Can DNA methylation contribute to explain morphological variability? The case of loggerhead turtle Caretta caretta

The loggerhead turtle Caretta caretta is a common and threatened sea turtle in the Mediterranean Sea. It is listed in the Red List of Threatened Species of the International Union for Conservation of Nature (IUCN 2012) and, although it is widely studied, little is currently known about the relationships between its phenotype and genetic variability. In the last few years some authors observed the presence of individuals, collected in different parts of the Mediterranean Sea, with a variable number of carapacial and plastron scutes. This variability, in some cases, makes difficult the sound identification of the species (Margaritoulis and Chiras, 2011; Turkozan et al. 2001; Oliver 2014). To date, no one has tried to correlate these morphological anomalies with the genetic diversity of the species, even if several studies indicate that, beyond genetic variation, natural phenotypic variations can be generated through a suite of epigenetic mechanisms. Here we present some results about the possible correlation among the variability of the number of carapacial and plastron scutes and cytosine DNA methylation. 12 specimens of Caretta caretta, collected from different parts of Sicily, were analysed. Morphological characters were used for the identification of the collected specimens according to the guidelines of Marquez (1990). Afterwards, all the individuals were subjected to genetic analysis by the sequencing of a fragment of 243 bp of the mitochondrial cytochrome b gene. Genomic DNA methylation level of cytosines was evaluated by dot blot analysis using antibodies against methylated cytosines. Four of the twelve individuals analyzed showed an unexpected number of carapacial and plastron scutes making difficult the immediate and sound identification of the specimens as C. caretta. Sequences of the mitochondrial cytochrome b gene of these four individuals did not show any difference with those obtained from the other eight loggerhead turtles and with those from Genbank, thus allowing us to assign the four individuals to the species Caretta caretta. Conversely, dot blot analysis of genomic DNA showed a reduced global level of methylated cytosines in these four “abnormal” individuals in respect to the other ones. We hypothesize that the variability of the number of scutes could be described as an epigenetic effect probably due to the environmental parameters experienced by the embryos during incubation. This finding could have implications in our understanding of the pathways of morphological evolution and diversification in the chelonians.