Molecular Ecology (2004) 13, 1025 – 1034 doi: 10.1111/j.1365-294X.2004.02114.x © 2004 Blackwell Publishing Ltd Blackwell Publishing, Ltd. On the neutrality of molecular genetic markers: pedigree analysis of genetic variation in fragmented populations C. VAN OOSTERHOUT,* M. K. VAN HEUVEN and P. M. BRAKEFIELD Section of Evolutionary Biology, Institute of Biology, Leiden University, P.O. Box 9516, 2300 RA Leiden, the Netherlands Abstract Many studies employ molecular markers to infer ecological and evolutionary processes, assuming that variation found at genetic loci offers a reliable representation of stochastic events in natural populations. Increasingly, evidence emerges that molecular markers might not always be selectively neutral. However, only a few studies have analysed how deviations from neutrality could affect estimates of genetic variation, using populations with known genealogy. We monitored changes in allozyme variation over eight generations in captive metapopulations of the butterfly Bicyclus anynana. Population demography was recorded by individually marking 35 000 butterflies and constructing pedigrees. We designed a computer program that simulated the inheritance of founder allozyme alleles in butterfly pedigrees. We thus tested whether the observed transmission of allozyme alleles could be explained by random genetic drift alone, or whether there was evidence for positive or negative selection. This analysis showed that in the smallest metapopulations the loss of allozyme variation exceeded the neutral rate. Possibly, linkage disequilibria between deleterious mutations and marker alleles resulted in background selection and a faster erosion of allozyme variation. In larger metapopulations, one locus (MDH) showed a significant heterozygote excess and smaller than expected loss in heterozygosity, observations con- sistent with (associative) overdominance. This study demonstrates that the neutrality of molecular markers cannot always be assumed, particularly in small populations with a high mutation load. Keywords: allozymes, associative overdominance, background selection, Bicyclus anynana, meta- population, temporal genetic analysis Received 30 September 2003; revision received 2 December 2003; accepted 11 December 2003 Introduction Molecular techniques have made an important contri- bution to population genetics and evolutionary biology in the last two decades. Throughout this period, a debate persisted on whether random genetic drift or selection is the dominant force in molecular evolution (Kimura 1983; Gillespie 1991). Considerable progress has been made in this ‘selectionist/neutralist debate’ through advances in statistical techniques (Kreitman 1996; Ford 2002). Estimates on the distribution of selective effects on genetic variation can be inferred from the distribution of polymorphisms between and within taxa, populations and individuals (Ford 2002). In these studies, the strict neutral theory has become the standard null hypothesis, even though this is commonly rejected. Unfortunately, a limitation in most studies is that the underlying demographic data can only be inferred from observed genetic variation of the markers themselves. A particular pattern of genetic variation can therefore be explained both by deviations from the neutral model, as well as by certain demographic processes (see, e.g. Bowcock et al . 1991). For instance, in populations with an island-model population structure, loci experiencing balancing selection will have allele frequencies more similar than expected under neutrality (Porter 2003). This might be interpreted incorrectly as evidence for gene flow. Similarly, fluctuation in population size may create a genetic signature identical to that observed after disruptive or Correspondence: Cock van Oosterhout. *Present address: Depart- ment of Biological Sciences, University of Hull, Hull, HU6 7RX, UK. E-mail: C.van-Oosterhout@hull.ac.uk