus (Laicharting, 1781) D S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 Bodilopsis rufa (Moll, 1782) D G 0 0 2 0 0 61 24 0 1 0 0 27 8 0 0 0 0 Bodilopsis sordida (Fabricius, 1775) D S 0 0 0 0 0 0 0 0 0 0 0 9 1257 5 0 0 0 Bodilus lugens (Creutzer, 1799) D S 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 Calamosternus granarius (Linnaeus, 1767) D G 0 0 0 1 0 0 0 0 0 0 1 0 43 0 0 35 0 Calamosternus hyxos (Petrovitz, 1962) D G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 Chilothorax distinctus (Müller, 1776) D G 0 0 0 0 4 0 6 0 0 0 1 51 6 0 0 0 0

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Species FG HS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Chilothorax lineolatus (Illiger, 1803) D S 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Table S5 continued. Species FG HS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Chilothorax melanostictus (Schmidt, 1840) D S 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 1 4 Chilothorax sp. D NA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 146 17 Colobopterus erraticus (Linnaeus, 1758) Ts S 111 0 4 29 4 0 2 0 52 97 0 14 0 19 1 0 0 Coprimorphus scrutator (Herbst, 1789) D S 5 0 0 0 0 0 0 0 1 0 0 10 64 0 0 0 0 Esymus merdarius (Fabricius, 1775) D S 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 0 0 Esymus pusillus (Herbst, 1789) D S 0 0 0 216 3 0 0 0 36 24 2 37 1 27 0 0 0 Eudolus quadriguttatus (Herbst, 1783) D S 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 Euheptaulacus sus (Herbst, 1783) D S 0 0 0 0 0 0 0 0 0 0 0 0 1569 0 0 0 0 Euorodalus paracoenosus (Balthasar & Hrubant, 1960) D G 0 0 0 0 0 0 0 0 0 0 0 90 431 0 0 0 0 Euorodalus tersus (Erichson, 1848) D S 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Eupleurus subterraneus (Linnaeus, 1758) D S 0 0 0 0 0 0 0 0 0 0 0 2 3 0 5 0 0 Labarrus lividus (Olivier, 1789) D S 0 0 0 0 0 0 0 0 0 0 0 0 264 0 0 0 0 Loraphodius suarius (Faldermann, 1832) D G 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 Melinopterus consputus (Creutzer, 1799) D S 0 0 0 0 4 1 0 0 0 0 0 1 47 0 0 0 0 Melinopterus prodromus (Brahm, 1790) D G 0 0 0 5 0 89 1667 0 0 1 0 14 0 0 0 1 0 Melinopterus reyi (Reitter, 1892) D NA 0 0 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 Melinopterus sphacelatus (Panzer, 1798) D G 0 0 16 0 0 68 42 0 0 0 0 0 0 0 0 0 0 Nialus varians (Duftschmid, 1805) D S 0 0 0 1754 0 0 0 0 0 0 0 1 4 0 2 0 0 Nimbus contaminatus (Herbst, 1783) D S 0 0 0 0 0 435 4737 0 0 0 0 0 0 0 0 0 0 Nimbus obliteratus (Panzer, 1823) D G 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 Nobiellus inclusus (Reitter, 1892) D S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 51 17 Nobius serotinus (Panzer, 1799) D G 0 0 0 0 0 0 0 0 0 0 0 0 70 0 0 0 0 Otophorus haemorrhoidalis (Linnaeus, 1758) D G 2 0 0 421 0 25 1 0 49 19 6 58 694 16 1 0 0 Oxyomus sylvestris (Scopoli, 1763) D G 0 0 0 1 0 0 0 0 0 5 0 0 70 1 0 0 0 Parammoecius gibbus (Germar, 1817) D S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 Phalacronothus biguttatus (Germar, 1824) D S 0 0 0 0 0 0 0 0 0 0 0 44 16 0 0 0 0 Planolinus fasciatus (Olivier, 1789) D S 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 Plagiogonus putridus (Geoffroy, 1785) D S 0 0 0 0 0 0 0 0 0 0 0 22 1 0 0 0 0

