Maximum‐likelihood phylogenetic tree of Slc5a aligned amino acid sequences in representative vertebrate taxa. Phylogenetic analysis used a LG + G + F substitution model. A maximum‐likelihood tree inferred using PhyML (see Section 2) rooted here on the CHT clade. Numbers at nodes indicate bootstrap support (among 100 replicates). Protein names omit ”Slc”. Human and stickleback proteins are highlighted in blue and red, respectively. Results identified three main clades: the NIS (sodium–iodide symporter) clade at the top, the SGLT (sodium–glucose cotransporter) clade in the middle, and the slc5a7 clade. Dre, Danio rerio; GAC, Gasterosteus aculeatus; Hsa, Homo sapiens; Loc, Lepisosteus oculatus; Mus, Mus musculus

Conserved synteny analysis of slc5a gene family members in the NIS clade. (a, c, e, g, i) Ortholog comparison plots for human (Hsa, top), gar (Loc, middle), and stickleback (Gac, bottom). Red lines link orthologous slc5a family members. (b, d, f, h, j) Dot‐plot analyses showing a human chromosome on the horizontal axis with the location of its SLC5A gene marked; stickleback orthologs and close paralogs of human genes are displayed directly above the location of the human gene on a row indicating the chromosome on which the stickleback gene resides. For example, “b” shows the SLC5A5 gene on human chromosome 19 and indicates its ortholog on stickleback linkage group GacVIII. Gene order in the horizontal dimension is that of the human chromosome. Chromosome regions that appear in duplicate due to the teleost genome duplication event are evident. (k) Dot plot of Hsa2 paralogs on other human chromosomes showing the location of NIS‐clade genes in paralogons. (l) Dot plot of Hsa22 paralogs on other human chromosomes showing the location of SGLT‐clade genes in paralogons

Colocalization of thyroid follicles and thyroglobulin protein at 8, 14, and 30 dpf in stickleback larvae. (a, c, e) Hematoxylin and eosin (H&E)‐stained horizontal sections of the pharynx showing thyroid follicles at 8–30 dpf. (b, d, f) Positive staining for thyroid antibody (anti‐TG [thyroglobulin]) is visible in and around the lumen of thyroid follicles at 8–30 dpf. Imaged at 400X. thy, thyroid follicles

Expression patterns of slc5a5 clade genes in the head of 18 dpf threespine stickleback. In situ hybridization of mRNA expression of (a) slc5a5, (b) slc5a6a, (c) slc5a6b, (d) slc5a8, (e) slc5a8l, and (f) slc5a8l sense strand as control, and (g) H&E‐stained histological sections showing teeth, eye, brain, and thyroid of larval stickleback. Imaged at 40X. br, brain; gcl, ganglion cell layer; inl, inner nuclear layer; pcl, photoreceptor cell layer; te, teeth; thy, thyroid follicles

Expression patterns of slc5a5 clade genes in the abdomen of 30 dpf threespine stickleback. (a) slc5a5, (b) slc5a6a, (c) slc5a6b, (d) slc5a8, (e) slc5a8l, and (f) slc5a8l sense strand as control, and (g) H&E‐stained histological sections showing gonad, liver, pancreas, and intestine imaged at 400X. g, gonad; int, intestine; liv, liver; pan, pancreas

Expression patterns of slc5a5 clade genes in the head of 30 dpf threespine stickleback. (a) slc5a5, (b) slc5a6a, (c) slc5a6b, (d) slc5a8, (e) slc5a8l, and (f) slc5a8l sense strand as control, and (g) H&E‐stained histological sections shown in the craniofacial skeleton, eye, thyroid, and pharyngeal teeth. Imaged at 400X. gcl, ganglion cell layer; inl, inner nuclear layer; pcl, photoreceptor cell layer; te, teeth; thy, thyroid follicles; s, skeletal tissue

Expression of slc5a5 clade genes in the abdomen of 30 dpf threespine stickleback. (a) slc5a5, (b) slc5a6a, (c) slc5a6b, (d) slc5a8, (e) slc5a8l, and (f) slc5a8l sense strand as control, and (g) H&E‐stained histological sections. Imaged at 400X. g, gonad; int, intestine; liv, liver; pan, pancreas

Expression of slc5a5 clade genes in the abdomen of 9 mpf threespine stickleback. (a) slc5a5, (b) slc5a6a, (c) slc5a6b, (d) slc5a8, (e) slc5a8l, and (f) slc5a8l sense strand as control, and (g) H&E‐stained histological sections. Imaged at 400X. int, intestine; kid, kidney; oo, oocyte; pan, pancreas; tes, testis

Single‐cell RNA‐seq analysis of 30 dpf zebrafish gonads. (a) tSNE plot with clusters numbered. Each dot represents a cell; (b) Cells from genetically female (red) and male (blue) gonads; (c) zp3f.2 eggshell protein gene expression, darker red indicates higher expression level according to the legend in the corner; (d) slc5a5 expression in maturing oocytes; (e) slc5a6a expression in a few immature ZW germ cells and in many maturing oocytes; (f) slc5a6b expression in a few immature ZW germ cells and in many maturing oocytes; (g) slc5a8l expression primarily in somatic cells of the gonad; and (h) pax2a expression in the same cluster as slc5a8l. The scale bar gives the natural log‐transformed feature counts for each cell divided by the total counts for that cell, multiplied by the scale factor (a value of 1 is added to each count before the calculation to avoid taking the log of 0)