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1 2 3 4 5 6 7 8 9 10 TADPOLES OF HYBRIDISING FIRE-BELLIED TOADS (B. BOMBINA AND B. VARIEGATA)
11 DIFFER IN THEIR SUSCEPTIBILITY TO PREDATION 12 13 14 15 16 17 Radovan Smolinský1,#, Vojtech Baláž2 and Beate Nürnberger1,* 18 19 20 21 22 1Research Facility Studenec, Institute of Vertebrate Biology, Czech Academy of Sciences, Brno, 23 Czech Republic 24 25 2Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical 26 Sciences, Brno, Czech Republic 27 28 #Current address: Department of Biology, Faculty of Education, Masaryk University, Brno, Czech 29 Republic 30 31 32 33 * corresponding author 34 E-mail: [email protected] (BN) 35 36 37 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
38
39 Abstract 40 41 The role of adaptive divergence in the formation of new species has been the subject of much
42 recent debate. The most direct evidence comes from traits that can be shown to have diverged
43 under natural selection and that now contribute to reproductive isolation. Here, we make the case
44 for differential adaptation of two fire-bellied toads (Anura, Bombinatoridae) to two types of aquatic
45 habitat. Bombina bombina and B. variegata are two anciently diverged taxa that now reproduce in
46 predator-rich ponds and ephemeral aquatic sites, respectively. Nevertheless, they hybridise
47 extensively wherever their distribution ranges adjoin. We show in laboratory experiments that, as
48 expected, B. variegata tadpoles are at relatively greater risk of predation from dragonfly larvae,
49 even when they display a predator-induced phenotype. These tadpoles spent relatively more time
50 swimming and so prompted more attacks from the visually hunting predators. We predict that
51 genomic regions linked to high activity in B. variegata are barred from introgression into the B.
52 bombina gene pool and thus contribute to gene flow barriers that keep the two taxa from merging
53 into one.
54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69
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70 Introduction 71 72 Adaptation to local environments is commonplace in nature [1] and drives the evolution of novel
73 ecotypes. Whether adaptive phenotypic divergence plays an important role in the origin of new
74 species has been the subject of much discussion [2–7]. In this case, adaptation to different habitats
75 should entail a marked fitness reduction for ecotypes in the 'wrong place' and for the hybrids
76 between them. Of particular interest are case studies in which the selective agents and the traits
77 under selection have been identified [7], because then the mechanisms that link adaptation to the
78 emergence of reproductive barriers can be studied. Long-term research programmes for example
79 on sticklebacks, sunflowers, Heliconius butterflies, lake whitefish and limpets have led the way in
80 this multi-disciplinary endeavour. Relative fitnesses of different phenotypes can be estimated in
81 different natural [8,9] or semi-natural habitats [10,11] or in experimental settings [12–14]. At the
82 genomic level, one can infer the strength of resulting gene flow barriers from the distribution of trait
83 effect sizes [15,16], investigate genetic linkage between traits under natural and/or sexual selection
84 [17,18] and ask whether the inferred barrier effects are supported by patterns of introgression.
85 Ecological divergence has long been assumed to generate barriers to gene flow in the hybrid zone
86 of European fire-bellied toads [19], for which genomic resources have recently been developed
87 [20]. Here we seek experimental evidence that differential adaptation of tadpoles to predators
88 contributes to this barrier.
89
90 In contrast to the very young taxa referenced above, the fire-bellied toad (Bombina bombina) and
91 the yellow-bellied toad (B. variegata) are more anciently derived [MRCA ~ 3.2 mya, 20].
92 Nevertheless, they interbreed wherever they meet and produce a wide range of recombinants in
93 typically narrow (2-7 km wide) hybrid zones [21]. Classic cline theory [22] suggests that the mean
94 fitness of intermediate hybrid populations is reduced by 42% compared to that of pure populations
95 on either side [23]. This figure presumably reflects the accumulation of diverse partial gene flow
96 barriers since the lineage split. There is experimental evidence for intrinsic selection against hybrid
97 embryos and tadpoles [24]. Profound ecological divergence is also strongly implicated. B. bombina
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98 reproduces in semi-permanent lowland ponds, such as flooded meadows and oxbows that fall dry
99 in winter [e.g. 25]. B. variegata lays eggs in more ephemeral habitat (‘puddles’) and sometimes
100 also in small steams [26], typically at higher elevations. Consequently, hybrid zones tend to be
101 located at ecotones. Plausible arguments can be made that a large number of documented taxon
102 differences result from adaptation to these different habitats. This applies to features ranging from
103 anatomy over life history to the mating system [19]. However, these hypotheses need to be tested
104 and the effects quantified to demonstrate that ecological divergence hinders gene flow.
105
106 Here we focus on tadpoles, because they experience the strongest habitat contrast. Faced with the
107 ever present risk of desiccation, B. variegata tadpoles hatch from relatively larger eggs, grow faster
108 and metamorphose earlier [27,28]. They also spend relatively more time swimming and feeding
109 [29,30]. The less active behaviour of B. bombina tadpoles should protect them against visually
110 hunting predators in ponds [29]. These taxon differences reflect a general pattern across anurans.
111 More permanent aquatic habitats have a greater abundance and diversity of predators [31–33].
112 Anuran species that reproduce in them have typically quiescent tadpoles [32,34–36] that take
113 longer to reach metamorphosis than those that breed in ephemeral sites [34,35,37]. More active
114 tadpoles species are typically more susceptible to predation [34,38–42]. This robust pattern has
115 been interpreted as a trade-off [34,37,43,44]: tadpoles can either be highly active and maximise
116 food intake for fast growth and development in temporary habitats or avoid predators through
117 quiescent behaviour in more permanent sites, but not both. Tadpole activity and growth rate covary
118 as expected in evolutionary contrasts [45] but evidence for a causal relationship is mixed at the
119 intra-specific level [46–51]. However, the trade-off between rapid growth and predator avoidance is
120 almost universal [52] irrespective of whether activity is the mechanism that links the two. We
121 therefore expect that B. variegata tadpoles are at greater risk of predation than B. bombina. Any
122 traits associated with such increased risk would therefore be unlikely to introgress into the B.
123 bombina gene pool and so represent a partial barrier to gene flow between the taxa.
124
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125 Nevertheless, B. variegata tadpoles do sporadically encounter predators such as newts, dragonfly
126 and dytiscid larvae, water scorpions and salamander larvae [26]. Thus, tadpoles would maximise
127 their chance of reaching metamorphosis by adjusting their phenotype to the local balance of risk.
128 Indeed, a wide range of anurans display phenotypic plasticity in response to predators. Almost
129 invariably, tadpoles reduce their activity level when they perceive chemical cues that indicate
130 predator presence [37,53,54]. This response can occur within minutes [55]. Longer term exposure
131 to such cues can cause an increase in refuge use [56,57], alter larval morphology [54], prompt the
132 development of colour spots on the tail [58,59], and slow growth and development [60,61]. These
133 effects are seen across venues from the laboratory to the field [62,63].