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Species FG HS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Rhodaphodius foetens (Fabricius, 1787) D S 0 0 0 0 0 34 30 0 0 0 0 23 1371 0 0 0 0 Sigorus porcus (Fabricius, 1792) D S 0 0 0 0 0 0 0 0 0 0 0 101 206 0 0 0 0 Table S5 continued. Species FG HS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Subrinus sturmi (Harold, 1870) D G 0 0 0 0 0 0 0 0 0 0 0 0 121 0 0 0 0 Subrinus vitellinus (Klug, 1845) D S 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Teuchestes fossor (Linnaeus, 1758) D G 0 0 1 19 0 7 4 0 3 32 0 0 0 24 0 0 0 Trichonotulus scrofa (Fabricius, 1787) D S 0 0 0 14 10 0 0 0 0 0 0 0 458 0 0 0 0 Volinus sticticus (Panzer, 1798) D G 0 0 0 0 0 30 9 0 0 0 2 4 1 0 0 0 0 Scarabaeinae Caccobius mundus (Ménétriès, 1838) Ts S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 Caccobius schreberi (Linnaeus, 1767) Ts S 7 0 0 0 0 0 0 0 0 0 0 514 440 0 21 0 0 Copris hispanus Linnaeus, 1764 Tl S 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Copris lunaris (Linnaeus, 1758) Tl S 0 0 0 0 0 0 0 0 0 0 0 3 127 0 0 0 0 Euoniticellus fulvus (Goeze, 1777) Ts S 15 0 0 13 1 0 0 0 0 0 0 168 664 0 27 0 0 Euoniticellus pallipes (Fabricius, 1781) Ts S 0 0 0 0 0 0 0 0 0 0 0 0 53 0 0 0 0 Euonthophagus amyntas (Olivier, 1789) Ts S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 53 0 Euonthophagus gibbosus (Scriba, 1790) Ts S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 Euonthophagus mostafatsairii Palestrini, Varola & Zunino, 1979 Ts S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 Gymnopleurus qurosh Montreuil, 2011 Rs S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 Onitis belial Fabricius, 1798 Ts S 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Onthophagus amirus Kabakov, 1982 Ts S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 Onthophagus coenobita (Herbst, 1783) Ts G 0 0 0 77 0 2 2 0 3 0 0 138 1 0 0 0 0 Onthophagus dorsosignatus D'Orbigny, 1898 Ts S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 Onthophagus emarginatus Mulsant, 1842 Ts S 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 Onthophagus fissicornis (Steven, 1809) Ts S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11 0 Onthophagus fracticornis (Preyssler, 1790) Ts G 0 0 0 0 180 0 0 0 0 0 0 2268 316 0 0 0 0 Onthophagus furcatus (Fabricius, 1781) Ts G 81 0 0 0 0 0 0 0 0 0 0 84 149 0 0 0 0 Onthophagus gibbulus (Pallas, 1781) Ts S 0 0 0 0 0 0 0 0 0 0 0 0 220 0 0 0 0 Onthophagus grossepunctatus Reitter, 1905 Ts S 0 0 0 0 46 0 0 0 0 0 0 2 0 0 0 0 0 Onthophagus illyricus (Scopoli, 1763) Ts S 0 0 0 0 0 0 0 0 0 0 0 242 59 0 0 0 0

