Supplementary figures Supplementary Figure S1. Orthology analysis. Bayesian phylogenetic analysis supporting orthology for genes investigated in this study. Red color refers to Xenacoelomorpha taxa and blue colour refers to N. vectensis. Bootstrap values were shown when equal or above 30%. Names of genes or proteins, if available, follow the name of organism(s); otherwise the accession number is written. Asterisks indicate genes with a spatial expression by WMISH. Supplementary Figure S2. Expression of amts, aquaporins, slc4 b, slc4 c, slc12 b, rootletin and plastin in I. pulchra. Whole mount in situ hybridization (WMISH) of (a) slc4 b (b) slc4 c (c) slc12 b (d) aquaporin a (e) aquaporin b (f) aquaporin c (g) aquaporin d (h) rootletin (i) amt 1 (j) amt 2 (k) amt 3 (l) amt 4 (m) amt 5 (n) amt 5 and (o) plastin in I. pulchra. The inset in panel d shows a different focal plane of the animal. Anterior is to the left. ad, anterior domain; ds, digestive syncytium; fg, female gonads; lpd, lateral parenchymal domain; pd, posterior domain; te, testis. Scale bar = 50 µm. Supplementary Figure S3. Expression of amt, aquaporins, slc4 b, slc4 c, v-atpase a2, v-atpase b2, stomatin/podocin c, stomatin/podocin d, rootletin and plastin in M. stichopi. Whole mount in situ hybridization (WMISH) of (a) stomatin/podocin d (b) stomatin/podocin e (c) slc4 b (d) slc4 c (e) slc26 b (f) aquaporin a (g) aquaporin b (h) aquaporin c (i) aquaporin d (j) aquaporin e (k) aquaporin f (l) rootletin (m) amt (n) v- ATPase a2 (o) v-ATPase b2 and (p) plastin in M. stichopi. The inset in panel j shows a lateral view of the indicated animal. Anterior is to the left. ad, anterior domain; ep, epidermis; dlr, distal lateral rows; fg, female gonads; glc, gut-lining cells; m, mouth; plr, proximal lateral rows; sc, subepidermal cells. Scale bar = 100 µm. Supplementary Figure S4. Expression of amts, cd2ap, zo1 and ca in N. vectencis juvenile polyps. Whole mount in situ hybridization (WMISH) of (a) amt1 (b) amt2 (c) amt3 (d) amt4 (e) amt5 (f) amt6 (g) ca a1 (h) ca a2 (i) ca a3 (j) cd2ap and (k) zo1 in N. vectensis. Panels a’-a’’, b’, c’, d’, e’ and f show a higher magnification of the indicated domains in panels a, b, c, d, e and f, respectively. Anterior is to the left. bw, ect, ectoderm; endodermal body wall; mes, mesenteries; oe, oral ectoderm; p, pharynx; pd, posterior gastrodermal domain; ten, tentacles. Scale bar = 100 µm. Supplementary Table 1. Summary of expression/role of excretion-related genes in metazoan and non metazoan taxa. Compilation of excretion-related gene expression/role data for metazoan and non metazoan taxa for genes investigated in this study. Data are based on this study unless stated otherwise. Question marks represent missing data. Supplementary Table 2. GenBank accession numbers. GenBank accession numbers used in this study are listed. S1 B.glabrata_Sns 9 1 H.sapiens_Nephrin X.tropicalis_Nphs1 H.miamia_98054641 C.submaculatums_c17370 8 7 D.gymnopharyngeus_c10580 D.longitubus_c37893 D.longitubus_c36603 D.gymnopharyngeus_c10380 H.miamia_98022051 I.pulchra_nephrin/kirrel b * 8 9 I.pulchra_nephrin/kirrel c * C.submaculatums_c17370 9 9 C.submaculatums_c15556 5 0 3 1 