134
135 The commonest and most closely analysed morphological response to predators is the
136 development of a relatively deeper tail fin [35,36,47,54], usually in combination with a shorter tail
137 and a shallower body. A deeper tail may facilitate rapid escape (faster burst swimming speed) or
138 lure attacks away from the more vulnerable body. While there is evidence that natural variation in
139 tail shape affects tadpole swimming performance [42,64], studies to date have given stronger
140 support to the lure hypothesis [65,66]. Irrespective of the particular mechanism, there is evidence
141 that predator-induced phenotypes are eaten less than predator-naive ones [67–70] and that
142 survival is correlated with measured traits that show a plastic response [70–72]. A recent study of
143 Rana temporaria [73] makes a particularly strong case for adaptive phenotypic plasticity in both
144 behaviour and morphology, including an assessment of the variability in predation risk within and
145 between ponds and over time that could bring about such a flexible strategy.
146
147 Both B. bombina and B. variegata respond to the presence of predators with reduced activity
148 [29,30] and by developing a relatively deeper tail fin [74, see also 54]. Exposure to chemical
149 predation cues slows their growth and development under laboratory conditions [74]. At present,
150 we do not know whether the induced phenotypes offer better protection from predators. Because
151 we want to know whether B. variegata would suffer higher predation than B. bombina in ponds, we
152 contrast induced phenotypes only. Two previous studies addressed the relative predation risk of
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153 these toads. In a comparison of predator-naïve tadpoles, B. variegata suffered relatively higher
154 predation from a range of predators [Aeshna, Dytiscus, Triturus; 29]. In the second study with
155 predator-induced tadpoles, a strong population effect overwhelmed any taxon differences [two
156 populations per taxon; 74] In both cases, significant effects were observed in the first half of the
157 larval period. Observations in outdoor mesocosms show that the overall rate of predation by
158 dragonfly larvae is highest during that time and drops off sharply afterwards [75]. Similarly
159 declining rates of dragonfly predation have been reported for two species of Rana [76].
160
161 Here, we estimate the relative susceptibility to predation of pure B. bombina and B. variegata
162 throughout the first four weeks post-hatching, i.e. until the first B. variegata tadpoles reach
163 metamorphosis. Dragonfly larvae were used as predators, because they are ubiquitous in ponds
164 and also found in puddles. We also assess the contributions of activity, body size and tail shape to
165 the observed differences in predation risk.
166
167 Materials and Methods
168
169 Animal collections, pairing scheme and toad care
170 Adult toads were collected from Moravia (Czech Republic) where the Carpathian lineage of B.
171 variegata meets the Southern lineage of B. bombina [77]. The experiment was carried out in 2017.
172 Starting in early May, aquatic habitats were visited regularly (14 known B. bombina sites and 19
173 known B. variegata sites). Due to an unusually cold spring, toads did not appear in appreciable
174 numbers until early June and were then promptly collected to ensure that females had not yet laid
175 eggs. Collection sites (Table 1) were located at least 6 km from known areas of hybridisation and
176 deemed 'pure' based on the taxon-specific spot pattern on the toads' bellies [19]. For each taxon,
177 minimum and maximum distances between sites were 1.5 and 43.6 km (B. bombina) and 0.7 and
178 14.9 km (B. variegata). B. bombina sites were ponds with a minimum width of 5 m and a maximum
179 depth greater than 1 m. They all contained predators (incl. Aeshna cyanea, Natrix natrix, fish) and,
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180 with one exception (site E), they had abundant aquatic vegetation. B. variegata sites were either
181 water-filled wheel ruts (max. depth < 30 cm) or in one case (site 2) the shallow end of an
182 abandoned swimming pool. They were devoid of aquatic vegetation and three of nine sites
183 contained no predators at all (see S1 Table for details).
184 185 Table 1. Collection sites and pairing scheme. 186 187 Taxon Site Coordinates (N) Coordinates (E) Family indices B. bombina E 49°15´10'' 15°41´35" 13 H 49°13´30.6" 16°02´53" 18, 19, 20 K 48°51´41.078" 15°43´39" 15, 16 M 49°13´59" 16°03´50.5" 11, 12, 17 B. variegata 1A 49°06´27.6" 17°13´52.3" 1, 2, 7(m) 1B 49°06´25.5" 17°13´51.9" 6, 7(f) 2 49°08´26" 17°21´51" 3 5 49°05´59.82" 17°13´11" 10(f) 6 49°10´04.6" 17°22´25.8" 8 16 49°05´11" 17°12´43" 4(m) 17 49°05´23.5" 17°13´26.7" 4(f) 19 49°05´52.3" 17°13´25.2" 9(m), 10(m) 20 49°06´00.1" 17°13´33.8" 9(f) 188 In four B. variegata families, the parents came from different sites: m and f in parentheses indicate 189 the origin of the male and the female, respectively. 190 191 Males and females were paired within sites whenever possible. However due to unequal sex ratios
192 per site, four B. variegata pairs involved toads from separate, but nearby sites (Table 1, maximum
193 distance between sites per pair: 0.96 km). Each pair was housed in a 52 x 35 cm plastic box with
194 7L of well water, a polystyrene island and three pieces of plastic aquarium plants as support for
195 egg batches. As a precaution against the potential spread of Batrachochytrium infection, all waste
196 water from the entire experiment was heated to 100º C and treated with UV (Eheim UV reflex
197 UV800) before disposal to kill any fungal spores.
198
199 On 20 June, a belly swab was taken from each toad to test for Batrachochytrium dendrobatidis
200 (Bden) and B. salamandrivorens (Bsal) infection. The former pathogen is widely distributed in the
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201 Czech Republic, but so far no population declines have been observed [78]. The qPCR followed
202 the protocol of Blooi et al. [79] using a Roche Light Cycler480. No evidence of either infection was
203 found. We note that samples in the same essay but from an unrelated study scored positive for
204 Bden infection. The adults were subsequently returned to their exact collection sites.
205
206 Predators
207 We used Aeshna cyanea larvae as predators. This dragonfly is widely distributed across Europe
208 and was present in most of the habitats we surveyed (see above). Two hundred larvae were
209 collected from one site near Jihlava (Vysočina County) and were kept in groups of 15 in 50L plastic
210 boxes. They were fed every other day with Tubifex worms before the predation trials started and
211 with tadpoles thereafter.
212
213 Oviposition
214 To induce reproduction, 250 I.U. of human chorionic gonadotropin, dissolved in 0.2 ml of
215 amphibian Ringer solution, were injected into the lymph sac of an outer thigh of both males and
216 females on 6 June. Most pairs produced egg clutches overnight. Over the following two days, 30-
217 40 eggs per family were transferred into the rearing set-up (see below). The remaining offspring
218 were raised in the oviposition boxes, with weekly water changes. They were used as food for the
219 dragonflies in order to condition the water in the rearing set-up. The adults were moved to new
220 boxes of the same size. Tadpoles hatched on 12-13 June. In the following, we refer to 13 June as
221 day 0 of the experiment.
222
223 Tadpole rearing
224 We constructed two gravity flow circuits. For each of them, five plastic tubs (52 x 37 x 16 cm) were
225 interconnected with silicone tubing such that water could be pumped from a 20 L bucket at floor
226 level into the top tub and then descend stepwise via the other four tubs before returning to the
227 bucket. Inflows and outflows in each tub were positioned diagonally opposite to maximise mixing.
