DOI: 10.5061/dryad.t76hdr80m
title: Data from: Predator specific responses and emergent multi-predator effects on oviposition site choice in gray treefrogs, Hyla chrysoscelis

description: 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.
description: 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
 m<sup>2</sup>) cylindrical mesocosms (N=45) laid out in
 isosceles trapezoids (Fig. 1a), crossing the presence/absence of three
 predatory fish: golden topminnows (<i>Fundulus chrysotus</i>),
 golden shiners (<i>Notemigonus crysoleucas</i>), and brown
 madtoms (<i>Noturus phaeus</i>)(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; <i>N. crysoleucas</i> is a small,
 pelagic, omnivorous-planktivorous, gape-limited species, <i>F.
 chrysotus</i> is a small topminnow that surface feeder that is also
 gape-limited, and <i>No. phaeus</i> 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 <i>H. chrysoscelis
 </i>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
 <i>Hyla</i> (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% (<i>F. chrysotus</i>), 94% (<i>No.
 phaeus</i>), and 91% (<i>N. crysoleucas</i>). Only two
 mesocosms did not fully hold treatment until the end of the experiment;
 one <i>F. chrysotus</i>×<i>N. phaeus</i> (FC×NP)
 mesocosm had no surviving <i>F. chrysotus</i>, and one
 three-species mesocosm (X3) also had no surviving <i>F.
 chrysotus</i>. 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<i>. </i>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 <i>Hyla</i> 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
 (<i>X+0.5</i>
 ). This provided the best fit (minimize
 c<sup>2</sup>/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
 (<i>X+0.5</i>
 ), 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 <i>a
 priori,</i> 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.