Adipose Fin Development and Its Relation to the Evolutionary 2 Origins of Median Fins 3 4 5 6 Thomas A

Home , Electrona, Mochokus, Sisor

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1 Adipose fin development and its relation to the evolutionary 2 origins of median fins 3 4 5 6 Thomas A. Stewart1*, Robert K. Ho1, and Melina E. Hale1 7 8 1Department of Organismal Biology and Anatomy 9 The University of Chicago 10 1027 E. 57th St, Chicago, IL 60637 11 12 13 14 *Correspondence to: Thomas A. Stewart, Department of Organismal Biology and Anatomy, The 15 University of Chicago, 1027 E. 57th St, Chicago, IL 60637; email: [email protected]; 16 phone: (315) 546-4175 17 bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

18 Abstract

19 The dorsal, anal and caudal fins of vertebrates are proposed to have originated by the partitioning

20 and transformation of the continuous median fin fold that is plesiomorphic to chordates.

21 Evaluating this hypothesis has been challenging, because it is unclear how the median fin fold

22 relates to the adult median fins of vertebrates. To understand how new median fins originate,

23 here we study the development and diversity of adipose fins. Phylogenetic mapping shows that in

24 all lineages except Characoidei (Characiformes) adipose fins develop from a domain of the larval

25 median fin fold. To inform how the larva’s median fin fold contributes to the adipose fin, we study

26 Corydoras aeneus (Siluriformes). As the fin fold reduces around the prospective site of the

27 adipose fin, a fin spine develops in the fold, growing both proximally and distally, and sensory

28 innervation, which appears to originate from the recurrent ramus of the facial nerve and from

29 dorsal rami of the spinal cord, develops in the adipose fin membrane. Collectively, these data

30 show how a plesiomorphic median fin fold can serve as scaffolding for the evolution and

31 development of novel, individuated median fins, consistent with the median fin fold hypothesis.

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33 Introduction

34 Fins have evolved repeatedly in vertebrates1-4 and, thus, provide a powerful system for

35 studying how new body parts originate. Primitively, chordates are characterized by a median fin

36 fold (MFF), a midline structure comprised of dorsal and ventral portions that meet posteriorly to

37 form a protocercal tail2. The extinct chordates Haikuichthys and Haikuella exhibit this condition,

38 with the ventral portion of the LMFF interrupted by the anus5,6. The extant cephalchordate

39 amphioxus also has a MFF, which passes to the right of the anus uninterrupted7. Spatially

40 differentiated, individuated median fins evolved later, in craniates1. These new fins are

41 hypothesized to have originated by the partitioning of the MFF into multiple fin modules4,8-12.

42 Specifically, the dorsal, anal and caudal fins are predicted to have evolved from the MFF by its

43 reduction in some positions and its retention in others. This ‘median fin-fold hypothesis’ is related

44 to the ‘lateral fin-fold hypothesis’ of paired pectoral and pelvic fin origin, which itself posits that

45 paired continuous fins along the flank were subdivided to create the pectoral and pelvic fins8-10.

46 Although the lateral fin-fold hypothesis has largely been abandoned in favor of a scenario where

47 pectoral fins evolved first and pelvic fins evolved secondarily1,13,14, the MFF hypothesis remains

48 influential.

49 In many fishes, ontogeny appears to recapitulate the phylogenetic transformational

50 scenario predicted by the MFF hypothesis. For example in zebrafish, Danio rerio (Cyprinidae), a

51 larval median fin fold (LMFF) encompasses the trunk early in development15. The LMFF develops

52 as the somites are forming; specification and outgrowth proceeds in a caudal-to-rostral direction,

53 driven by Fgf signaling15. The LMFF is composed of an epithelial bilayer medial to which are

54 actinotrichia (tapered collagenous rods organized approximately parallel to the fin’s proximodistal

55 axis), which sandwich a core of mesenchyme16. Later in development, spatially discontinuous

56 adult median fins—the dorsal, anal, and caudal fins—form17,18, and the LMFF is reduced by

57 apoptosis in positions that do not bear adult fins19.

58 Adult median fins are described as developing from the LMFF15,20-24. However, D. rerio

59 mutants suggest that the development of adult median fins does not depend on proper formation

60 of the LMFF25. Further, most tissues that comprise adult fins (e.g., dermal and endoskeleton,

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61 musculature, and fin-associated innervation) are not derived from tissues in the LMFF, but from

62 other sources, including paraxial mesoderm20,26,27. Thus, while the LMFF might function as

63 scaffolding for the morphogenesis of adult fins (e.g., actinotrichia guiding the migration of

64 osteogenic mesenchyme that forms lepidotrichia28,29), the relationship between the LMFF and

65 adult fins is not one of straightforward ontogenetic transformation. This poses a challenge to

66 recapitulist30 arguments for the MFF hypothesis8-10.

67 Here, to inform hypotheses of (1) phylogenetic transformation from MFFs into

68 individuated fins and (2) ontogenetic transformation of the LMFF into adult fins, we study the

69 diversity and development of adipose fins. These appendages have evolved repeatedly within

70 teleosts3 and are positioned on the dorsal midline between the dorsal and caudal fins. Adipose

71 fins have been studied as models of how form and function evolves in vertebrate appendages3,31-

72 33 and might also inform how development evolves to generate novel appendages. Descriptions

73 of adipose fin morphogenesis are scattered throughout the literature—in taxonomies of larval

74 fishes, staging papers for select taxa, and a study of early development of these fins34. We

75 aggregate the data on adipose fin development from the literature and analyze them in a

76 phylogenetic context. Additionally, we characterize adipose fin development in the South

77 American armored catfish Corydoras aeneus (Gill 1858) (Siluriformes, Callicithyidae), focusing on

78 the development of the adipose fin skeleton and sensory anatomy. Collectively, these data reveal

79 that adipose fins can evolve and develop by retention and elaboration of a domain of the LMFF.

