Predators affect prey through both consumptive and non-consumptive effects, and prey typically face threats from multiple simultaneous predators. While different predators have a variety of non-consumptive effects on prey, little is known regarding effects of simultaneous multiple predators on demographic habitat selection. Demographic habitat selection is unique among non-consumptive effects, especially in discrete habitat patches; decisions directly affect both distribution and abundance of species across habitat patches, rather than simply abundance and performance within patches. Our goal was to determine strength of avoidance responses to multiple species/species combinations of predatory fish, and responses to predator richness. We assessed responses of ovipositing gray treefrogs (Hyla chrysoscelis) to three predatory fish species and substitutive combination of species. In single-species treatments, treefrogs avoided only one species, Notemigonus crysoleucas. All two-species combinations, and the three-species combination, were avoided, including the Fundulus chrysotus × Noturus phaeus combination, of which neither were avoided alone. This suggests emergent properties of multiple predators, with potential interactive effects among cues themselves or in the perception of cues by treefrogs. Our results indicate effects of multiple predators are not predictable based on individual effects and illustrate the importance, and complexity, of effects of demographic habitat selection on distribution and abundance.

Our experiment was conducted in a large, old field at the University of Mississippi Field Station (UMFS), Lafayette County, MS. We set up five arrays (blocks), each with nine, 1300 L (2.54 m²) cylindrical mesocosms (N=45) laid out in isosceles trapezoids (Fig. 1a), crossing the presence/absence of three predatory fish: golden topminnows (Fundulus chrysotus), golden shiners (Notemigonus crysoleucas), and brown madtoms (Noturus phaeus)(Fig. 1b). These species are among the most frequently encountered fish and co-occur in ponds at UMFS. All three are generalist mesopredators, and each represents a different habitat/foraging strategy; N. crysoleucas is a small, pelagic, omnivorous-planktivorous, gape-limited species, F. chrysotus is a small topminnow that surface feeder that is also gape-limited, and No. phaeus is a small, benthic foraging catfish with a larger terminal size and size-specific gape the other two (Chan & Parsons 2000). All three feed primarily on aquatic insects and other invertebrates. Like most predatory fish in North America, they feed opportunistically, and all three represent a significant threat to the eggs and larvae of H. chrysoscelis relative to fish-free controls.

Mesocosms were filled with well-water from 11–13 May 2017, received 1 kg of dried mixed hardwood leaf litter each, and were quickly colonized by zooplankton and numerous small dipterans whose adults and/or eggs/larvae can pass through the screens (1.3 × 1.13 mm mesh used to separate predators and insect colonists/frog eggs) (Fig. S1), providing the fish a resource base. High overall survival and positive growth of all three fish supports presence of an adequate food base. Six fish were added to each mesocosm on 14 May: 6/species (single species), 3/3 (two-species), and 2/2/2 (three-species). Each block also contained two fishless controls. Density is on the lower end of biomass density from previous experiments and natural ponds, but above the threshold eliciting avoidance in Hyla (0.5g/100 l)(Rieger et al. 2004; unpubl. data). To equalize biomass within blocks, we created pairs comprised of 1 “large” and 1 “small” individual for each species (by eye to minimize stress), and randomly assigned pairs within blocks, thus establishing initial equal density, approximate biomass, and size-structure within blocks. On 15 May the experiment was begun by submerging screen lids to allow oviposition and efficient collection of eggs, and to separate fish from treefrogs – there were no consumptive effects possible here.

