Fig. 2. Comparative analyses reveal fms-dependence of hybrid pigment pattern formation across Danio species. (A) Phylogenetic relationships. The traditional grouping of danios comprises at least two major clades, `Danio' and `Devario' (Kullander, 2001; Fang, 2003). Heterospecific danios with names in color were used for interspecific complementation tests with fms^j4e1 mutant D. rerio. Colors of names indicate fms-dependence of hybrid (or heterozygous) pigment pattern: red, strong fms-dependence; green, mild fms-dependence; blue, no apparent fms-dependence (see text). (B,E,H,K,N,Q) Danio wild-type pigment patterns. (C,F,I,L,O,R) Control hybrids between wild-type D. rerio and heterospecific danios. (D,G,J,M,P,S) Hybrids between fms^j4e1 mutant D. rerio and heterospecific danios. (B-D) Danio albolineatus lack distinctive stripes whereas control hybrids develop stripes with irregular borders; fms mutant hybrids lack stripes. (E-G) Danio aff. albolineatus resembles D. albolineatus but has a reduced interstripe region posteriorly and the anal fin lacks a melanophore stripe; these fish may represent a divergent population of D. albolineatus or a closely related species (Fang and Kottelat, 2000). Control hybrids develop stripes with irregular borders like D. albolineatus control hybrids; fms mutant hybrids lack distinctive stripes. (H-J) Danio `hikari' resembles D. kerri (Parichy and Johnson, 2001): melanophore stripes are broad and diffuse and include very few xanthophores; interstripe regions are narrow and irregular by comparison with D. rerio (Fig. 1A). Control hybrids develop distinctive melanophore stripes and interstripes with regular borders. Tester fms mutants have fewer xanthophores than controls. (K-M) Danio aff. kyathit resembles D. rerio initially but develops fissures in melanophore stripes as the fish grows; D. aff. kyathit also exhibit red erythrophores, particularly in the fins. Control hybrids lack stripe fissures, and fms mutant hybrids are not discernibly different (minor differences between control and tester hybrids are within the range of variation exhibited by different families within genotypes). (N-P) Danio choprae transiently develop horizontal stripes, then lose these stripes as a uniform pattern of melanophores emerges; vertical bars of melanophores arise in adults. Control hybrids develop and maintain horizontal stripes resembling those of D. rerio whereas fms mutant hybrids have fewer melanophores and xanthophores and less organized patterns. (Q-S) Danio aff. dangila adults have melanophore stripes interrupted by lighter spots and interstripes, and are morphologically indistinguishable from D. dangila (Parichy and Johnson, 2001). Control hybrids resemble D. rerio, and fms mutant hybrids exhibit no clear difference from controls. Data not shown: Danio nigrofasciatus exhibit well-defined stripes and control hybrids are intermediate between D. rerio and D. nigrofasciatus parental species (Parichy and Johnson, 2001; Quigley et al., 2004); tester hybrids do not differ from controls for either fms^j4blue (Parichy and Johnson, 2001) or fms^j4e1. Tester fms^j4e1 hybrids could not be obtained for D. kerri; tester fms^j4blue hybrids resemble control hybrids, similar to tester fms^j4blue hybrids with D. albolineatus, likely reflecting modifier loci in the fms^j4blue background (see text). Hybrids with species outside of the Danio clade (A) were not viable, consistent with previous observations (Parichy and Johnson, 2001). All fish are between 25-35 mm standard length, except D. dangila and its hybrids, which are 50-80 mm. GenBank accession numbers for 12S and 16S sequences used in phylogeny reconstruction were: AY707450, AY707456; U21372, U21381; AF322658, AF322663; AY707446, AY707452; AF322663; AY707449, AY707455; AY707447, AY707453; AF322656, AF322661; U21376, U21384; AF322659, AF322664; U21377, U21377; U21375, U21370; AY707448, AY707454; U21553, U21554; AF322660, AF322665; U21378, U21386; AY707445, AY707451; AY37481, AY37482; AY37483, AY37484.