C.macropyga_5188.1 C.macropyga_2490.1 N.westbladi_51630 8 6 4 2 M.stichopi_nephrin/kirrel c * X.bocki_12500.1 4 2 8 6 X.profunda_c18867 H.sapiens_KIRREL1 9 3 H.sapiens_KIRREL2 5 3 H.sapiens_KIRREL3 3 6 X.tropicalis_Kirrel 9 9 D.melanogaster_Dumbfounded 3 6 D.melanogaster_C-roughest C.elegans_SIG2 M.stichopi_nephrin/kirrel a * C.elegans_SIG1 I.pulchra_nephrin/kirrel a * 5 3 C.macropyga_2933.1 E.macrobursalium_c4372 3 0 C.submaculatums_c17306 D.longitubus_c38278 M.stichopi_nephrin/kirrel b * 3 1 X.profunda_13792.1 9 1 X.profunda_14967.1 X.bocki_5576.1 N.westbladi_48390.0 8 8 D.melanogaster_Hibris D.melanogaster_Sns 9 9 IG-like domain H.sapiens_IGF1R D.melanogaster_InR 0.8 X.bocki_20883.1 5 1 A.queenslandica_I1EJ07 4 9 D.melanogaster_Q9W1F7 9 9 100 X.tropicalis_stoml2 3 2 H.sapiens_stomatin 2 3 0 N.vectensis_91851 3 2 C.elegans_stoml-1 100 STOMATIN-2 A.queenslandica_I1FUT2 A.queenslandica_I1FXM9 7 7 100 D.longitubus_c33065 H.miamia_9800988 C.elegans_un24 9 6 100 H.miamia_9803130 6 8 C.macropyga_10774 100 H.sapiens_stomatin 1 STOMATIN-1 9 9 X.tropicalis_stoml1 N.vectensis_247670 3 2 7 4 D.melanogaster_Q8MZ13 D.melanogaster_mec2 100 N.vectensis_218685 N.vectensis_147236 X.bocki_1047.1 D.melanogaster_band7 5 6 M.stichopi_stomatin/podocin e * 9 2 N.westbladi_35920.0 N.westbladi_47675.0 100 X.tropicalis_nphs2 H.sapiens_podocin C.elegans_stom2 H.sapiens_band7 9 1 H.sapiens_stomatin H.sapiens_stoml-3 X.tropicalis_stoml-3 N.vectensis_98301 8 4 N.vectensis_41366 N.vectensis_164373 N.vectensis_98373 7 4 C.elegans_mec-2 C.elegans_stom4 C.elegans_stom6 5 6 C.elegans_stom5 C.elegans_unc-1 C.elegans_stom3 D.gymnopharyngeus_c13414 STOMATIN-3/PODOCIN D.gymnopharyngeus_c2158 SLIPINS 7 0 H.miamia_9800107 H.miamia_9800318 H.miamia_9803220 D.longitubus_c30579 30 D.longitubus_c36799 I.pulchra_stomatin/podocin c 8 0 C.macropyga_229.1 * C.submaculatum_c18531 E.macrobursalium_c8407 A.queenslandica_I1FPX0 C.elegans_stom1 3 0 I.pulchra_stomatin/podocin a * 9 0 I.pulchra_stomatin/podocin b * C.macropyga_14931.1 C.submaculatum_c17900 M.stichopi_stomatin/podocin a 7 4 * M.stichopi_stomatin/podocin b * M.stichopi_stomatin/podocin c * M.stichopi_stomatin/podocin d * 100 E.macrobursalium_c6060 C.macropyga_729.1 C.submaculatum_c19891 Sterreria_c17109 X.bocki_12758.1 6 3 N.westbladi_52579.0 N.westbladi_25494.0 5 9 H.miamia_9800081 H.miamia_9803037 A.queenslandica_I1FPW9 H.miamia_9800396 100 C.albicans_paraslipin 100 P.tetraurelia_paraslipin T.thermophila_paraslipin PROKARYOTIC PARASLIPINS 2.0 T.adhaerens_B3S7T9 N.vectensis_zo1 * C.elegans_zo1 4 3 100 D.melanogaster_pyd S.purpuratus_782687.2 X.bocki_3789.1 5 8 C.macropyga_5972.1 4 7 3 4 D.gymnopharyngeus_c12822 D.gymnopharyngeus_c13251 100 D.longitubus_c38032 C.submaculatum_c19373 4 4 I.pulchra_zo1 * H.miamia_98057262 M.stichopi_zo1 9 7 * N.westbladi_51947.0 H.sapiens_ZO-1 9 9 H.sapiens_ZO-2 H.sapiens_ZO-3 H.sapiens_Dlg1 100 Disks large D.melanogaster_Dlg1 0.2 H.sapiens_cd2ap A.queenslandica_XP_003386167.1 9 9 100 N.vectensis_cd2ap * X.bocki_1192.1 4 6 4 6 M.stichopi_cd2ap * 5 1 N.westbladi_47339.0 9 0 7 4 N.westbladi_51312.0 D.melanogaster_cindr 4 7 I.pulchra_cd2ap 4 1 * D.gymnopharyngeus_c14992 H.sapiens_Spectrin_a chain 9 4 H.sapiens_Spectrin_b