Kammerer’s Alytes experiments: design and results
Four publications report on Kammerer’s experimental work with Alytes (Kammerer 1906, 1909, 1911 and 1919). The data that inspired Vargas (2009) and Vargas et al. (2016) to suggest an epigenetic explanation for the experiments are described in the 1909 and 1911 publications, that we here summarize. Note that the terms ‘eggs’ and ‘embryos’ (fertilized eggs) are here used interchangeably.
The 1909 publication is based on experiments in terrariums with three experimental groups, each initiated with 15 males and 15 females. One group was kept in an unheated room, one group was kept in a room at temperatures of 25 - 30 °C and one group was placed in a room kept at 35 - 40 °C. The latter group was soon abandoned, as the high temperature turned out to be lethal for the experimental animals. No bookkeeping was done on the identity of the parents of the clutches. No data are provided on mortality and the number of offspring successfully reared. A fourth group of animals was kept in an outdoors terrarium and served as a source of new experimental animals. Experiments were continued for at least four generations. No data were provided on the numbers of animals in the experimental groups of the new generations. Kammerer claimed that the experiments in which he exposed midwife toads to temperatures between 25 and 30 °C resulted in the following changes in comparison to free living animals or animals kept in an unheated room:
- Behavioural changes. Males exposed to temperatures between 25 and 30 °C gave up parental care of the embryos, the sexes paired in the water instead of on land (Kammerer, 1909), in subsequent reproductive cycles even without the stimulus provided by a higher temperature (Kammerer, 1919).
- Changes in egg size and egg number. Females exposed to temperatures between 25 and 30 °C laid larger clutches of smaller eggs.
- Changes in resistance of the eggs. Initially, the majority of eggs laid in water succumbed due to infection with Oomyceta such as Saprolegnia, but eggs seemed to become resistant against these moulds, already in later reproductive cycles by the same parents, and more so in subsequent generations.
- Changes in development. Water-born larvae emerge before the horny external beaks develop, with external gills and a distinct yolk-sac. These larvae develop into adults in several months, whereas larvae emerging from land-eggs take over a year to reach adulthood.
- Morphological changes. Males of midwife toads raised at temperatures between 25 and 30°C gradually developed nuptial pads, i.e. in two to four generations. Eggs produced at these temperatures developed thicker gelatinous coats and had smaller yolk masses.
In 1911 Kammerer reports the data of his crossing experiments between water- and land-breeding midwife toads. He claims that when the father is land-breeding and the mother water-breeding, all animals of the F1 -offspring are land-breeding and that three quarters of the F2 -offspring is land-breeding and one quarter is water-breeding. Likewise, when the father is water-breeding, all animals in the F1 are water-breeding, three quarters of the F2 -offspring is water-breeding and one-quarter is land-breeding. Importantly, these results seem to be in agreement with a “parent of origin effect”, in this case operating through the father and appear to be the main reason for Vargas et al. (2016) to suggest that epigenetics could explain Kammerer’s (1911) results. Kammerer (1911) treats “land-breeding” and “water-breeding” as alleles of a single gene. Vargas et al. (2016) do not question this and do not discuss how a different genetic architecture might affect the interpretation of Kammerer’s data.
In nature, male midwife toads have been observed to call to attract females at temperatures up to 22 °C (Llusia et al., 2013). This begs the question how a moderate increase in temperature could provoke all these simultaneous changes. Vargas et al. (2016) suggest that the mechanism for the reactivation consists of epigenetic switches that act in concert on the genes involved. All Alytes species breed on land and have male parental care. They diverged from the other, water-breeding discoglossid frogs such as in the genera Bombina and Discoglossus ca. 140 million years ago. The reproductive mode of terrestrial breeding with paternal care pre-dates the most recent common ancestor of the five extant Alytes-species, which amounts to ca. 15 Ma (Arntzen & García-París, 1995; Martínez-Solano et al., 2004). The hypothesis of Vargas et al. (2016) implies that the genetic architecture underlying all the changes listed above has been conserved since the split between Alytes and the other discoglossid frogs. Characters that are either not expressed or not adaptive are subject to genetic degeneration, as, for instance, the loss of scleral ossification and eyesight in cave fish (Yoshizawa et al., 2012; Meng et al., 2013; O’Quinn et al., 2015) Thus, such a scenario is only plausible when switching from land-breeding to water-breeding at temperatures above 25 °C (and the reverse at temperatures below 25 °C) would be adaptive. Vargas et al. (2016) do not provide any evidence for this. Moreover, they accept Kammerer’s (1911) data as genuine, without any further analysis.
In the following we will: (1) provide evidence in favour of a multigenic architecture at the basis of all the changes reported by Kammerer – if these were to exist – and show how this affects the interpretation of Kammerer’s (1911) genetic data, (2) address the question if a mechanism as suggested by Vargas et al. (2016) would have been favoured and maintained by natural selection, (3) discuss the possible role of selection in Kammerer’s experiments, and (4) look critically at some of Kammerer’s (1911, 1913) numerical data and address the question if they are genuine.