Sex differentiation in grayling ( Salmonidae ) goes through an all - male stage and is 1 delayed in genetic males who instead grow faster 2 3

27 Fish can be threatened by distorted sex ratios that arise during sex differentiation. It is 28 therefore important to understand sex determination and differentiation, especially in river29 dwelling fish that are often exposed to environmental factors that may interfere with sex 30 differentiation. However, sex differentiation is not sufficiently understood in keystone taxa 31 such as the Thymallinae, one of the three salmonid subfamilies. Here we study a wild 32 grayling (Thymallus thymallus) population that suffers from distorted sex ratios. We found 33 sex determination in the wild and in captivity to be genetic and linked to the sdY locus. We 34 therefore studied sex-specific gene expression in embryos and early larvae that were bred and 35 raised under different experimental conditions, and we studied gonadal morphology in five 36 monthly samples taken after hatching. Significant sex-specific changes in gene expression 37 (affecting about 25,000 genes) started around hatching. Gonads were still undifferentiated 38 three weeks after hatching, but about half of the fish showed immature testes around seven 39 weeks after hatching. Over the next few months, this phenotype was mostly replaced by the 40 “testis-to-ovary” or “ovaries” phenotypes. The gonads of the remaining fish, i.e. 41 approximately half of the fish in each sampling period, remained undifferentiated until six 42 months after fertilization. Genetic sexing of the last two samples revealed that fish with 43 undifferentiated gonads were all males, who, by that time, were on average larger than the 44 genetic females (verified in 8-months old juveniles raised in another experiment). Only 12% 45 of the genetic males showed testicular tissue six months after fertilization. We conclude that 46 sex differentiation starts around hatching, goes through an all-male stage for both sexes 47 (which represents a rare case of “undifferentiated” gonochoristic species that usually go 48 through an all-female stage), and is delayed in males who, instead of developing their gonads, 49 grow faster than females during these juvenile stages. 50 51 peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/135194 doi: bioRxiv preprint first posted online May. 8, 2017;