The Balearic Islands were last connected to the continent during the Messinian regression, when the Mediterranean basin was dried out, allowing faunal exchanges between the islands and the continental lands [ for calibrating the DNA molecular clock of caprines.
Previously, only dates outside caprines (the emergence of the family Bovidae 18.5 Mya), or dates that are too recent for accurate estimations (common ancestry of some domestic sheep breeds a few thousand years ago) could be used .
For this purpose, we decided to retrieve the complete cytochrome bone, obtained in 2002 from Cova des Gorgs, that looked macroscopically very well preserved and therefore was suggestive of DNA survival.
The putative presence of nuclear mt DNA insertions is very unlikely, since we proceed designing the L primers for the next fragment in the sequence already retrieved from the previous fragment, and the H primers in a consensus Caprinae sequence.
The 12S gene sequence (305 base pairs) was retrieved in three overlapping fragments (see primers in Table ), and clearly different to the human one and to the cow, a suggested source of contamination because of the use of BSA (bovine serum albumine) in ancient DNA amplifications.
The number of lineages in each node is show in logarithmic scale.
The horizontal scale bar represents 1 Mya for both the tree and the lineages through time plot.Moreover, no other Bovids, extinct or living, had been analyzed in the same laboratory when the extraction, amplification, cloning and sequencing of the ) showed that two of the sequences (about 20% of the clones) were human contaminants; this accounts for the noise observed in the direct sequencing of the PCR product.