To prevent the influences of other specimens, each specimen was individually put into a cup with a different combination of salinity and temperature.
First, when we analyze the three Neandertal bones Vi33.16, Vi33.25, and Vi33.26 separately, we obtain consistent values of the D statistics, which is unlikely to arise under the hypothesis of contamination because each specimen was individually handled and was thus unlikely to have been affected by the same degree of contamination (SOM Text 15).
Briefly, tpsRelw uses a generalized Procrustes analysis (GPA) to compute a consensus shape with a minimum sum of squared Procrustes distances to other specimens wherein each specimen is individually superimposed onto the average of previous specimens until all specimens are averaged.
The genotyping-by-sequencing approach which generated these data used restriction enzymes to cut up the genome into DNA fragments that are distributed across the genome, sequenced tens of millions of these fragments on the Illumina next-generation sequencing platform, and aligned the fragments to discover genetic variation (facilitated by each specimen being individually barcoded).
The specimens were individually positioned perpendicularly to the tester tip, and the VHN values were recorded by a software program (CAMSTM_WIN program - Newage Testing Instruments, Southampton, PA, USA).
In groups G2, G3 and G4, the specimens were individually coupled to a metallic circular matrix and polished with SiC sandpaper (#1200) for 5 s to remove the outer resin layer, obtain a standardized and stable surface.
Standard COI barcodes for all specimens were individually Sanger sequenced and compared against the Barcode of Life Data Systems for taxonomic confirmation.
Collected mite specimens were individually rehydrated for two hours in 200 μl of sterile water before being crushed in liquid nitrogen.
Specimens were individually placed in microcentrifuge tubes containing 500 μL of tissue lysis buffer (QIAGEN, Valencia, CA, USA) and 20 μL of proteinase K (QIAGEN), bisected with a sterile blade, and incubated at 56°C for ≥1 h.
Specimens were individually immersed in 10 mL of demineralizing solution (2.2 mM CaCl2, 2.2 mM NaH2PO4, 50 mM acetic acid adjusted pH of 4.8) for 8 h and in the same volume of remineralizing solution (1.5 mM CaCl2, 0.9 mM NaH2PO4, 0.15 mM KCl adjusted pH of 7.0) for 16 h [ 10].
