DOI: 10.5061/dryad.9s4mw6mhn
title: Genetic data from the extinct giant rat from Tenerife (Canary Islands)

description: 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.
description: 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.
description: Cb10 include Mitochondrial Capture Data of individual
 10. Cb4 include Shotgun Sequencing Data of individual
 4.