Evolution of vertebrate endemics in oceanic islands follows a predictable pattern, known as the island rule, according to which gigantism arises in originally small-sized species and dwarfism in large ones. Species of extinct insular giant rodents are known from all over the world. In the Canary Islands, two examples of giant rats, Canariomys bravoi and Canariomys tamarani, endemic to Tenerife and Gran Canaria islands, respectively, disappeared soon after human settlement. The highly derived morphological features of these insular endemic rodents hamper the identification of their mainland relatives and the reconstruction of their evolutionary histories. We have retrieved partial mitochondrial (mtDNA) data from C. bravoi and used this information to explore its evolutionary history. The resulting dated phylogeny confidently places C. bravoi within the African grass rat clade (Arvicanthis niloticus). The estimated divergence time, 650,000 years ago, is roughly coincident with the onset of the Günz-Mindel interglacial stage. C. bravoi ancestors would have reached the island via passive rafting and then underwent a yearly increase of mean body mass calculated between 0.0015 g and 0.0023 g; this corresponds to fast evolutionary rates (in darwins [d], ranging from 7.09 d to 2.78 d) that are well above those observed for non-insular mammals.

All DNA extraction and initial library preparation steps (prior to amplification) were performed in a dedicated clean lab, physically-isolated from the laboratory used for post-PCR analyses, at the Biomedical Research Park (PRBB) in Barcelona. Strict protocols were followed to minimize the amount of human DNA in the ancient DNA laboratory, including the wearing a full body suit, sleeves, shoe covers, clean shoes, facemask, hair net and double gloving. All lab surfaces consumables, disposables, tools and instruments are wiped with bleach and ethanol, and UV irradiated before and after use.

First, teeth samples were UV irradiated (245 nm) for 10 min and the outermost surface of the teeth was scrapped off with a drill engraving cutter, followed by another UV irradiation for 10 more minutes in order to exclude the surface DNA contamination. Second, approximately 30 mg of tooth cementum were drilled into a fine powder by drilling at low speed (5000 rpm) with a new engraving cutter.

DNA extraction from teeth powder was performed following the method proposed by Dabney et al. (2013). The teeth powder samples, including an extraction blank, were added to 1 ml of extraction buffer (final concentrations: 0.45 M EDTA, 0.25 mg/ml Proteinase K, pH 8.0), resuspended by vortexing and incubated at 37°C overnight (24h) on rotation (750-900 rpm). The remaining tooth powder was then pelleted by centrifugation in a bench-top centrifuge for 2 min at maximum speed (16,100 × g). The supernatant was added to 10 ml of binding buffer (final concentrations: 5 M guanidine hydrochloride, 40% (vol/vol) isopropanol, 0.05% Tween-20, and 90 mM sodium acetate (pH 5.2)) and purified on a High Pure Extender column (Roche). DNA samples were eluted with 45 μl of low EDTA TE buffer (pH 8.0).

A total of 35 μl of each DNA extract was used for library preparation in three sequential reactions: end‐repair, adapter ligation, and nick fill‐in; following the BEST protocol (Carøe et al. 2018). Sample CB-4 was converted into an Illumina sequencing library by PCR amplification with two uniquely barcoded primers and used for shotgun sequencing. Both libraries were purified with a 1x AMPure clean (Beckman Coulter) and eluted in 25 μl of low EDTA TE buffer (pH 8.0). Library size and concentration was determined with the Agilent DNA 7500 Kit on the 2100 BioAnalyzer instrument. The libraries were sequenced in a Hiseq400 of Illumina platform (Macrogen).

After library preparation, sample CB-10 was enriched for mitochondrial DNA sequences with the use of commercially biotinylated probes for mouse mtDNA (MYbaits®). Prior to hybridization, the DNA library (∼500 ng) was brought to 7 μl using a Speedvak concentrator. Two consecutive hybridizations were conducted with the myBaits Capture Kit (Arbor Biosciences) according to the manufacturer’s manual v 4.01. The hybridization reaction was carried out at 65 °C for 24 h in a final volume of 30 μl. Captured targets were recovered with Dynabeads® MyOne Streptavidin C1 magnetic beads (Invitrogen), followed by bead-bait binding and washing according to the manufacturer’s recommendations. After the first round of enrichment, post captured amplification was performed using PCR primers IS5 and IS6. All of the captured material was concentrated to 7 µl and used for the second round of hybridization. The second hybridization was performed under the same conditions and the final captured pool was amplified with P5 and P7 indexed primers compatible for Illumina sequencing (Kircher et al. 2012). Several attempts to capture the mitochondrial DNA from sample CB-4 failed.

Cb10 include Mitochondrial Capture Data of individual 10.

Cb4 include Shotgun Sequencing Data of individual 4.