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228 Each tub held 27L of water at a depth of 14 cm. In order to maintain uniform water quality, the
229 pumps (Eheim StreamON+ 2000) were operated 24 hours per day at a low flow rate and per circuit
230 7 L of water were replaced daily. Throughout the experiment, water temperature was 22-24°C and
231 the air temperature was 24-26°C. All water changes for adults and tadpoles were carried out with
232 well water heated to 22°C and sterilised with UV (Eheim UV reflex UV800).
233
234 The upper four tubs in each circuit were subdivided with fibreglass insect mesh into eight
235 compartments (size: 18 x 11.5 x 16.5 cm, 64 compartments in total). A plastic aquarium plant was
236 added to each compartment for structural complexity. At the start of the experiment, each
237 compartment held 10 hatchlings from a single family. Families (nine per taxon) were distributed
238 over either three or four compartments in total and either one or two compartments in each circuit.
239 Exactly half of the compartments in each circuit were assigned to each taxon. Placement of
240 families within circuits was decided by random draws.
241
242 Tadpoles were fed boiled nettle (Urtica dioica) leaves ad libitum and uneaten food was removed
243 every day. In order to simulate predator presence, we let chemical cues of tadpole predation
244 circulate from day 0 onwards. For this, we used the fifth tub in each circuit to feed 1-2 non-
245 experimental tadpoles (depending on size) to one dragonfly larva every other day.
246
247 Predation trials
248 Predation trials started on day 2 when the tadpoles were active. They were carried out in two
249 plastic boxes subdivided with fibreglass insect mesh into four compartments (26 x 26 x 18 cm),
250 each with two plastic aquarium plants (akin to Myriophyllum). They contained a 1:1 mixture of well
251 water and water from the rearing circuits. For each trial, four tadpoles (2 B. bombina, 2 B.
252 variegata) were placed into a given compartment and allowed to acclimate for 30 min. A dragonfly
253 that had not been fed on the previous day was then added and the trial was stopped when the first
254 tadpole had been eaten (maximum duration: 45 min). The identity of the missing tadpole was
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255 determined from digital photographs of the animals taken before the trial (see below). The
256 survivors were returned to their rearing compartments. The trials were recorded with a Microsoft
257 LifeCam VX-2000 video camera suspended over each experiment tub.
258
259 Predation trials were carried out on 18 days as follows: two consecutive experiment days were
260 followed by one ‘off-day’ (1 exception); this pattern was repeated nine times until day 29 post
261 hatching, when the first B. variegata tadpoles entered metamorphosis. There were 20 trials per day
262 between 10:00 and 15:00 hours. A custom Perl script was used to haphazardly draw the
263 composition of each trial per day such that (a) four different families were represented, (b) one
264 tadpole per taxon came from circuit 1 and the other from circuit 2 and (c) each rearing
265 compartment contributed at least one tadpole on a given day. However towards the end of the
266 experiment, a few rearing compartments were empty and the contribution of others had to be
267 increased accordingly. No individual tadpole or dragonfly was used more than once per experiment
268 day.
269
270 Morphometrics
271 Digital images were taken by placing a tadpole into a glass cuvette and photographing it from the
272 side with a Canon EOS 400D camera (lens: Canon EF 28-90mm). TPS Dig (Software by F. James
273 Rohlf) software was used to take the following measurements from these images: total length, tail
274 length, body height and maximum tail height.
275
276 Behaviour
277 From the video footage of the predation trials, we recorded the behaviour of a focal tadpole during
278 one 30 sec interval per trial for half of the trials on a given day. A custom Perl script provided the
279 pseudo-random start point of the observation interval and allowed behaviour (swimming, feeding,
280 resting) to be recorded with separate key strokes. From one trial to the next, we alternated
281 between a B. bombina and B. variegata tadpole. Taxon identification was based on size in the
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282 early trials. Morphological differences were used as soon as they became apparent. Footage from
283 the first two days was not used, because the taxa could not be reliably distinguished. We discarded
284 all observation intervals that overlapped with a predator attack. We also recorded for 68 predator
285 attacks (four on each on 17 experiment days) whether they were initiated by and directed towards
286 an actively swimming tadpole.
287
288 Collection permits were granted by the relevant regional offices (South Moravia and Zlín) of the
289 Czech Ministry for the Environment. The experimental protocol was approved by the ethics
290 committee of the Institute of Vertebrate Biology, CAS, permit no. 15/2016.
291
292
293 Statistical analyses
294 Estimates of predation risk
295 We estimated predation risk per rearing compartment as the number of tadpoles eaten divided by
296 the number of valid trials in which that compartment was represented. Especially with a small
297 number of trials, this estimate may be biased if tadpoles from a certain compartment happen to be
298 assigned to trials with, say, particularly well defended congeners. There is a range of statistical
299 methods to estimate an individual's skill from a number of contests with opponents of varying
300 strength (as e.g. in chess), starting with the model of Bradley and Terry [80]. However, none of
301 these match our trial design of a single winner (or rather: looser) and a 'tie' for the other three. For
302 each rearing compartment, we therefore computed the expected 'risk context' from a large number
303 of simulated trials and compared this estimate with the mean risk context in the actual trials. A
304 close match indicates the absence of bias. Risk context is here defined as the mean predation risk
305 of the other three rearing compartments represented per trial, averaged over all trials in which the
306 focal compartment was represented. Using the same custom script as before, we simulated 2550
307 trials and used these to compute the expected risk context of all 64 rearing compartments.
308
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309 We investigated the effects of taxon, circuit and family on predation risk per rearing compartment.
310 Circuit was treated as a fixed effect, because it has only two levels. Family was modeled as a
311 random effect nested within taxon. We did not include a population term in the model, because
312 populations were unevenly represented and some families had parents from different sites. Any
313 differences between populations should appear as family effects. Linear mixed models were fitted
314 with the R module lme4 [81]. Model comparisons were carried out with likelihood ratio tests.
315
316 Morphological correlates of survival
317 Logistic models were used to investigate the effects of taxon, tadpole size and tail shape on
318 survival per trial. The input was generated by sorting all valid trials in chronological order and
319 picking from them alternately (a) a surviving tadpole at random or (b) the non-surviving tadpole.
320 For the entire dataset, the four morphological measurements were log-transformed to improve
321 normality and entered into a principal component analysis (PCA). We treat the first principal
322 component as a measure of tadpole size. From a regression of log(tail height) on PC1 we
323 extracted the residuals as a measure of relative tail height (≈ tail shape). To ensure shared
324 allometry between the taxa, we inspected pairwise plots of the four measured variables [82]. For
325 the focal tadpole per trial, PC1 and residual log(tail height) were expressed as a deviation from the
326 trial mean. All analyses were carried out in R v.3.4.4.
