Genetic determination (with the possibility of epigenetics37;) and stochasticity were probably the most responsible factors explaining the registered variability in tolerance, because neither trends within breeding seasons nor correlations with any field or laboratory environmental parameter were found. However, this lack of patterns did not fully exclude the occurrence of phenotypic plasticity and maternal effects, which occur commonly in some species of amphibians2,38. The embryos of some species can express such maternal effects, depending on the size of the yolk in the eggs; eggs with larger yolk would hatch larger larvae38. It is documented that female frogs show variability in egg’s yolk size, for different egg masses, but only for populations inhabiting very environmental unstable environments38,39; which was not the case of the reference site visited in the present work.

The aim of this work was to test the recessive tolerance inheritance (working-) hypothesis. To support its worst-case scenario – (full) recessivity – each of the tested egg masses should fall in one of the following categories: (i) all eggs being highly sensitive, with small relative spread (at least one parent being homozygous dominant); approximately matched by egg masses C, D, F, and H for AMD and by egg masse B14 for copper; (ii) all eggs being highly tolerant, with small relative spread (both parents being homozygous recessive); matched by no egg mass (Figs. S1, S2, S4); (iii) eggs, within each egg mass, being either highly tolerant or highly sensitive in similar (~50%) proportions, with very large relative spread (the crossing of a heterozygous with a homozygous recessive frog); matched by no egg mass for AMD and approximately matched by egg masse K14 for copper; (iv) similar to the previous category but with a much higher proportion (~80%) of highly sensitive eggs, with large relative spread (both parents being heterozygous); approximately matched by egg mass A, E and G for AMD and mass E14 for copper (Figs. S1, S2, S4). The first and second categories are also possible outcomes of all other tolerance inheritance patterns (dominance, overdominance, underdominance, and incomplete dominance). The third category may also be a possible outcome of all patterns except the incomplete dominance.

Only six out of 21 egg masses exposed to AMD and only three out of 40 egg masses exposed to copper were found to possibly support the (full) recessivity mechanism for tolerance. Only egg masses matching the fourth category (3 out of 21 egg masses exposed to AMD and one out of 40 egg masses exposed to copper) fully support tolerance being recessive because this category is a possible outcome of no other inheritance pattern.

If tolerance is inherited as a (fully) dominant, overdominant or underdominant trait, then neither a partially lethal nor even an almost fully lethal input of AMD or copper would eliminate alleles from the impacted population, since either the heterozygote would be maximally tolerant (dominance and overdominance) or both homozygotes were the most tolerant individuals (underdominance). In the AMD assay, eight (egg masses A, C, D, E, F, G, H, T, and U) out of 21 (38%) egg masses could support (full) dominance, overdominance or underdominance (Figs. S1 and S2). Whereas in the copper assays, nine (B14, E14, K14, P14, R14, R16, T14, Q16, R16, and T16) out of 40 (22.05%) egg masses could match those pattern of tolerance inheritance (Figs. S1 and S4).

Incomplete dominance was almost fully supported: egg masses presented broad ranges of egg tolerance with a unimodal distribution (Tables T3 and T4, Figs. S2 and S4). All possible unimodal tolerance patterns could be explained by incomplete dominance (up to 95% of the collected egg masses for AMD up to 97.5% for copper). A partially lethal pulse of AMD or copper would wipe out the most sensitive genotypes, although not fixing in the population the allele conferring tolerance. Allele’s fixation would happen only after exposure to an almost fully lethal concentration (only the homozygous tolerant genotypes would survive), depending on the degree of incomplete dominance, encompassing from almost full recessivity to almost full dominance. Nevertheless, neither of the alternative scenarios discussed above (full recessivity, full dominance, overdominance or underdominance) can be totally excluded. These less likely patterns could be supported by 38% of the egg masses exposed to AMD and by 22% of those exposed to copper. This 16% difference between the two contaminants can be due to the fact that tolerance to acid mine drainage may involve different physiological mechanisms than that to copper, probably because of the different pH, the former being notably acid while the latter rather neutral. Indeed, pH can influence metallic ions speciation (biotic-ligand-model theory)40. At least for fish (but arguably for others aquatic vertebrates breathing through gills), increasing pH decrease copper ions absorption40. However, because the acid mine drainage contains much more than copper alone29, its toxicity is possibly due also to other ions.

In all the possible patterns of tolerance inheritance, very tolerant and very sensitive egg masses should present a small relative spread: intermediately tolerant egg masses should present higher spread. Because the latter egg masses would comprise only very sensitive and very tolerant eggs, with the exception of the incomplete dominance pattern for which the latter egg masses include intermediately sensitive and tolerant eggs. Thus, when comparing the relationship between relative spread and LT 50 values, the only expected shape for the distribution would be that of a crystal clear inverted-U. Which would be much less evident only if tolerance inheritance is due to incomplete dominance. This was found in the present study, for both AMD and copper (Figs. S3 and S5), further supporting incomplete dominance as the mechanism of tolerance inheritance.

A reasonable assumption is that each egg mass was fertilized by a one male. However, amplexi involving more than one male were reported for other amphibian species41. Some studies described multiple paternity in some anuran species, but the offspring of polyandrous mating were always a small proportion. In Rana temporaria, where high clutch piracy was reported (84% of clutches), the secondary males fertilized only 26% of the clutches and, in these clutches, only 24% of embryos were fertilized by pirate males42. Basically only 5%, or less, of all eggs collected at that breeding season were sired by a secondary male42. This is also the case in Rana dalmatina, in which only 4% of all eggs were sired by a secondary male43. It is reasonable that the effect and occurrence of polyandry, if present at all, would be negligible; however, secondary fertilization might result in egg masses with a possibly broader genetic makeup. Such situation could lead to an egg mass with a very large relative spread, which matches egg mass A and masses K14, Q14, S14, P16, and Q16 (Tables T3 and T4, Figs. S2 and S4). Those masses could also result from the simultaneous presence of extremely tolerant and sensitive eggs and the almost or full absence of intermediately tolerant eggs. This could also be determined by tolerance being a trait other than incompletely dominant or, at much less extent, arise from a polyandric mating41, especially in the cases of masses which show a bimodal distribution. However, as far as we are aware of, there are no evidences of multiple paternities in P. perezi. In the closest relatives for which information is available, the percentage of eggs sired by a secondary father is very low, with offspring of polyandrous mating being always a small proportion42,43. Furthermore, bimodality is clearly the exception rather than the rule in our data (Figs. S2 and S4).

The low occurrence of safely tolerant egg masses (S, T and U: only three out of 20 for AMD; O14, P14, R14, S14, R16, S16 and T16: only seven out of 40 for copper) compared with those critically sensitive (B, C, D, E, F, G, H: seven out of 20 for AMD; A14, B14, C14, D14, E14, F14, A16, C16, D16, E16: 17 out of 40 for copper), could be explained by the lack of past selection for tolerance to metal contamination.