DOI: 10.5061/dryad.w9ghx3fn2
title: Dataset for: Numbers, neighbours and hungry predators: what makes chemically defended aposematic prey susceptible to predation?

description: Many chemically defended aposematic species are characterised by
 relatively low toxin levels, which enables predators to include them in
 their diets under certain circumstances. Knowledge of the conditions
 governing the survival of such prey animals – especially in the context of
 the co-occurrence of similar but undefended prey, which may result in
 mimicry-like interactions – is crucial for understanding the initial
 evolution of aposematism. In a one-month outdoor experiment using fish
 (the common carp Cyprinus carpio) as predators, we examined the survival
 of moderately defended aposematic tadpole prey (the European common toad
 Bufo bufo) with varying absolute densities in single-species prey systems
 or varying relative densities in two-species prey systems containing
 morphologically similar but undefended prey (the European common frog Rana
 temporaria). The density effects were investigated in conjunction with the
 hunger levels of the predator, which were manipulated by means of the
 addition of alternative (non-tadpole) food. The survival of the B. bufo
 tadpoles was promoted by increasing their absolute density in the
 single-species prey systems, increasing their relative density in the
 two-species prey systems, and providing ample alternative food for the
 predator. Hungry predators eliminated all R. temporaria individuals
 regardless of their proportion in the prey community; in treatments with
 ample alternative food, high relative B. bufo density supported R.
 temporaria survival. The results demonstrated that moderately defended
 prey did benefit from high population densities (both absolute and
 relative), even under long-term predation pressure. However, the
 physiological state of the predator was a crucial factor in the survival
 of moderately defended prey. While the availability of alternative prey in
 general should promote the spread and maintenance of aposematism, the
 results indicated that the resemblance between the co-occurring defended
 and undefended prey may impose mortality costs on the defended model
 species, even in the absence of actual mimicry.
description: The experiments were conducted in concrete ponds (6 × 6 m,
 adjustable water depth set at 75 cm) at the Muchocin experimental station
 of the Poznań University of Life Sciences (52°37′14.47″ N; 15°50′36.84″ E)
 from 2016–18. The experimental setup consisted of mesh enclosures (100 x
 100 x 100 cm, submerged up to 75 cm) with cube-shaped steel frames for
 internal support, placed in the ponds. The ponds, with 4 evenly
 distributed enclosures in each, were individually supplied with water from
 the neighbouring river (nutrient range values: 0.25–1.02 mg
 PO4<sup>–</sup> L<sup>–1</sup>; 0.1–0.5 mg
 NO3<sup>–</sup> L<sup>–1</sup>; total nitrogen
 2.16–7.37 mg N L<sup>–1</sup>) that passed through inlets
 fitted with fine-mesh screens. The walls of the enclosure (1 mm mesh size)
 were inserted into the bottom of the basin, giving the tadpoles and fish
 free access to the sandy substrate. A submerged PVC mesh cylinder (100 cm
 in length, 10 cm in diameter, 10 mm mesh size) was fastened to the bottom
 in each enclosure to create habitat complexity and to provide a refuge
 structure for tadpoles. The enclosures were covered with PVC mesh (10 mm
 mesh size) on the top to prevent predatory insects from entering. All
 enclosures were inoculated with zooplankton by adding 7 L of natural pond
 water. The study investigated changes in the survival
 of chemically defended <em>B. bufo</em> tadpoles and in their
 fitness-related traits (mass at metamorphosis) along a gradient of tadpole
 density (both absolute and relative), crossed with two levels of
 availability of alternative food for the predators, with some replication
 across the gradient. The study consisted of 2 experiments. In both
 experiments, the response variables were survival of <em>B.
 bufo</em> tadpoles to metamorphosis and their mass at metamorphosis.
