1 Sex chromosomes can evolve during the evolution of genetic sex determination (GSD) 2 from environmental sex determination (ESD). Despite theoretical attention, early mechanisms 3 involved in the transition from ESD to GSD have yet to be studied in nature. No mixed ESD-4 GSD animal species have been reported, except for some species of Daphnia, small freshwater 5 crustaceans in which sex is usually determined solely by the environment, but in which a 6 dominant female sex-determining locus is present in some populations. This locus follows 7Mendelian single-locus inheritance, but has otherwise not been characterized genetically. We 8 now show that the sex-determining genomic region maps to the same low-recombining peri-9 centromeric region of linkage group 3 (LG3) in three highly divergent populations of D. magna, 10 and spans 3.6 Mb. Despite low levels of recombination, the associated region contains signs of 11 historical recombination, suggesting a role for selection acting on several genes thereby 12 maintaining linkage disequilibrium among the 36 associated SNPs. The region carries numerous 13 genes involved in sex differentiation in other taxa, including transformer2 and sox9. Taken 14 together, the region determining the NMP phenotype shows characteristics of a sex-related 15 supergene, suggesting that LG3 is potentially an incipient W chromosome despite the lack of 16 significant additional restriction of recombination between Z and W. The occurrence of the 17 female-determining locus in a pre-existing low recombining region illustrates one possible form 18 of recombination suppression in sex chromosomes. D. magna is a promising model for studying 19 the evolutionary transitions from ESD to GSD and early sex chromosome evolution. 20 animal groups (Ohno 1967;Pokorná and Kratochvíl 2016). Although transition from ESD to 44 GSD may be gradual, involving shifting genotype-specific thresholds for male vs. female 45 development under fluctuating environmental conditions (Van Dooren and Leimar 2003), a 46 scenario similar to that of the transition from hermaphroditism to separate sexes is also plausible: 47Supplementary Material S1). In all three crosses, the fully linked markers mapped between cM 129 positions 87.8 cM and 94.0 cM of LG3 in the reference genetic map. This region also contains 130 the centromere (at 90.8 cM). We call this region the "NMP region" (Fig. 2). A Marey map of 131LG3 (Dukič et al. in press) shows that the NMP-region corresponds to a large (~3 Mb) non-132 recombining region around the centromere (Fig. 2). Non-recombining regions around the 133 centromeres are found on all linkage groups of D. magna, and are not a sign of reduced 134