Unexplainable aspects in Kammerer’s experiments
Resistance of the eggs to aquatic mouldsnext section
Many researchers have tried to breed Alytes larvae from eggs placed in water, but they failed as all eggs died from Oomycete infections (De L'Isle du Dreneuf, 1873; Fischer-Sigwart, 1885; Boulenger, 1912; Nijs, 1985). Midwife toads in nature breed on land and are normally not exposed to the water dwelling moulds. Moreover, soon after an egg is laid, the exterior gelatinous layer toughens and forms a cover that protects the egg from fungi. So, under natural conditions, they are not exposed to the fungi that attack amphibian eggs in water. Alytes has no recent co-evolutionary history with aquatic fungi such as Saprolegnia and, as a consequence, no resistance against them. Kammerer admits that mortality of Alytes-eggs placed in water is high: “Ultimately, the eggs stay behind in the water, where, indeed, most of them perish (in the first breeding attempt of that kind), but a few of them then still continue their development.” (Kammerer, 1919:326). He claims that mortality decreases with subsequent breeding attempts of the same animals: “Later, the results improve considerably; in later generations of animals with completed instinct variation, the mortality of water eggs is hardly higher than that of other frog eggs that are normally laid in water.” (Kammerer, 1919:356). The only possible mechanism for such a rapid decrease in egg mortality would be that eggs became resistant to Oomycetes. There is substantial evidence that resistance of amphibian eggs to Oomycetes has a genetic basis and that maternal effects do not represent a major contribution to variation in infection of eggs and embryos (Sagvik et al., 2008a, b; Ault et al., 2012; Urban et al., 2015). We fail to recognize any plausible mechanism for how subsequent clutches of the same mother could become increasingly resistant. Kammerer’s (1909) experiments were started with a moderate number of adults, the F1 generation suffered massive mortality, resulting in a genetic bottleneck. Thus, selection in his experiment cannot have resulted in massive resistance against a community of Oomycetes species within one or two generations. It is even more difficult to understand how the epigenetic activation or the inactivation of one or more genes could confer resistance of eggs against pathogens to which they have not been exposed before. Hence, it is simply hard to believe that Kammerer succeeded in breeding the large numbers of water-breeding midwife toads he mentions in 1906 and in his crossing experiments (Kammerer, 1911).
It is unclear why a rise in temperature would have induced the midwife toads in Kammerer’s experiment to move to the water basin and spend most of their time in the water. Kammerer (1909:462) says “… it is the unusually high temperature that forces them to spend much more time in the water…” and he explains this by saying that ”... animals that have to endure disagreeable temperatures go into the water to cool themselves …”. This cannot be true for his experiments, as the water in the basin must have had the same elevated temperature as the room and the terrarium. Later, Kammerer (1919:325) adds a different explanation: “Alytes, as most nocturnal amphibians, likes it cool, and looks for a cool place and shelter against desiccation of the skin in the water as soon as the air becomes too warm.” (italics are ours). It is unlikely that the elevated temperature would have caused a low air humidity, as there was a water basin in the terrarium and there was only little ventilation. Moreover, breeders report difficulties in breeding Alytes obstetricans (Laurenti, 1768) indoors even under circumstances mimicking the natural world. The Iberian congeneric A. cisternasii Boscá, 1879 and A. muletensis (Sanchiz & Adrover, 1977) appear easier in this respect but were not available to Kammerer (Tonge and Bloxam, 1989; Bloxam and Tonge, 1995; Michaels et al., 2016).
Egg size, number, and development
Water-breeding frogs and toads lay smaller eggs and in larger numbers than the land-breeding midwife toads. Based on this observation Kammerer expected midwife toads to develop the same adaptations, when forced to breed in water. Egg size and female fecundity are heritable traits, hence selection on egg size in experiments is possible. However, in Kammerer’s experiments there was no direct selection on egg size and number. It is hard to see how an increase in temperature could have selected for smaller eggs and higher fecundity.
Kammerer describes morphological changes of the eggs: they develop more voluminous gelatinous coats and yolk decreases with subsequent breeding cycles and generations. Embryonic development in water is heterochronous in comparison to eggs developing on land. This change involves a suite of adaptations, of which it is unlikely that they can be brought to expression by a mild increase in temperature.
Mating behaviour and nuptial pads
It is hard to understand how the experiment described in the Kammerer (1919) paper could have resulted in F1 water-breeding offspring without parental care, irrespective of the temperature regime applied. Selection in one generation cannot have resulted in the disappearance of the normal breeding behaviour, and the animals continuously kept at lower temperatures after exposure to the experimental temperatures might be expected to have resumed the original land-breeding behaviour.
According to Kammerer, water-breeding males developed nuptial pads, but these did not appear until the third generation. At that stage of Lamarckian evolution, the pads were scarcely visible and only they became fully expressed in the fifth generation. An epigenetic explanation of the appearance of the nuptial pads must also explain why the expression of this character increases gradually over the generations. If Kammerer really produced male midwife toads with nuptial pads in his experiments, the most likely explanation is that he had used a wild-caught male with nuptial pads (as are rarely found in nature, see Kändler, 1924) and, with this very male, raised offspring also in possession of nuptial pads.
The variance in the genetic data
Kammerer explicitly states that he predicts Mendelian ratios to apply for the offspring in his crossing experiments (Kammerer, 1911). Actually, the numbers of water- and land-breeding F2 -offspring provided by Kammerer (1911:101-104) are remarkably close to the expected 3 to 1 Mendelian ratio. In the four Alytes-experiments, with sample sizes in the 20-28 range, the differences between the observed values and those suggested by the Mendelian ratio are -0.5, 0, 0 and 0.5 (see Appendices I and II). However, for unbiased sampling at sample sizes of ≥ 20 the expected deviates (|observation minus the mean|) have an average well in excess of unity. This raised our suspicion as to the nature of Kammerer's data. We hence investigated other results of which Kammerer said they were in support of the 3 to 1 Mendelian ratio and found 11 observations on crosses of different phenotypes of the fire salamander, namely the spotted ‘typical form’ and the striped ‘taeniata’ form (Kammerer, 1913:131), that are currently known as the subspecies Salamandra salamandra salamandra Linnaeus, 1758 and Salamandra salamandra terrestris Lacépède, 1788. The argument is reminiscent of the Mendel-Fisher controversy and for all possible intricacies and subtleties involved in the conscious or unconscious mechanisms operating in data gathering that could explain certain biases we refer to Franklin et al. (2008) and references therein. As for Kammerer's results, we found our concern vindicated (Figure 2). The chance for finding results as close as observed to the Mendelian ratio are 0.510, i.e. P<0.001 for the fire salamander and 0.514, i.e. P<0.0001 for the two data sets combined. We propose that there can be no reasonable doubt that Kammerer's data are too good to be genuine.
Figure 2. Cumulative probabilities of the binomial distribution for the 3 to 1 Mendelian ratio at sample sizes ≤ 40. Published data are shown by open square symbols (N=4, midwife toad; Kammerer, 1911:101-104) and by open round symbols (N=11, fire salamander; Kammerer, 1913:131). Unbiased data would be equally distributed over the four quartiles, whereas the given data all fall in the second and third quartiles (light shading) and none in the first or fourth quartile (dark shading), suggesting an anomaly of kinds. The small solid dots represent a data ambiguity, presumably a typographical error (for an explanation see Appendix II).