Gene targeting techniques have led to the phenotypic characterization of numerous genes; however, many genes show minimal to no phenotypic consequences when disrupted, despite many having highly conserved sequences. The standard explanation for these findings is functional redundancy. A competing hypothesis is that these genes have important ecological functions in natural environments that are not needed under laboratory settings. Here we discriminate between these hypotheses by competing mice (Mus musculus) whose Hoxb1 gene has been replaced by Hoxa1, its highly conserved paralog, against matched wildtype controls in seminatural enclosures. This Hoxb1 A1 swap was reported as a genetic manipulation resulting in no discernible embryonic or physiological phenotype under standard laboratory tests. We observed a transient decline in first litter size for Hoxb1 A1 homozygous mice in breeding cages, but their fitness was consistently and more dramatically reduced when competing against controls within seminatural populations. Specifically, males homozygous for the Hoxb1 A1 swap acquired 10.6% fewer territories and the frequency of the Hoxb1 A1 allele decreased from 0.500 in population founders to 0.419 in their offspring. The decrease in Hoxb1 A1 frequency corresponded with a deficiency of both Hoxb1 A1 homozygous and heterozygous offspring. These data suggest that Hoxb1 and Hoxa1 are more phenotypically divergent than previously reported and support that sub-and/or neofunctionalization has occurred in these paralogous genes leading to a divergence of gene function and incomplete redundancy. Furthermore, this study highlights the importance of obtaining fitness measures of mutants in ecologically relevant conditions to better understand gene function and evolution.KEYWORDS fitness assay; functional redundancy; Hoxa1; Hoxb1; intraspecific competition; subfunctionalization G ENE targeting techniques have led to the phenotypic characterization of thousands of genes across eukaryotes (for reviews see Thorneycroft et al. 2001;Capecchi 2005;Collins et al. 2007) and this characterization continues as this invaluable technology develops (e.g., Meyer et al. 2012;Hsu et al. 2014). However, an estimated 10-15% of mouse genes show minimal to no phenotypic consequences when disrupted (mouse appears normal), despite many having highly conserved sequences (Barbaric et al. 2007). One explanation for these findings is functional redundancy-genes throughout the genome, typically paralogs of disrupted genes, code for the same, or at least overlapping, functions (Nowak et al. 1997;Kafri et al. 2009). A competing explanation for "no phenotype" gene disruptions is that these genes have important ecological functions in natural environments that are not needed, or are of minimal importance, within laboratory settings. Here we discriminate between these hypotheses by using mice that have experienced a manipulation previously reported to have no embryonic or physiological phenotype, wherein the coding sequence of the Hoxb1 gene has ...

Here we assess the fitness consequences of the replacement of the Hoxa1 coding region with its paralog Hoxb1 in mice (Mus musculus) residing in semi-natural enclosures. Previously, this Hoxa1B1 swap was reported as resulting in no discernible embryonic or physiological phenotype (i.e., functionally redundant), despite the 51% amino acid sequence differences between these two Hox proteins. Within heterozygous breeding cages no differences in litter size nor deviations from Mendelian genotypic expectations were observed in the outbred progeny; however, within semi-natural population enclosures mice homozygous for the Hoxa1B1 swap were out-reproduced by controls resulting in the mutant allele being only 87.5% as frequent as the control in offspring born within enclosures. Specifically, Hoxa1B1 founders produced only 77.9% as many offspring relative to controls, as measured by homozygous pups, and a 22.1% deficiency of heterozygous offspring was also observed. These data suggest that Hoxa1 and Hoxb1 have diverged in function through either sub- or neo-functionalization and that the HoxA1 and HoxB1 proteins are not mutually interchangeable when expressed from the Hoxa1 locus. The fitness assays conducted under naturalistic conditions in this study have provided an ultimate-level assessment of the postulated equivalence of competing alleles. Characterization of these differences has provided greater understanding of the forces shaping the maintenance and diversifications of Hox genes as well as other paralogous genes. This fitness assay approach can be applied to any genetic manipulation and often provides the most sensitive way to detect functional differences.