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The present paper deals with the estimation of inbreeding and substructure levels in a set of ten (later regrouped as eight) African-derived quilombo communities from the Ribeira River Valley in the southern portion of the state of São Paulo, Brazil. Inbreeding levels were assessed through F values estimated from the direct analysis of genealogical data and from the statistical analysis of a large set of 30 molecular markers. The levels of population substructure found were modest, as well as the degree of inbreeding: in the set of all communities considered together, F values ranged from 0.00136 to 0.00248, when using raw and corrected data from their complete genealogical structures, respectively, to 0.027 to 0.036, when using the information taken from the statistical analysis of all 30 loci and of 14 loci of SNPs respectively. The overall frequency of consanguineous marriages in the set of all communities considered together was around 2%. Although modest, the values of the estimated parameters are much larger than those obtained for the overall Brazilian population and in general much smaller than the ones recorded for other Brazilian isolates. To circumvent problems related to heterogeneity sampling and virtual absence of reliable records of biological relationships we had to develop or adapt several methods for making valid estimates of the prescribed parameters.