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Species FG HS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Onthophagus joannae Goljan, 1953 Ts G 3 0 0 199 641 0 0 0 0 6 0 0 0 0 0 0 0 Onthophagus lemur (Fabricius, 1781) Ts S 0 0 0 0 717 0 0 0 0 0 0 0 0 0 0 0 0 Onthophagus medius (Kugelann, 1792) Ts S 0 0 0 7 375 0 0 0 3 0 0 0 0 0 0 0 0 Table S5 continued. Species FG HS 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Onthophagus nuchicornis (Linnaeus, 1758) Ts S 0 0 0 0 0 0 0 0 0 0 0 23 433 0 0 0 0 Onthophagus opacicollis Reitter, 1892 Ts G 431 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Onthophagus ovatus (Linnaeus, 1767) Ts G 0 0 0 1045 0 0 0 0 134 1 0 232 0 0 1 0 0 Onthophagus pygargus Motschulsky, 1845 Ts S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 46 0 Onthophagus ruficapillus Brullé, 1832 Ts S 0 0 0 0 519 0 0 0 0 0 0 111 246 0 0 7 0 Onthophagus similis (Scriba, 1790) Ts G 0 0 0 0 0 28 116 0 0 0 0 0 0 0 0 0 0 Onthophagus taurus (Schreber, 1759) Ts S 5 0 0 487 0 0 0 0 0 0 0 353 2188 0 102 0 0 Onthophagus vacca (Linnaeus, 1767) Ts G 11 0 0 60 0 0 0 0 0 0 0 21 18 0 0 0 0 Onthophagus verticicornis (Laicharting, 1781) Ts G 0 0 0 18 84 0 0 0 0 0 0 2 0 0 0 0 0 Onthophagus sp. Ts NA 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 0 0 Sisyphus schaefferi (Linnaeus, 1758) Rs S 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 Total number 691 569 40 4480 3064 885 7315 3 309 254 51 4760 11855 136 173 371 38 dwellers (D) 24 569 36 2545 478 844 6592 3 117 148 15 575 6858 117 16 240 38

small rollers (Rs) 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 2 0

large tunnellers (Tl) 2 0 0 0 6 11 603 0 0 2 36 13 210 0 0 0 0

small tunnellers (Ts) 665 0 4 1935 2568 30 120 0 192 104 0 4172 4787 19 157 129 0 Species richness 16 4 7 23 18 15 17 1 12 13 8 36 44 9 12 18 3 dwellers (D) 6 4 6 14 6 12 11 1 8 8 7 20 29 8 6 8 3

small rollers (Rs) 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0

large tunnellers (Tl) 1 0 0 0 1 1 3 0 0 2 1 2 3 0 0 0 0

small tunnellers (Ts) 9 0 1 9 10 2 3 0 4 3 0 14 12 1 6 9 0 * Aphodius fimetarius s.l. should be regarded as a species complex of two distinct species, as genomic and morphologic differences exist between Aphodius fimetarius and Aphodius pedellus (Miraldo, Krell, Smalén, Angus, & Roslin, 2014), or more recently A. fimetarius and Aphodius cardinalis (Fery & Rössner, 2015) have been described.

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Table S6 – Diversity indices for biogeographic region (Udvardy, 1975) and study site (with mapping codes between brackets, see figure 1). Nt= number of baited pitfall traps, Ni= number of individuals, S= number of species, H'= Shannon-Weaver index (using the effective number of species transformation (Jost, 2009)), J= evenness (Hill (1973)).

Biogeographic region Study site Nt Ni S H' J Atlantic Le Chesnoy (4) 48 4480 23 6.23 0.59 The Zwin (6) 72 885 15 5.93 0.66

Kalmthout (7) 84 7315 17 2.94 0.38

Steinbühl (9) 30 309 12 5.05 0.65 Boreonemoral Tähtvere parish (14) 36 136 9 6.23 0.83 British islands Moor House NNR (2) 36 569 4 2.10 0.54 Swindon (3) 12 40 7 4.44 0.77

Caucaso-Iranian highlands Shahrekord (16) 84 371 18 6.55 0.65 Tange Sayad (17) 36 38 3 2.61 0.87 Central European highlands Bayreuth(10) 54 254 13 6.36 0.72 Bavarian Forest NP (11) 35 51 8 3.03 0.53

Iberian highlands Castillo de Viñuelas (1) 6 691 16 3.60 0.46 Mediterranean sclerophyl INRA, La Fage (5) 10 3064 18 6.89 0.67 Middle European forest Vácrátót (12) 54 4760 36 8.25 0.59 Pannonian Bugac (13) 90 11855 44 15.80 0.73 Pontian steppe Braila Islands (15) 36 173 12 4.01 0.56 West Eurasian taiga Lygra (8) 36 3 1 1.00 -

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Table S7 – Results of the generalized linear models with dung beetle abundance or species richness as the response variable and geographic and climate variables as predictor variables. The estimated coefficients, standard errors (SE), t-values and p- values are shown of those variables which were retained after a combined forward and backward model selection procedure based on AIC.