chain 100 D.melanogaster_Spectrin_a chain 7 3 D.melanogaster_Spectrin_b chain 0.4 S.mediterranea_slc13a3 SLC1 S.mediterranea_slc1a-4 S.mediterranea_slc1a-2 H.sapiens_SLC1A2 I.pulchra_slc1b 33 4 7 * M.stichopi_scl1 S.mediterranea_slc1a-3 30 I.pulchra_slc1a 9 9 * I.pulchra_slc1c * N.vectensis_230013 3 8 H.sapiens_SLC1A5 9 2 H.sapiens_SLC1A4 N.vectensis_slc1a2_A7S331 3 7 H.sapiens_SLC1A7 H.sapiens_SLC1A6 9 0 H.sapiens_SLC1A3 H.sapiens_SLC1A1 S.mediterranea_slc1a-1 S.mediterranea_slc1a-5 H.sapiens_SLC13A1 9 7 SLC13 H.sapiens_SLC13A4 H.sapiens_SLC13A3 H.sapiens_SLC13A2 8 5 H.sapiens_SLC13A5 M.stichopi_slc13 a * M.stichopi_slc13 b 8 7 * M.stichopi_slc13 c * 5 0 M.stichopi_slc13 d * 9 7 N.vectensis_1563873 N.vectensis_1563939 I.pulchra_slc13 * S.mediterranea_slc13a2 S.mediterranea_slc13a4 8 4 S.mediterranea_slc13a5 S.mediterranea_slc13a6 S.mediterranea_slc13a7 S.mediterranea_slc12a1 SLC12 6 7 S.mediterranea_slc12a4a 7 9 I.pulchra_Slc12 b * 8 6 H.sapiens_SLC12A6 H.sapiens_SLC12A4 8 9 H.sapiens_SLC12A5 H.sapiens_SLC12A7 3 7 S.mediterranea_slc12a4b 7 9 M.stichopi_Slc12 c S.mediterranea_slc12a3 9 4 S.mediterranea_slc12a2 H.sapiens_SLC12A9 M.stichopi_Slc12 a * 9 3 S.mediterranea_slc12a5 H.sapiens_SLC12A8 N.vectensis_238013 30 6 5 N.vectensis_XM_0016413 N.vectensis_104399 H.sapiens_SLC12A1 6 3 H.sapiens_SLC12A2 H.sapiens_SLC12A3 I.pulchra_Slc12 a * 5 3 M.stichopi_Slc12 b * S.mediterranea_slc5a-1 SLC5 S.mediterranea_slc5a-3 S.mediterranea_slc5a-4 8 3 H.sapiens_SLC5A5 H.sapiens_SLC5A6 H.sapiens_SLC5A8 H.sapiens_SLC5A12 7 9 H.sapiens_SLC5A7 S.mediterranea_slc5a-2 N.vectensis_89958 30 N.vectensis_31622 4 7 I.pulchra_SLC5A * M.stichopi_SLC5A * H.sapiens_SLC5A10 8 0 H.sapiens_SLC5A9 H.sapiens_SLC5A1 H.sapiens_SLC5A2 H.sapiens_SLC5A4 N.vectensis_SLC5a1_A7SQZ6 H.sapiens_SLC5A3 N.vectensis_1228981 8 2 N.vectensis_16302 I.pulchra_Slc5B * M.stichopi_Slc5B * H.sapiens_SLC5A11 5.0 H.sapiens_SLC8A2 SLC8 H.sapiens_SLC8A1 M.stichopi_slc8 * 3 5 S.mediterranea_slc8a3b 6 4 S.mediterranea_slc8a-2 S.mediterranea_slc8a4a 5 4 S.mediterranea_slc8a4b S.mediterranea_slc8a-5 4 3 S.mediterranea_slc8a-1 S.mediterranea_slc8a3a 3 6 I.pulchra_slc8 * 9 0 N.vectensis_gi|1563681 N.vectensis_gi|1563970 N.vectensis_gi|1564001 H.sapiens_SLC8A3 M.stichopi_slc9 * SLC9 I.pulchra_slc9 * N.vectensis_XP_0016262 6 5 100 N.vectensis_XM_0016261 4 6 H.sapiens_SLC9A1 H.sapiens_SLC9A2 6 0 H.sapiens_SLC9A3 H.sapiens_SLC9A4 H.sapiens_SLC9A5 S.mediterranea_slc9a-1 3 4 7 7 S.mediterranea_slc9a-4 6 4 S.mediterranea_slc9a-7 S.mediterranea_slc9a-8 9 9 S.mediterranea_slc9a-9 S.mediterranea_slc9a-3 9 3 S.mediterranea_slc9a-5 H.sapiens_SLC9A8 3 8 S.mediterranea_slc9a-2 H.sapiens_SLC9A6 5 2 6 4 H.sapiens_SLC9A7 H.sapiens_SLC9A9 3 3 N.vectensis_XP_0016327 5 4 N.vectensis_XM_0016326 N.vectensis_XM_0016350 N.vectensis_XP_0016351 4.0 S.mediterranea_slc4a-3 9 5 SLC4 S.mediterranea_slc4a-6 4 0 S.mediterranea_slc4a-7 I.pulchra_SLC4B * 7 7 I.pulchra_SLC4A * I.pulchra_SLC4C * M.stichopi_SLC4B * 5 4 9 3 N.vectensis_XM_0016378 N.vectensis_XM_0016238 H.sapiens_SLC4A1 9 4 H.sapiens_SLC4A2 H.sapiens_SLC4A3 H.sapiens_SLC4A4 8 0 H.sapiens_SLC4A9 H.sapiens_SLC4A5 4 9 H.sapiens_SLC4A7 8 9 H.sapiens_SLC4A8 6 2 H.sapiens_SLC4A10