Determination of Human Caucasian Mitochondrial DNA Haplogroups by Means of a Hierarchical Approach CRISTINA SANTOS, 1,2 RAFAEL MONTIEL, 1,3 NATA ` LIA ANGLE ´ S, 1 MANUELA LIMA, 3 PAOLO FRANCALACCI, 4 ASSUMPCIO ´ MALGOSA, 1 AUGUSTO ABADE, 2 AND M. PILAR ALUJA 1 Abstract In this paper we propose a hierarchical approach that allows the screening of mitochondrial DNA (mtDNA) haplogroups in populations that have essentially West Eurasian mtDNA backgrounds but that could have some non–West Eurasian contributions. To develop and validate this scheme, we used data on 18 coding region polymorphisms (17 analyzed by RFLP analysis and 1 by sequencing) and sequences of hypervariable segment I (HVSI) of the mtDNA control region from the Azores Islands (Portugal) population. The proposed scheme allows the characterization of almost all West Eurasian and African major clusters by means of RFLPs. Furthermore, the scheme includes information on situations in which sequencing is perti- nent to defining a particular haplogroup. The validity of the scheme is en- sured by (1) using relatively stable polymorphic positions, (2) screening more than one position to define a specific haplogroup, and (3) typing con- firmatory positions. Dubious samples can be resolved by sequencing. The robustness of this approach was assessed by sequencing all samples for HVSI, taking advantage of the previously established relationships between RFLPs and control region sequence polymorphisms. The use of this hierar- chical approach avoids the screening of unnecessary control region polymor- phisms and therefore results in a more rapid and cost-efficient screening than one in which all polymorphic positions are analyzed. Even if this approach leads to a lower level of phylogeographic resolution than the sequencing of all samples, it allows us to define population movements on a continental level and can be applied, unlike sequencing all samples, with a low cost in any laboratory. 1 Anthropology Unit, Department BABVE, Faculty of Sciences, Autonomous University of Barcelona, 08193 Bellaterra (Barcelona), Spain. 2 Department of Anthropology, University of Coimbra, 3000 Coimbra, Portugal. 3 CIRN and Department of Biology, University of the Azores, 9500 Ponta Delgada, S. Miguel, Azores, Portugal. 4 Department of Zoology and Biological Anthropology, University of Sassari, 07100 Sassari, Italy. Human Biology, June 2004, v. 76, no. 3, pp. 431–453. Copyright  2004 Wayne State University Press, Detroit, Michigan 48201-1309 KEY WORDS: MTDNA, HAPLOGROUPS, RFLP, AZORES, HIERARCHICAL APPROACH TO HAPLOGROUP SCREENING.