Response variable Functional group Predictor variable Estimate SE t-value p-value Abundance all groups intercept 9.914 2.033 4.875 <0.001 latitude -0.091 0.040 -2.243 0.027 longitude -0.045 0.016 -2.894 0.005 total monthly precipitation -0.012 0.004 -2.776 0.006 dwellers intercept 7.692 1.947 3.952 <0.001 latitude -0.055 0.039 -1.405 0.163 longitude -0.041 0.015 -2.753 0.007 total monthly precipitation -0.016 0.004 -3.589 <0.001 small tunnellers intercept 12.670 2.047 6.190 <0.001 latitude -0.195 0.042 -4.664 <0.001 large tunnellers intercept 4.861 1.021 4.761 <0.001 mean temperature -0.144 0.054 -2.652 0.010 total monthly precipitation -0.010 0.005 -2.127 0.038 resource specialists intercept 0.585 0.103 5.687 <0.001 latitude -0.022 0.007 -3.309 0.002 longitude 0.007 0.003 2.220 0.010 total monthly precipitation -0.003 0.001 -2.445 0.038 Richness all groups intercept 2.626 0.537 4.894 <0.001 longitude -0.019 0.007 -2.475 0.015 mean temperature 0.056 0.027 2.055 0.042 total monthly precipitation -0.008 0.003 -2.871 0.005

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Table S8 – Results of the generalized linear models with dung removal in complete dung fauna assemblages as response variable and geographic and climate variables as predictor variables. The estimated coefficients, standard errors (SE), t- values and p-values are shown of those variables that were retained after a combined forward and backward model selection procedure based on AIC.

Predictor variable Estimate SE t-value p-value intercept 1.064 0.135 7.903 <0.001 latitude -0.012 0.003 -4.304 <0.001 longitude -0.005 0.001 -5.487 <0.001 mean temperature -0.006 0.002 -2.292 0.022 total monthly precipitation 0.001 0.000 4189 <0.001

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Figure S1 – Seed dispersal ratio vs. dung removal ratio by seed size classes (with green= small-sized, blue= medium-sized, and red= large-sized seeds).