327
328 Results
329
330 Taxon differences in predation risk
331 In total, there were 360 trials. Of these, no tadpole was eaten in 96 trials, more than one was eaten
332 in five trials and in four trials the two taxa were not evenly represented. Discarding these, 256 valid
333 predation trials remain. The proportion of valid trials in which a B. variegata tadpole was eaten was
334 0.65. There was also a slightly higher risk for tadpoles from circuit 1 as opposed to circuit 2
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335 (proportion = 0.56). This effect was largely restricted to B. bombina (see below). The number of
336 trials in which no tadpole was eaten was larger in the second half of the experiment (tadpole age:
337 16-29 days, n = 66) than in the first half (tadpole age: 2 - 15 days, n = 30).
338
339 Predation risk was computed per rearing compartment as the number of tadpoles eaten during
340 valid predation trials over the number valid trails in which this compartment was represented. The
341 observed risk context (as defined in the Materials and Methods) closely matched the prediction
342 from simulated trials (푛 per compartment = 159.9): for 56 of 64 compartments the observation
343 deviated by less than 10% from the expectation (max. deviation 14%) and in all cases it was well
344 within one standard deviation of the prediction (S1 Fig). We therefore use predation risk per rearing
345 compartment as the response variable in the following analyses. The estimates are plotted in Fig 1.
346 347 Fig 1. Predation risk per rearing compartment arrayed by family. B. variegata: families 1-4 and 348 6-10, B. bombina: families 11 -13 and 15 - 20. 349 350 The full linear mixed model (Table 2, model 1) included taxon, family (nested within taxon) and
351 circuit as main effects and also interactions between taxon and circuit and between family and
352 circuit. Model comparison showed that neither of interaction terms were significant (models 2 vs 1:
353 p = 0.0829; models 3 vs 1: p = 1) and so both were dropped (model 4). The main effect of taxon
354 was overwhelmingly significant (models 5 vs 4: p = 1.911e-05), providing clear evidence that B.
355 variegata tadpoles were at relatively greater risk of predation by Aeshna cyanea larvae. There was
356 also a significant circuit effect (models 6 vs 4: p = 0.0278). Zero variance was assigned to the
357 family effect in models 3 - 6. Two-way ANOVAs of taxon and circuit closely reproduced the
358 patterns of significance for these two factors (analyses not shown). While there is clear evidence
359 for a taxon effect and a minor effect of circuit, a large amount of unexplained variation remains (Fig
360 1).
361 362 363
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364 365 366 Table 2. Linear mixed models of predation risk per rearing compartment. 367 No. Model 1 asin_riska ~ taxon + circuit + taxon*circuit + (1 + circuit | taxon/family)b 2 asin_risk ~ taxon + circuit + (1 + circuit | taxon/family) 3 asin_risk ~ taxon + circuit + taxon*circuit + (1 | taxon/family)c 4 asin_risk ~ taxon + circuit + (1 | taxon/family) 5 asin_risk ~ circuit + (1 | family) 6 asin_risk ~ taxon + (1 | taxon/family) 368 aarcsine(sqrt(risk)). 369 bfamily is a random effect nested within taxon, slope of 'circuit' is allowed to vary among families. 370 cfamily is a random effect nested within taxon. 371 372 Tadpole size and shape
373 Throughout the experiment, B. bombina was on average smaller with a shallower tail (Figs 2A and
374 1B). But after correction for body size, B. bombina's tail fin was relatively higher than that of B.
375 variegata (Fig 2C). We note that our shearing approach to correct for body size is validated by the
376 shared allometry in both taxa (S2 Fig). If size and/or tail shape are functionally important, we would
377 expect to see within-taxon correlations with survival as well. The logistic regression analysis (Table
378 3, see Materials and Methods for details) confirmed the significantly lower survival of B. variegata
379 and also showed a strong interaction of tadpole size (PC1) and taxon. There were no significant
380 effects associated with relative tail height.
381 382 Fig 2. Morphological development by taxon. Error bars represent one standard deviation. PC1 383 = 1. principal component ≈ body size, res.log(tailht) = residual log(tail height) ≈ size-corrected tail 384 height. 385 386 Table 3. Coefficients of the logistic model of survival. 387 Estimate Std. Error z value Pr(>|z|) (Intercept) 0.9110 0.3036 3.001 0.00269** taxon (B.v.) -1.0405 0.4161 -2.501 0.01240* PC1 1.9817 0.6327 3.132 0.00173** resid(LTH)a 5.1895 3.6139 1.436 0.15100
14 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
taxon*PC1 -2.4135 0.8963 -2.693 0.00709** taxon*resid(LTH) -2.2078 4.7176 -0.468 0.63978 388 Survival is coded as 1 (yes) and 0 (no). Morphological measurements were expressed as 389 deviations from the trial mean. See legend of Fig 2 for an explanation of the independent variables. 390 aresidual log(tail height). 391 392 Fig 3A illustrates that larger B. bombina tadpoles survived better than smaller ones (t = 3.5993, df
393 = 85.589, p-value = 0.0005). But this effect is not consistent across the whole dataset. B. variegata
394 tadpoles are overall larger and more at risk of predation, whereas smaller size is associated with
395 greater risk in B. bombina. There is therefore no evidence that a general preference of Aeshna
396 larvae for larger prey causes higher mortality in B. variegata tadpoles. The analogous plot for
397 relative tail height hints at an advantage for higher tail fins (Fig 3B), but the present dataset
398 provides no statistical support for this hypothesis.
399
400 Fig 3. Means of body size and tail shape by taxon and survival. Plotted are the means from the 401 logistic regression dataset for PC1 (A) and residual log(tail height) (B). Error bars indicate one 402 standard error of the mean. 403 404 Tadpole activity
405 During the 30 sec observation intervals, both taxa spent most of their time either resting or,
406 seemingly, feeding. No food was provided in these trials. So, the tadpoles' persistent scraping of
407 surfaces with their mouthparts, especially during the first half of the experiment, probably provided
408 little, if any nutrition. Swimming typically occurred in short bursts of a few seconds separated by
409 inactive intervals. Ninety-two percent of recorded predator attacks were directed at actively
410 swimming tadpoles.
411
412 The distributions of the time spent swimming were strongly skewed to the right in both taxa (Fig 4).
413 As expected, B. variegata spends more time swimming than B. bombina (means: 4.60 and 1.75
414 sec, respectively; unpaired Wilcoxon rank sum test, W = 2234.5, p-value = 0.0096). Focal tadpoles
415 were chosen based on their clear visibility during the 30 sec observation interval. Their survival
15 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
416 status is unknown. We can therefore not investigate within-taxon correlations between activity and
417 survival.
418 419 Fig 4. Time spent swimming during 30 sec observation intervals by taxon. Number of 420 intervals: 76 and 75 for B. variegata and B. bombina, respectively. 421 422 Predation rates over time
423 A logistic model with taxon and experiment day as independent variables produced only a non-
424 significant taxon x day interaction (analyses not shown). There is thus no indication that the relative
425 survival of the two taxa changed over the course of the experiment. Fig 5 illustrates the proportion
426 of B. variegata tadpoles consumed and the number of valid trials over time. Not only were there
427 fewer valid trials in the second half of the experiment (tadpole age: 16-29 days), the mean duration
428 of trials also increased from 9.7 min to 13.6 min.
