Background: Artificial selection of postembryonic features is known to have established morphological variation in goldfish (Carassius auratus). Although previous studies have suggested that goldfish and zebrafish are almost directly comparable at the embryonic level, little is known at the postembryonic level. Results: Here, we categorized the postembryonic developmental process in the wild‐type goldfish into 11 different stages. We also report certain differences between the postembryonic developmental processes of goldfish and zebrafish, especially in the skeletal systems (scales and median fin skeletons), suggesting that postembryonic development underwent evolutionary divergence in these two teleost species. Conclusions: Our postembryonic staging system of wild‐type goldfish paves the way for careful and appropriate comparison with other teleost species. The staging system will also facilitate comparative ontogenic analyses between wild‐type and mutant goldfish strains, allowing us to closely study the relationship between artificial selection and molecular developmental mechanisms in vertebrates. Developmental Dynamics 244:1485–1518, 2015. © 2015 Wiley Periodicals, Inc.

Background Twin‐tail ornamental goldfish have “bifurcated median fins,” a peculiar morphology known to be caused by a mutation in the chdA gene. However, several ambiguities regarding the development of the phenotype remain due to a paucity of detailed observations covering the entire developmental timeframe. Results Here, we report a detailed comparative description of embryonic and postembryonic development for two representative twin‐tail ornamental goldfish strains and single‐tail common goldfish. Our observations reveal a polymorphic developmental process for bifurcated median fins; disrupted axial skeletal development at early larval stages; and modified bilateral location of the pelvic fin. Conclusions Variations in development of bifurcated median fins and disrupted axial skeletal patterns reflect how artificial selection for adult morphological features influenced molecular developmental mechanisms during the domestication of twin‐tail ornamental goldfish. The polymorphic appearance of bifurcated median fins also implies that, unlike previously proposed hypotheses, the development of these structures is controlled by molecular mechanisms independent of those acting on the pelvic fin. Our present findings will facilitate further study of how modifications of preexisting developmental systems may contribute to novel morphological features. Developmental Dynamics 248:251–283, 2019. © 2019 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Twin-tail goldfish strains are examples of drastic morphological alterations that emerged through domestication. Although this mutation is known to be caused by deficiency of one of two duplicated chordin genes, it is unknown why equivalent mutations have not been observed in other domesticated fish species. Here, we compared the chordin gene morphant phenotypes of single-tail goldfish and common carp (close relatives, both of which underwent chordin gene duplication and domestication). Morpholino-induced knockdown depleted chordin gene expression in both species; however, while knockdown reproduced twin-tail morphology in single-tail goldfish, it had no effect on common carp morphology. This difference can be explained by the observation that expression patterns of the duplicated chordin genes overlap completely in common carp, but are sub-functionalized in goldfish. Our finding implies that goldfish drastic morphological changes might be enhanced by the subsequent occurrence of three different types of evolutionary event (duplication, sub-functionalization, and selection) in a certain order.

The twin‐tail of ornamental goldfish provides unique evolutionary evidence that the highly conserved midline localization of axial skeleton components can be changed by artificial selection. This morphological change is known to be caused by a nonsense mutation in one of the recently duplicated chordin genes, which are key players in dorsal–ventral (DV) patterning. Since all of the multiple twin‐tail ornamental goldfish strains share the same mutation, it is reasonable to presume that this mutation occurred only once in domesticated goldfish. However, zebrafish with mutated szl gene (another DV patterning‐related gene) also exhibit twin‐tail morphology and higher viability than dino/chordin‐mutant zebrafish. This observation raises the question of whether the szl gene mutation could also reproduce the twin‐tail morphology in goldfish. Here we show that goldfish have at least two subfunctionalized szl genes, designated szlA and szlB, and depletion of these genes in single‐fin goldfish was able to reproduce the bifurcated caudal fin found in twin‐tail ornamental goldfish. Interestingly, several phenotypes were observed in szlA‐depleted fish, while low expressivity of the twin‐tail phenotype was observed in szlB‐depleted goldfish. Thus, even though szl gene mutations may produce twin‐tail goldfish, these szl gene mutations might not be favorable for selection in domestic breeding. These results highlight the uniqueness and rarity of mutations that are able to cause large‐scale morphological changes, such as a bifurcated axial skeleton, with high viability and expressivity in natural and domesticated populations.

The twin tail of ornamental goldfish is known to be caused by a nonsense mutation in one chordin paralogue gene. Our previous molecular studies in goldfish revealed that the ancestral chordin gene was duplicated, creating the chdA and chdB genes, and the subsequent introduction of a stop codon allele in the chdA gene ( chdA E127X ) caused the twin‐tail morphology. The chdA E127X allele was positively selected by breeders, and the allele was genetically fixed in the ornamental twin‐tail goldfish population. However, little is known about the evolutionary history of the chdB paralogue, begging the question: are there the functionally distinct alleles at the chdB locus, and if so, how did they evolve? To address these questions, we conducted molecular sequencing of the chdB gene from five different goldfish strains and discovered two alleles at the chdB gene locus; the two alleles are designated chdB 1 and chdB 2 . The chdB 1 allele is the major allele and was found in all investigated goldfish strains, whereas the chdB 2 allele is minor, having only been found in one twin‐tail strain. Genetic analyses further suggested that these two alleles are functionally different with regard to survivability ( chdB 1 > chdB 2 ). These results led us to presume that in contrast to the chdA locus, the chdB locus has tended to be eliminated from the population. We also discuss how the chdB 2 allele was retained in the goldfish population, despite its disadvantageous function. This study provides empirical evidence of the long‐term retention of a disadvantageous allele under domesticated conditions.