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References

Buse, J., Šlachta, M., Sladecek, F. X. J., Pung, M., Wagner, T., & Entling, M. H. (2015). Relative importance of pasture size and grazing continuity for the long-term conservation of European dung beetles. Biological Conservation, 187, 112-119. doi:10.1016/j.biocon.2015.04.011 Carpaneto, G. M., Mazziotta, A., Pittino, R., & Luiselli, L. (2011). Exploring co-extinction correlates: the effects of habitat, biogeography and anthropogenic factors on ground squirrels–dung beetles associations. Biodiversity and Conservation, 20(13), 3059-3076. Fery, H., & Rössner, E. (2015). Notes on the Aphodius (s. str.) fimetarius-complex–morphology, taxonomy, nomenclature and worldwide distribution (with emphasis on the Iberian Peninsula, Austria and Germany) (Scarabaeoidea: Scarabaeidae: Aphodiinae). Linzer biolologische Beiträge, 47(1), 459-489. Frolov, A., & Akhmetova, L. (2013). A contribution to the study of the Lower Volga center of scarab beetle diversity in Russia: checklist of the tribe Aphodiini (Coleoptera, Scarabaeidae) of Dosang environs. Biodiversity Data Journal, 1, e979. Gharakloo, M. M., & Ziani, S. (2010). Iranian dung beetles (Coleoptera: Scarabaeoidea) associated with rodent burrows: a list of collected species and some additional comments. Boletín de la Sociedad Entomologica Aragonese (S.E.A.), 47(2010), 165-170. Hill, M. O. (1973). Diversity and evenness: a unifying notation and its consequences. Ecology, 54(2), 427-432. IUCN (2016). The IUCN Red List of Threatened Species. Retrieved from http://www.iucnredlist.org IUSS Working Group (2006). World reference base for soil resources 2006: A framework for international classification, correlation and communication. Rome, Italy: Food and Agriculture Organization of the United Nations. Janssens, A. (1960). Insectes Coléoptères Lamellicornes. Brussels, Belgium: Institut Royal des Sciences Naturelles de Belgique. Jessop, L. (1986). Dung beetles and chafers (Vol. 5). London: Royal Entomological Society of London. Jost, L. (2009) Partitioning diversity into independent alpha and beta components. Ecology, 88, 2427- 2439. Löbl, I., & Löbl, D. (2016). Catalogue of Palaearctic Coleoptera. Volume 3. Scarabaeoidea - Scirtoidea - Dascilloidea - Buprestoidea - Byrrhoidea. Revised and updated edition. Leiden-Boston: Brill. Miraldo, A., Krell, F. T., Smalén, M., Angus, R. B., & Roslin, T. (2014). Making the cryptic visible– resolving the species complex of Aphodius fimetarius (Linnaeus) and Aphodius pedellus (de Geer) (Coleoptera: Scarabaeidae) by three complementary methods. Systematic Entomology, 39(3), 531-547. NOAA (2016). Climate maps. Retrieved from http://gis.ncdc.noaa.gov/maps/ Udvardy, M. D. F. (1975). A classification of the biogeographical provinces of the world. Morges, Switserland: IUCN Occasional paper 18, World Conservation Union.

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A C Full assemblage all included

Tunneller exclusion all tunnellers

large tunnellers

Roller exclusion all rollers *

large rollers *

Combined all tunnellers & tunneller & all rollers * roller exclusion

large tunnellers & large rollers *

B all tunnellers & cattle dung large rollers *

large tunnellers & all rollers * optional dung type 1

Combined dweller, all dwellers, tunneller & tunnellers & optional dung type 2 roller exclusion rollers

all biota >1mm

D

experimental unit uncovered soil top view sampling unit fine mesh ground scr

fence coarse mesh ground

standardized dung pat fine mesh wall

sampling pot (0.2L/1L) coarse mesh wall side view fine mesh roof chicken wire

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Figure 2 – GLM model results showing relationships between species richness, dung beetle abundance or the proportion of resource specialist (y-axes) and latitude (a, e, i), longitude (b, f, j), mean temperature (c, g, k), and total monthly precipitation (d, h, l). Model-predicted means (solid lines) and standard errors are plotted for significantly correlated variables. Full model results are provided in table S7.

535x403mm (300 x 300 DPI)

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Figure 3 – Dung removal in complete dung fauna assemblages in the Western Palaearctic. Graphs show the relationship between the percentage of dung removed after one month and geographic (latitude (a), longitude (b)) and climatic variables (mean temperature (c), total monthly precipitation (d)). Significantly correlated variables after GLM analysis are plotted as solid lines with standard errors (grey). Full model results are provided in table S8.

257x257mm (300 x 300 DPI)

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Figure 4 – Hierarchical partitioning results for dung removal by dwellers, tunnellers, rollers and soil macro invertebrates in the Western Palaearctic. Displayed is the percentage of independent effect contributed by each functional group in each study site. Study sites are coded according to the codes in figure 1 and table S1 and are ordered by longitude. Asterisks indicate whether the contribution of a functional group is significant (p<0.050) in a particular study site. The level of significance of the independent contribution of each functional group was estimated using randomization tests based on 1000 permutations. On top of each bar R² values of the total model is shown for each study site.

338x203mm (300 x 300 DPI)

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