429 430 Fig 5. Relative predation risk and number of valid trials per day. A. The proportion of tadpoles 431 consumed that were B. variegata. B. The number of valid trials per experiment day (out of 20). 432 433
434 Discussion
435
436 In our experimental setting, tadpoles of the puddle breeder B. variegata are at greater risk of
437 predation from dragonfly larvae than those of B. bombina that develop in semi-permanent ponds.
438 This result confirms our expectation based on similar species comparisons [35]. It also agrees with
439 the findings of Kruuk and Gilchrist [29] who conducted predation trials with larger groups of
440 predator-naīve B. bombina and B. variegata tadpoles that could only adjust their behaviour. Here,
441 we allowed tadpoles to develop a more complete predator-induced phenotype. Nonetheless, the
442 survival difference between the taxa persisted and it was particularly strong during the first two
443 weeks of larval development (Fig 5), when risk from dragonfly predation is highest [75]. Vorndran
444 et al. [74] also reared tadpoles in the presence of chemical predation cues and found a marked
16 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
445 survival difference between tadpoles from the two replicate B. variegata populations. The two
446 collection sites in Romania also differed in predator abundance and proximity to the hybrid zone,
447 observations that warrant more detailed population comparisons in that area [83].
448
449 Before we interpret our findings more fully, we consider potential limitations of our experimental
450 design. Many tadpoles were used in more than one trial. Later trials may therefore have involved
451 individuals that benefited from experience gained and/or were inherently better at avoiding
452 predators. We believe that this resembles the situation in nature more closely than the alternative
453 of shielding tadpoles from predator encounters until the day of their one experimental test. As
454 pointed out by Melvin and Houlahan [84], typical tadpole mortality in nature (approx. 95%
455 according to their review) may be non-random with regard to traits under study. The typically much
456 more benign conditions in the laboratory may thus produce experimental cohorts that are not
457 representative of the population of origin. While we have not reproduced the selective regime of
458 any semi-permanent pond, repeated exposure to dragonfly larvae adds realism to our study.
459 Nevertheless, we consider this close-up view of predator-prey interactions in the laboratory as a
460 piece in a mosaic that should be complemented with experiments in semi-natural settings and
461 using additional predator species.
462
463 If B. bombina and B. variegata were reproductively isolated, these results would address the
464 question why B. variegata reproduces only rarely in semi-permanent, predator-rich ponds [85].
465 Whether or not B. bombina tadpoles are present in these habitats may not matter, because there is
466 little evidence for competition among tadpoles in ponds [33,35,38]. Predators tend to keep tadpole
467 numbers sufficiently low so that competition is negligible. B. bombina then simply provides a
468 benchmark for a successful 'pond strategy'. B. variegata's greater susceptibility to predation shows
469 that its tadpoles fall short of this benchmark even when they display the predator induced
470 phenotype. Other traits such as relatively fewer eggs per clutch [27] may also limit B. variegata's
471 reproductive success in ponds.
472
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473 Hybridisation shifts the focus away from a comparison between distinct types and towards
474 selection on traits in recombinants. These genotypes predominate in Bombina hybrid zones, while
475 F1 individuals are very rare, if present at all [21,23]. Each generation, associations between traits
476 are broken up by hybrid matings but also replenished by the influx of pure types from the
477 periphery. Theory predicts a dynamic equilibrium between these processes that determines how
478 strongly the hybrid zone functions as a genetic barrier to gene flow [86,87]. Any trait that increases
479 the susceptibility of tadpoles to predation is unlikely to introgress into the B. bombina genepool and
480 it should impede the introgression of traits with which it is genetically linked. In this way, traits that
481 mediate adaptation to either ponds or puddles contribute to partial reproductive isolation in
482 Bombina.
483
484 We considered traits that are expected to affect the risk of being detected by predators and the
485 ability to escape once attacked. Prey activity alerts visually hunting predators and correlates
486 strongly with tadpole predation rates in inter-specific comparisons (see Introduction) and also
487 within species [67,88]. In our experiments, B. variegata spent on average more time swimming
488 than B. bombina. Since swimming was the single most important trigger of dragonfly attacks,
489 activity contributes importantly to the difference in predation rate between the taxa. There was no
490 evidence that relatively higher tail fins caused better survival in either taxon. The large variances
491 that rendered differences between group means (taxon, survival) non-significant (Figs 2C and 3B)
492 may reflect heritable variation among families in the amplitude and type of morphological plasticity
493 [89]. This trait warrants further study using more detailed morphometrics [64]. Finally, our analyses
494 disproved the hypothesis that an overall preference of Aeshna cyanea larvae towards larger prey
495 contributed to B. variegata's greater mortality.
496
497 Is higher predation risk in B. variegata a direct consequence of adaptation to ephemeral habitat?
498 Population studies have shown that successful metamorphosis can be restricted in a given year to
499 less than 50% of sites in which eggs have been laid (depending on annual rainfall) and that
500 desiccation is the main cause of reproductive failure [90–92]. This is reflected in the growth rate of
18 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
501 B. variegata tadpoles which was the fastest in a set of 15 European and NE North American
502 anurans [60]. Intra-specific competition should be commonplace as tadpoles grow and puddles
503 shrink [93]. The high activity of tadpoles growing in this type of habitat supports this [94]. In the lab,
504 large B. variegata tadpoles displace smaller conspecifics from food patches with their powerful tail
505 movements [95]. If indeed B. variegata’s larval development has been mainly shaped by time
506 constraints and competition, there may be trade-offs that limit the effectiveness of anti-predator
507 strategies. Indeed predator-induced plasticity in Bombina entails slower growth and delayed
508 metmorphosis [74]. Based on studies with Rana tadpoles [96,97], we expect all of these responses
509 to be mediated by the neuroendocrine stress axis and by levels of corticosterone in particular.
510
511 While phenotypic plasticity involves the same traits in both taxa, the differences between the
512 induced phenotypes are not just a matter of degree. There are qualitative differences as in the
513 body size effect on predation risk that is present in B. bombina but not in B. variegata (see above).
514 Moreover in B. variegata, absolute tail height shows no response to predator presence. The
515 increase in relative tail height reflects reduced growth rates of other body parts. In contrast, all
516 measured dimensions showed reduced growth in B. bombina, with an allometric change in tail
517 height [74]. Finally, B. variegata frequently develops dark spots on the tail fin that may divert
518 attacks away from the body, whereas B. bombina's tail fin is always uncoloured [98]. Assuming
519 that tail spots provide protection, the genomic regions linked to them may even show adaptive
520 introgression. As B. bombina and B. variegata adapt to different breeding habitats, they may
521 evolve somewhat different strategies to counter common selection pressures [99]. This could
522 reduce the fitness of hybrids in which these strategies are broken up. Based on this study, we
523 expect that higher activity in B. variegata tadpoles acts as a barrier to gene flow, but other
524 'component traits' of predation risk may also be under selection in the hybrid zone und some may
525 be conduits rather than barriers of gene flow.