Gray treefrogs lay eggs in floating packets of ~25–50 eggs spread on the water surface, thus, unlike other species, it is impossible to determine the number of clutches deposited, though we estimate based on mean clutch size. Use of mesocosms and submerged screens (Fig. S1) is essential, as the eggs begin to sink rapidly as they develop, often overnight. Thus, it is virtually impossible to assay oviposition in individual natural ponds, much less across a gradient of ponds, except by proxy measures that have proved unreliable (i.e., larval abundance). Dead fish (18/210) were replaced until 20 May, after which there was no observed mortality. The experiment was checked each morning for eggs until 1 September 2017; eggs were removed, photographed, and counted using ImageJ (Schneider et al. 2012; Bohenek et al. 2017), and placed in rearing tanks or fishless ponds. Fish survival from 20 May to the end of the entire experiment (8 December) was 91%: 89% (F. chrysotus), 94% (No. phaeus), and 91% (N. crysoleucas). Only two mesocosms did not fully hold treatment until the end of the experiment; one F. chrysotus×N. phaeus (FC×NP) mesocosm had no surviving F. chrysotus, and one three-species mesocosm (X3) also had no surviving F. chrysotus. We could not track survival during the experiment, so cannot determine when mortality occurred. Since neither mesocosm was an outlier or otherwise unusual, our assumption was that they held their respective treatments for most of the period treefrog oviposition (see Results). Given the overall low fish mortality, this is not unreasonable. Thus, we included both in the final analyses as their original treatment.

This experiment was also colonized by a diverse assemblage of aquatic insects (5961 insects of 66 species), mostly dytiscid and hydrophilid beetles, and hemipterans (Resetarits et al. in press). Insects (except the very smallest) were separated from and incapable of interacting with fish due to the screen lids. Many of the colonists, or their larvae, are potentially significant predators of larval anurans, however, just as we removed eggs daily, we removed colonizing insects weekly (from above the screens), so there was no ongoing community assembly to dampen fish effects on Hyla oviposition (Kraus and Vonesh 2010).  

We used a randomized complete block design crossing presence/absence of three fish species in a De Wit replacement series (De Wit 1960). Each of five blocks consisted of 9 mesocosms, one replicate of 7 treatments and two replicates of fishless controls (Fig. 1a,b). Controls contain extra replicates because greater precision in estimation of the control disproportionately increases the power of the test, which compares all individual treatments to that control (Dunnett 1955; Hsu 1992). We could not equalize position relative to the forest edge, so we included row (inner vs outer) nested within block as a measure of relative proximity, since treefrogs ultimately arrive from the forest (Fig. 1). Our primary response variable was mean total eggs/patch and we partitioned mean total eggs into two components, total oviposition nights per patch (eggs in a patch on a night = hits) and mean deposition per patch (eggs/hit). For both mean total eggs and mean deposition we used generalized linear mixed model ANOVA in PROC GLIMMIX (SAS) with treatment as a fixed effect and block, and row nested within block, as random effects, with a quasi-Poisson distribution and a log link function (Steele et al. 1997), on square root transformed counts (X+0.5 ). This provided the best fit (minimize c²/df) to egg count data. For hits we used general linear mixed model ANOVA in PROC MIXED (SAS) with treatment as a fixed effect and block, and row nested within block, as random effects, on squareroot transformed counts (X+0.5 ), which, again, provided the best fit to the data. All responses to fish by colonizing/ovipositing organisms that utilize our mesocosms in dozens of prior experiments have been either negative or neutral, which informed our hypothesis that fish effects would manifest as reduced oviposition, hence the more powerful one-tailed test appropriate to a directional hypothesis (Rice and Gaines 1994; Ruxton and Neuhäuser 2010). Treatment means were compared using a one-tailed Dunnett’s Procedure (with Dunnett-Hsu correction), asking whether fish treatments received fewer eggs than Controls, and three a priori, non-orthogonal, one-tailed contrasts to examine specific hypotheses relating to multiple predator effects, with the expectation that oviposition would decline with predator richness. Contrasts were (1) single species vs all multiple species treatments, (2) single species vs species pairs, and (3) single species vs all three species (Fig. 2). We also analyzed final body size of each of the three fish species. Because this was designed to inform us as to the possible dynamics of the habitat decisions, we used individual mass rather than mesocosm means to gain more insight into the species interactions. We regressed total eggs/patch vs total fish biomass/patch to examine effects of overall fish biomass. All ANOVA-based analyses used SAS v. 9.4 (SAS Institute 2016) with Type III sums of squares and α = 0.05.