Co-amplification of mitochondrial pseudogenes in Calomys musculinus (Rodentia, Cricetidae): a source of error in phylogeographic studies

GONZÁLEZ ITTIG, R. Y GARDENAL, C.N.

GENOME

NATL RESEARCH COUNCIL CANADA-N R C RESEARCH PRESS

Año: 2008 vol. 51 p. 73 - 78

0831-2796

Abstract In a previous phylogeographic study in the rodent Calomys musculinus, 24 haplotypes of the mitochondrial DNA D-loop region were detected using the restriction fragment length polymorphism technique (PCR-RFLP). Seven percent of the individuals showed patterns in which the sum of the sizes of the restriction fragments exceeded that of the original PCR product. In the present paper we analyze possible causes of these atypical haplotypes. PCR products were cloned; two or three different clones from a single individual were detected by their RFLP patterns. Nine clones with different restriction patterns were selected for sequence analyses. A maximum parsimony phylogenetic analysis revealed two well supported paraphyletic groups; one of them comprised sequences showing low nucleotide divergence compared with the most common haplotypes detected in the phylogeographic study. The other group was basal to the three species of Calomys other than C. musculinus included in the study; the mutations in the short portion of the cytochrome b amplified correspond to 12 amino acid substitutions. The results suggest that two independent insertions of mtDNA sequences into the nucleus occurred; these sequences would co-amplify in the PCR procedure. Identification of pseudogenes is crucial to get a reliable reconstruction of the intraspecific genealogy in phylogeographic studies.