526
527
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528 Acknowledgments
529 We thank Oliver Gast and Zuzana Hiadlovská for help in the field and with the experiments,
530 respectively. Jarrod Hadfield, Lumír Gvoždík and Crispin Jordan offered advice on the
531 experimental design and statistics. We thank Günther Gollmann, Jacek M. Szymura and Josh Van
532 Buskirk for comments on an earlier version of this manuscript.
533
534 References
1. Hereford J. A quantitative survey of local adaptation and fitness trade-offs. Am Nat. 2009;173:
579–588.
2. Schluter D. Ecology and the origin of species. Trends Ecol Evol. 2001;16: 372–380.
3. Schluter D. Evidence for ecological speciation and its alternative. Science. 2009;323: 737–
741. doi:10.1126/science.1160006
4. Hendry AP. Ecological speciation! Or the lack thereof? Can J Fish Aquat Sci. 2009;66: 1383–
1398.
5. Nosil P. Ecological Speciation. Oxford: Oxford University Press; 2012.
6. Bierne N, Gagnaire P-A, David P. The geography of introgression in a patchy environment
and the thorn in the thigh of ecological speciation. Curr Zool. 2013;59: 72–86.
7. Faria R, Renaut S, Galindo J, Pinho C, Melo-Ferreira J, Melo M, et al. Advances in ecological
speciation: an integrative approach. Mol Ecol. 2014;23: 513–521. doi:10.1111/mec.12616
8. Janson K. Selection and migration in two distinct phenotypes of Littorina saxatilis in Sweden.
Oecologia. 1983;59: 58–61.
20 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
9. Donovan LA, Ludwig F, Rosenthal DM, Rieseberg LH, Dudley SA. Phenotypic selection on
leaf ecophysiological traits in Helianthus. New Phytol. 2009;183: 868–879.
doi:10.1111/j.1469-8137.2009.02916.x
10. Hatfield T, Schluter D. Ecological speciation in sticklebacks: environment-dependent hybrid
fitness. Evolution. 1999;53: 866–873.
11. Arnegard ME, McGee MD, Matthews B, Marchinko KB, Conte GL, Kabir S, et al. Genetics of
ecological divergence during speciation. Nature. 2014;511: 307–311.
doi:10.1038/nature13301
12. Rogers SM, Gagnon V, Bernatchez L. Genetically based phenotype-environment association
for swimming behavior in Lake Whitefish ecotypes (Coregonus clupeaformis Mitchill).
Evolution. 2002;56: 2322–2329. doi:10.1111/j.0014-3820.2002.tb00155.x
13. Welch ME, Rieseberg LH. Habitat divergence between a homoploid hybrid sunflower species,
Helianthus paradoxus (Asteraceae), and its progenitors. Am J Bot. 2002;89: 472–478.
doi:10.3732/ajb.89.3.472
14. Le Pennec G, Butlin RK, Jonsson PR, Larsson AI, Lindborg J, Bergström E, et al. Adaptation
to dislodgement risk on wave-swept rocky shores in the snail Littorina saxatilis. PLOS ONE.
2017;12: e0186901. doi:10.1371/journal.pone.0186901
15. Lexer C, Welch ME, Durphy JL, Rieseberg LH. Natural selection for salt tolerance quantitative
trait loci (QTLs) in wild sunflower hybrids: Implications for the origin of Helianthus paradoxus,
a diploid hybrid species. Mol Ecol. 2003;12: 1225–1235. doi:10.1046/j.1365-
294X.2003.01803.x
16. Jones FC, Grabherr MG, Chan YF, Russell P, Mauceli E, Johnson J, et al. The genomic basis
of adaptive evolution in threespine sticklebacks. Nature. 2012;484: 55–61.
doi:10.1038/nature10944
21 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
17. Kronforst MR, Young LG, Kapan DD, McNeely C, O’Neill RJ, Gilbert LE. Linkage of butterfly
mate preference and wing color preference cue at the genomic location of wingless. Proc Natl
Acad Sci USA. 2006;103: 6575–6580.
18. Bay RA, Arnegard ME, Conte GL, Best J, Bedford NL, McCann SR, et al. Genetic coupling of
female mate choice with polygenic ecological divergence facilitates Stickleback speciation.
Curr Biol. 2017;27: 3344-3349.e4. doi:10.1016/j.cub.2017.09.037
19. Szymura JM. Analysis of hybrid zones with Bombina. In: Harrison RG, editor. Hybrid zones
and the evolutionary process. New York: Oxford University Press; 1993. pp. 261–289.
20. Nürnberger B, Lohse K, Fijarczyk A, Szymura JM, Blaxter ML. Para-allopatry in hybridizing
fire-bellied toads (Bombina bombina and B. variegata): Inference from transcriptome-wide
coalescence analyses. Evolution. 2016;70: 1803–1818. doi:10.1111/evo.12978
21. Yanchukov A, Hofman S, Szymura JM, Mezhzherin S, Morozov-Leonov SY, Barton NH, et al.
Hybridization of Bombina bombina and B. variegata (Anura, Discoglossidae) at a sharp
ecotone in Western Ukraine: comparisons across transects and over time. Evolution.
2006;60: 583–600.
22. Barton NH, Gale KS. Genetic analysis of hybrid zones. In: Harrison RG, editor. Hybrid zones
and the evolutionary process. Oxford: Oxford University Press; 1993. pp. 13–45.
23. Szymura JM, Barton NH. The genetic structure of the hybrid zone between the fire-bellied
toads Bombina bombina and B. variegata: comparison between transects and between loci.
Evolution. 1991;45: 237–261.
24. Kruuk LEB, Gilchrist JS, Barton NH. Hybrid dysfunction in Fire-Bellied Toads (Bombina).
Evolution. 1999;53: 1611–1616. doi:10.2307/2640907
25. Philippi D, Gollmann G. On the status of the fire-bellied toad, Bombina bombina, in Lobau
(Vienna, Donau-Auen National Park). Acta ZooBot Austria. 2014;150/151: 25–32.
22 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
26. Gollmann B, Gollmann G. Die Gelbbauchunke: von der Suhle zur Radspur. Bielefeld: Laurenti
Verlag; 2002.
27. Rafińska A. Reproductive biology of the fire-bellied toads, Bombina bombina and B. variegata
(Anura: Discoglossidae): egg size, clutch size and larval period length differences. Biol J Linn
Soc. 1991;43: 197–210.
28. Nürnberger B, Barton NH, MacCallum C, Gilchrist J, Appleby M. Natural selection on
quantitative traits in the Bombina hybrid zone. Evolution. 1995;49: 1224–1238.
29. Kruuk LEB, Gilchrist JS. Mechanisms maintaining species differentiation: predator mediated
selection in a Bombina hybrid zone. Proc R Soc Lond B. 1997;264: 105–110.
30. Reichwaldt E. Der Einfluss von Prädatoren auf die Kaulquappen hybridisierender Unken
(Bombina, Discoglossidae, Amphibia): Entwicklung, Morphologie und Verhalten. Diploma
Thesis. University of Munich. 1999.
31. Babbitt KJ, Baber MJ, Tarr TL. Patterns of larval amphibian distribution along a wetland
hydroperiod gradient. Can J Zool. 2003;81: 1539–1552.
