Table 1.

Stages in the evolution of sex-linked regions and sex chromosomes, with approaches that can be used to detect them. For each stage, the approaches listed for earlier stages can also be helpful and are therefore not repeated (for example, discovering sex-linked genes that are not the sex-determining genes, e.g. visible colour markers, tells one that a sex-linked region exists). The table uses the case of male heterogamety, but the same basic stages can also be found in ZW systems.

age of sex-determining system	type of sex chromosomes	non-recombining region	approaches for detection	references to examples of use of the approaches listed
very young	homomorphic	none (linked male- and female-determining loci, or single sex-determining locus)	genetic mapping in families to locate the sex-determining locus	[23]
young	usually homomorphic or micro-heteromorphic	offten small most genes still present on both homologues, only slightly diverged in sequence	searches for male-specific variants at sites in genes other than the sex-determining genes but that show LD with the SEX locus, using males and females: (a) RAD-Seq of samples from populations or families (b) analysis of heterozygote frequencies to detect sequences with variants heterozygous in all males, but not females (Fis analysis) (c) genetic studies of RNA-Seq variants in families to detect sex-linked inheritance patterns	(a) [11] (b) [24] (c) [25]
old	heteromorphic XY or ZW pair may be cytologically detectable in males	region includes multiple genes many genes present only in XX females due to loss from the Y chromosome Y-linked sequences diverged at many sites, sometimes making them ‘invisible’ (null alleles)	analysis of coverage in whole genome sequences, to detect hemizygous regions in one sex, indicating degenerated Y-linked regions in X/Y males, or highly diverged Y-linked sequences that are not assembled with the X-linked gene (or similar differences in ZW systems)	[26]