 In Experiment 1, <em>B. bufo</em> tadpole survival in
 single-species prey communities with varying absolute density was tested
 in the presence of a fish predator. Two independent variables were
 manipulated: B. bufo tadpole density (5/30/40/50/80 individuals per
 enclosure) and the level of alternative non-tadpole food (feed pellets)
 provided for the fish. Availability of alternative food was considered as
 a proxy for hunger level in the predators; ‘high food’ (100 g twice a
 week) versus ‘low food’ (2 g twice a week). In Experiment 2, <em>B.
 bufo</em> tadpole survival in two-species prey communities with R.
 temporaria was tested in the presence of a fish predator, with
 manipulation of the relative (but not absolute) density of <em>B.
 bufo</em>. The density of <em>B. bufo</em> tadpoles was
 kept constant (30 individuals/enclosure), while <em>R. temporaria
 </em>tadpole density varied (5/10/30/50/60 individuals per
 enclosure), as did the level of alternative, non-tadpole food provided for
 the fish (same as in Experiment 1). In both experiments, each enclosure
 contained one 1-year-old specimen of <em>C. carpio</em> (total
 length 100–130 mm) as a predator. The experimental design did not include
 predator-free treatments; this was based on our previous result, i.e. that
 the mortality of tadpoles at Gosner stage 25 or above was low in the
 absence of predation (Kaczmarek et al., 2018). The
 tadpoles used in the experiment originated from amplexed pairs of
 <em>B. bufo</em> and <em>R. temporaria</em>
 collected from ponds in Wielkopolska province (NW Poland) and housed for
 approximately one week in separate pens, 1 m3, partially submerged in
 water. The obtained egg masses and hatched larvae remained there until
 <em>B. bufo</em> tadpoles reached Gosner stage 25. At this
 stage, the tadpoles were collected (tadpoles of R. temporaria were
 slightly larger than those of B. bufo as a result of hatching earlier in
 the season), randomly assigned to treatments, and stocked into
 experimental enclosures. All anurans used in the study were later returned
 to their original habitats. The fish used in the experiment originated
 from semi-natural ponds and had no experience with anuran tadpoles as
 prey. For one week prior to the experiment, the fish were kept indoors in
 large fibreglass tanks and provided <em>ad libitum</em> with
 commercial fish feed (pellets: 35% total protein, 9% crude lipid) that was
 also later used during the experiment. Individual fish were placed singly
 in experimental enclosures one day after the enclosures were stocked with
 tadpoles. Apart from the feed, the fish could also forage on the natural
 invertebrate prey present in the substrate and in the water column. The
 mean increases in total fish body length in the low- and high-food
 treatments during the experiment were measured in 2018, equalling 18% and
 31%, respectively. The enclosures were stocked in early May of each year
 (range: 5–12 May), and the metamorphs were collected after 4–5 weeks. The
 emerging metamorphs (Gosner stage 46) were collected by dip netting and
 were counted and weighed to the nearest 0.01 g. The individuals that
 failed to complete metamorphosis during the experiment were included in
 the analysis of survival but not that of body mass. The
 study was conducted over three consecutive years, in uniform environmental
 conditions. During each year of the research, Experiment 1 and Experiment
 2 were conducted in different ponds. Numbers of replicates were uneven
 between treatments as well as seasons. Most replicates were performed in
 2017 and 2018; four replicates for each experiment were carried out in
 2016. Moreover, in 2017 and 2018, marsh frogs <em>Pelophylax
 ridibundus</em> oviposited in several of the ponds and, as some
 spawn remained undetected, the freshly hatched tadpoles were able to
 intrude, entering the enclosures through the mesh walls; the invaded
 enclosures were excluded from the data analysis. However, we believe that
 the unbalanced design of the experiments should not greatly affect the
 results; since we were investigating tadpole survival along density
 gradients and not across categorical levels (except for the manipulation
 of alternative non-tadpole food availability for fish predators), we were
 more interested in obtaining a broad gradient of different densities or
 proportions of defended/undefended tadpoles than in complete replication
 of all treatment combinations. The final analyses were performed on a set
 of 17 (Experiment 1) and 48 (Experiment 2) enclosures.