32. Richter-Boix A, Llorente GA, Montori A. A comparative study of predator-induced phenotype
in tadpoles across a pond permanency gradient. Hydrobiologia. 2007;583: 43–56.
doi:10.1007/s10750-006-0475-7
33. Werner EE, Skelly DK, Relyea RA, Yurewicz KL. Amphibian species richness across
environmental gradients. Oikos. 2007;116: 1697–1712.
34. Woodward BD. Predator-prey interactions and breeding pond use of temporary pond species
in a desert anuran community. Ecology. 1983;64: 1549–1555.
35. Smith DC, Van Buskirk J. Phenotyic design, plasticity, and ecological performance in two
tadpole species. Am Nat. 1995;145: 211–233.
23 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
36. Relyea RA. Morphological and behavioural plasticity of larval anurans in response to different
predators. Ecology. 2001;82: 523–540.
37. Lawler SP. Behavioural responses to predators and predation risk in four species of larval
anurans. Anim Behav. 1989;38: 1039–1047.
38. Morin PJ. Predation, competition, and the composition of larval anuran guilds. Ecol Monogr.
1983;53: 119–138.
39. Azevedo-Ramos C, Van Sluys M, Hero J-M, Magnusson WE. Influence of tadpole movement
on predation by odonate naiads. J Herpetol. 1992;26: 335–338.
40. Chovanec A. The influence of tadpole swimming behaviour on predation by dragonfly
nymphs. Amphib-Reptil. 1992;13: 341–349.
41. Relyea RA. The Relationship between predation risk and antipredator responses in larval
Anurans. Ecology. 2001;82: 541–554.
42. Dayton GH, Saenz D, Baum KA, Langerhans RB, DeWitt TJ, Lindström J. Body shape, burst
speed and escape behavior of larval Anurans. Oikos. 2005;111: 582–591.
43. Werner EE, Anholt BR. Ecological consequences of the trade-off between growth and
mortality-rates mediated by foraging activity. Am Nat. 1993;142: 242–272.
44. Cressler CE, King AA, Werner EE. Interactions between behavioral and life‐history trade‐offs
in the evolution of integrated predator‐defense plasticity. Am Nat. 2010;176: 276–288.
doi:10.1086/655425
45. Richardson JML. The relative roles of adaptation and phylogeny in determination of larval
traits in diversifying Anuran lineages. Am Nat. 2001;157: 282–299. doi:10.1086/319196
46. Skelly DK, Werner EE. Behavioral and life-history responses of larval American toads to an
odonate predator. Ecology. 1990;71: 2313–2322.
24 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
47. McCollum SA, Van Buskirk J. Costs and benefits of a predator-induced polymorphism in the
gray treefrog Hyla chrysoscelis. Evolution. 1996;50: 583–593.
48. Laurila A, Kujasalo J, Ranta E. Predator induced changes in life history in two anuran
tadpoles: effects of predators on diet. Oikos. 1998;83: 307–317.
49. Relyea RA, Werner EE. Quantifying the relation between predator-induced behavior and
growth performance in larval anurans. Ecology. 1999;80: 2117–2124.
50. Steiner UK. Linking antipredator behaviour, ingestion, gut evacuation and costs of predator-
induced responses in tadpoles. Anim Behav. 2007;74: 1473–1479.
doi:10.1016/j.anbehav.2007.02.016
51. Orizaola G, Richter‐Boix A, Laurila A. Transgenerational effects and impact of compensatory
responses to changes in breeding phenology on antipredator defenses. Ecology. 2016;97:
2470–2478. doi:10.1002/ecy.1464
52. Lima SC, Dill LM. Behavioral decisions made under the risk of predation: a review and
prospectus. Can J Zool. 1991;268: 619–640.
53. Anholt BR, Werner E, Skelly DK. Effect of food and predators on the activity of four larval
ranid frogs. Ecology. 2000;81: 3509–3521. doi:10.1890/0012-
9658(2000)081[3509:EOFAPO]2.0.CO;2
54. Van Buskirk J. A comparative test of the adaptive plasticity hypothesis: Relationships
between habitat and phenotype in Anuran larvae. Am Nat. 2002;160: 87–102.
doi:10.1086/340599
55. Dijk B, Laurila A, Orizaola G, Johansson F. Is one defence enough? Disentangling the
relative importance of morphological and behavioural predator-induced defences. Behav Ecol
Sociobiol. 2016;70: 237–246. doi:10.1007/s00265-015-2040-8
25 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
56. Teplitsky C, Plénet S, Joly P. Tadpoles’ responses to risk of fish introduction. Oecologia.
2003;134: 270–277. doi:10.1007/s00442-002-1106-2
57. Kurali A, Pásztor K, Hettyey A, Tóth Z. Resource-dependent temporal changes in
antipredator behavior of common toad (Bufo bufo) tadpoles. Behav Ecol Sociobiol. 2018;72:
91. doi:10.1007/s00265-018-2503-9
58. McCollum SA, Leimberger JD. Predator-induced morphological changes in an amphibian:
predation by dragonflies affects tadpole shape and color. Oecologia. 1996;109: 615–621.
59. Touchon JC, Warkentin KM. Fish and dragonfly nymph predators induce opposite shifts in
color and morphology of tadpoles. Oikos. 2008;117: 634–640. doi:10.1111/j.0030-
1299.2008.16354.x
60. Van Buskirk J. The costs of an inducible defense in anuran larvae. Ecology. 2000;81: 2813–
2821.
61. Relyea RA. Getting out alive: how predators affect the decision to metamorphose. Oecologia.
2007;152: 389–400. doi:10.1007/s00442-007-0675-5
62. Van Buskirk J. Natural variation in morphology of larval Amphibians: Phenotypic plasticity in
nature? Ecol Monogr. 2009;79: 681–705.
63. Michel MJ. Spatial dependence of phenotype-environment associations for tadpoles in natural
ponds. Evol Ecol. 2011;25: 915–932. doi:10.1007/s10682-010-9441-y
64. Arendt J. Morphological correlates of sprint swimming speed in five species of spadefoot toad
tadpoles: Comparison of morphometric methods. J Morphol. 2010;271: 1044–1052.
doi:10.1002/jmor.10851
65. Van Buskirk J, Anderwald P, Lüpold S, Reinhardt L, Schuler H. The lure effect, tadpole tail
shape, and the target of dragonfly strikes. J Herpetol. 2003;37: 420–424.
