Mitochondrial DNA genotyping
We genotyped all individuals at the hypervariable region I of the
mitochondrial control region using primers 5’-TACCTCGGTCTTGTAAACCG-3’
and 5’-AGGTAGGAACCAGATGCCG-3’, newly designed on the basis of
mitochondrial genomes of New World monkeys available in Genbank. PCR
reactions with a total volume of 30 µl contained 1 U BiothermTaq 5000
(Genecraft), 1x reaction buffer, 0.16 mM of each dNTP, 0.33 µM of each
primer and ca. 100ng total DNA. PCRs were performed with initial
denaturation at 95 °C for 2 min, 40 cycles of denaturation at 95 °C for
1 min, annealing at 58 °C for 1 min, extension at 72 °C for 1 min and a
final extension at 72 °C for 5 min. PCR products were run on 1%
agarose gels, excised from the gel and then purified with the Monarch
DNA Gel Extraction Kit (New England BioLabs) and sequenced on an ABI
3130xL sequencer using both amplification primers and the BigDye Cycle
Sequencing Kit (Applied Biosystems). Sequence electropherograms were
checked with 4Peaks 1.8
(https://nucleobytes.com/4peaks/index.html) and manually edited
and assembled in SeaView 4.5.4; all haplotypes were 567 bp long.