26 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
66. Johnson JB, Burt DB, DeWitt TJ. Form, function, and fitness: Pathways to survival. Evolution.
2008;62: 1243–1251. doi:10.1111/j.1558-5646.2008.00343.x
67. Van Buskirk J, McCollum SA. Functional mechanisms of an inducible defence in tadpoles:
morphology and behaviour influence mortality risk from predation. J Evol Biol. 2001;13: 336–
347. doi:10.1046/j.1420-9101.2000.00173.x
68. Teplitsky C, Plenet S, Léna J-P, Mermet N, Malet E, Joly P. Escape behaviour and ultimate
causes of specific induced defences in an anuran tadpole. J Evol Biol. 2005;18: 180–190.
doi:10.1111/j.1420-9101.2004.00790.x
69. Kraft PG, Wilson RS, Franklin CE, Blows MW. Substantial changes in the genetic basis of
tadpole morphology of Rana lessonae in the presence of predators. J Evol Biol. 2006;19:
1813–1818. doi:10.1111/j.1420-9101.2006.01185.x
70. Innes-Gold AA, Zuczek NY, Touchon JC. Right phenotype, wrong place: predator-induced
plasticity is costly in a mismatched environment. Proc R Soc B Biol Sci. 2019;286: 20192347.
doi:10.1098/rspb.2019.2347
71. Van Buskirk J, McCollum SA, Werner EE. Natural selection for environmentally induced
phenotypes in tadpoles. Evolution. 1997;51: 1981–1990.
72. Van Buskirk J, Relyea RA. Selection for phenotypic plasticity in Rana sylvatica tadpoles. Biol
J Linn Soc. 1998;65: 301–328. doi:10.1111/j.1095-8312.1998.tb01144.x
73. Van Buskirk J. Spatially heterogeneous selection in nature favors phenotypic plasticity in
anuran larvae. Evolution. 2017;71: 1670–1685. doi:10.1111/evo.13236
74. Vorndran IC, Reichwaldt E, Nürnberger B. Does differential susceptibility to predation in
tadpoles stabilize the Bombina hybrid zone? Ecology. 2002;83: 1648–1659.
27 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
75. Fischer N. Performance of pure, natural hybrid and F1 Bombina tadpoles in semi-natural
enclosures: Morphology, behaviour, growth, survival and development time. Diploma Thesis,
University of Munich. 2006.
76. Eklöv P, Werner EE. Multiple Predator Effects on Size-Dependent Behavior and Mortality of
Two Species of Anuran Larvae. Oikos. 2000;88: 250–258.
77. Fijarczyk A, Nadachowska K, Hofman S, Litvinchuk SN, Babik W, Stuglik M, et al. Nuclear
and mitochondrial phylogeography of the European fire-bellied toads Bombina bombina and
Bombina variegata supports their independent histories. Mol Ecol. 2011;20: 3381–3398.
78. Baláž V, Vojar J, Civiš P, Šandera M, Rozínek R. Chytridiomycosis risk among Central
European amphibians based on surveillance data. Dis Aquat Organ. 2014;112: 1–8.
doi:10.3354/dao02799
79. Blooi M, Pasmans F, Longcore JE, Spitzen-van der Sluijs A, Vercammen F, Martel A. Duplex
real-time PCR for rapid simultaneous detection of Batrachochytrium dendrobatidis and
Batrachochytrium salamandrivorans in Amphibian samples. J Clin Microbiol. 2013;51: 4173–
4177. doi:10.1128/JCM.02313-13
80. Bradley R, Terry M. Rank analysis of incomplete block designs. I. The method of paired
comparisons. Biometrika. 1952;39: 324–345.
81. Bates D, Mächler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. J
Stat Softw. 2015;67: 1–48. doi:10.18637/jss.v067.i01
82. McCoy MW, Bolker BM, Osenberg CW, Miner BG, Vonesh JR. Size correction: comparing
morphological traits among populations and environments. Oecologia. 2006;148: 547–554.
doi:10.1007/s00442-006-0403-6
28 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
83. Dittrich C, Drakulić S, Schellenberg M, Thein J, Rödel M-O. Some like it hot? Developmental
differences in Yellow-bellied Toad (Bombina variegata) tadpoles from geographically close
but different habitats. Can J Zool. 2016;94: 69–77. doi:10.1139/cjz-2015-0168
84. Melvin SD, Houlahan JE. Tadpole mortality varies across experimental venues: do laboratory
populations predict responses in nature? Oecologia. 2012;169: 861–868.
doi:10.1007/s00442-012-2260-9
85. Van Buskirk J. Habitat partitioning in European and North American pond-breeding frogs and
toads. Divers Distrib. 2003;9: 399–410. doi:10.1046/j.1472-4642.2003.00038.x
86. Barton NH. Multilocus clines. Evolution. 1983;37: 454–471.
87. Barton NH, Bengtsson B-O. The barrier to genetic exchange between hybridising populations.
Heredity. 1986;56: 357–376.
88. Anholt BR, Werner EE. Interaction between food availability and predation mortality mediated
by adaptive behavior. Ecology. 1995;76: 2230–2234.
89. Touchon JC, Robertson JM. You cannot have it all: Heritability and constraints of predator-
induced developmental plasticity in a Neotropical treefrog. Evolution. 2018;72: 2758–2772.
doi:10.1111/evo.13632
90. Barandun J, Reyer H-U. Reproductive Ecology of Bombina variegata: Development of Eggs
and Larvae. J Herpetol. 1997;31: 107–110. doi:10.2307/1565337
91. Köhler SC. Mechanisms of partial reproductive isolation in a Bombina hybrid zone in
Romania. Ph.D. Thesis. University of Munich. 2003.
92. Hartel T, Nemes S, Mara G. Breeding phenology and spatio-temporal dynamics of pond use
by the Yellow-Bellied Toad (Bombina variegata) population: The importance of pond
29 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
availability and duration. Acta Zool Litu. 2007;17: 56–63.
doi:10.1080/13921657.2007.10512816
93. Newman RA. Effects of density and predation on Scaphiopus couchi tadpoles in desert
ponds. Oecologia. 1987;71: 301–307.
94. Relyea RA. Competitor-induced plasticity in tadpoles: Consequences, cues, and connections
to predator-induced plasticity. Ecol Monogr. 2002;72: 523–540. doi:10.1890/0012-
9615(2002)072[0523:CIPITC]2.0.CO;2
95. Lemcke C. Verwandtenerkennung bei Kaulquappen der Gelbbauchunke, Bombina variegata.
Diploma Thesis, University of Munich. 2002.
96. Fraker ME, Hu F, Cuddapah V, McCollum SA, Relyea RA, Hempel J, et al. Characterization
of an alarm pheromone secreted by amphibian tadpoles that induces behavioral inhibition and
suppression of the neuroendocrine stress axis. Horm Behav. 2009;55: 520–529.
doi:10.1016/j.yhbeh.2009.01.007
97. Maher JM, Werner EE, Denver RJ. Stress hormones mediate predator-induced phenotypic
plasticity in amphibian tadpoles. Proc R Soc B. 2013;280: 20123075.
doi:10.1098/rspb.2012.3075
98. Thiesmeier B. Fotoatlas der Amphibienlarven Deutschlands, Österreichs und der Schweiz.
Bielefeld: Laurenti Verlag; 2019.
99. Richardson JML. A comparative study of phenotypic traits related to resource utilization in
anuran communities. Evol Ecol. 2002;16: 101–122. doi:10.1023/A:101638112286
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537
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30 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
539 Supporting Information
540 S1 Fig. Risk context per rearing compartment.
541 S2 Fig. Pairwise plots of morphological traits.
542 S1 Table. Ecological features of the collection sites.
31 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.04.02.021618; this version posted April 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.