80 We discuss how these data inform hypotheses of median fin origin in early vertebrates.

83 Results

84 Diversity of adipose fin development

85 Analysis of the literature yielded information on adipose fin development for twenty-four

86 species belonging to five orders of fishes (Suppl. Table 1). Two patterns of adipose fin

87 development are observed, consistent with previous descriptions34,35 (Fig. 1 a). In Characoidei

88 (Characiniformes), adipose fins develop de novo as buds following the reduction of the LMFF. In

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89 two Characoidei genera, Brycinus and Phenacogrammus, the adipose fin grows out before the

90 LMFF has been completely reduced34. In all other clades for which data is available, adipose fins

91 appear to develop by the retention of a domain of the LMFF between the dorsal and caudal fin

92 (Fig 1 b).

94 Adipose fin development in Corydoras aeneus

95 Corydoras aeneus (Suppl. Fig. 1) exhibits fold-associated adipose fin development.

96 Prior to adipose fin development in C. aeneus, the LMFF appears undifferentiated between the

97 dorsal and caudal fins (Fig. 2 a). Once the larvae have grown to approximately 8 mm standard

98 length (SL), the LMFF begins reducing both immediately posterior to the dorsal fin and anterior to

99 the caudal fin, and a condensation forms at the future site of the adipose fin, midway along the

100 proximodistal axis of the LMFF (Fig. 2 b). At the anterior boundary of this condensation, an

101 ossification forms that will become the adipose fin spine (Fig. 2 c). The ossification is unpaired,

102 positioned on the midline, and it grows both proximally and distally, parallel to actinotrichia in the

103 LMFF (Fig. 2 d-f, Fig. 3). Once the spine has extended proximally to just dorsal to the epaxial

104 musculature, its base widens into a saddle shape that wraps laterally around the trunk

105 musculature. Three scutes develop anterior to the adipose fin spine, their order of ossification

106 proceeding from posterior to anterior (Fig. 3 g-l). The LMFF continues to reduce, leaving a

107 domain posterior to the adipose fin spine that will constitute the membrane of the adipose fin. The

108 LMFF has finished reducing and the adipose fin has fully developed by the time C. aeneus reach

109 1.2 cm SL. Odontodes, small dermal denticles, develop on the scutes of C. aeneus36. In the

110 adipose fin, odontodes begin mineralizing before the adipose fin spine has ossified (Fig. 3 b).

111 The adipose fin of C. aeneus has extensive sensory innervation33. Therefore, we also

112 studied the ontogeny of sensory anatomy and innervation in the adipose fin. Before adipose fin

113 development is observed, the LMFF is innervated by nerve fibers entering from the midline and

114 from the trunk epithelium, and these nerves terminate as free nerve endings (Fig. 4 a). Nerve

115 fibers persist while the LMFF reduces, extending to the LMFF’s distal margin (Figs. 4 b, c and 5

116 a, b). Adipose fin-associated nerve fibers are observed posterior to the fin spine by the time the

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117 spine has extended to reach the trunk musculature (Fig. 5 a, b). These fibers appear to originate

118 from the dorsal rami of the spinal cord and dorsal projections of the recurrent ramus of the facial

119 nerve, which extends into the caudal fin.

120 Most of the adipose fin innervation enters the fin as a ramus immediately posterior to the

121 adipose fin spine that ramifies repeatedly to extend branches posteriorly into the adipose fin

122 membrane (Fig. 5 a, b). Additional nerves enter the adipose fin membrane posterior to the fin

123 spine and do not clearly correspond to somatic boundaries. Nerve fibers in the adipose fin

124 membrane are organized approximately parallel to the actinotrichia (Fig. 5 c-f). Nerve branches

125 also pass anteriorly through the adipose fin spine (Fig. 5 g, h).

126 Two kinds of putative sensory structures, diagnosed by their morphologies, are observed

127 on the adipose fin of C. aeneus. The first kind, observed on the fin spine, are columnar

128 projections with a rounded apex and a base composed of a cluster of globular cells (Fig. 5 i,

129 Suppl. Fig. 2). This morphology is consistent with previous descriptions of superficial

130 neuromasts37. These structures are also observed on the lateral scutes of C. aeneus and can be

131 distinguished from lateral line sensory cells, which have a filamentous tip (Suppl. Fig. 3). The

132 second kind of sensory structure, observed on the adipose fin membrane, are hillock shaped and

133 lack a pronounced apex (Fig. 5 j). These are diagnosed as taste buds by their size, shape, and

134 distribution38-40. In catfishes, taste buds are distributed across the body and commonly found on

135 both the trunk and fins, including the LMFF and adipose fin. These structures are composed of a

136 cluster of globular cells and are innervated by branches of the adipose fin nerves (Suppl. Fig. 4).

137 Nerves in the adipose fin membrane also terminate as free nerve endings (Fig. 5 k).

138

139

140 Discussion

141 Comparative studies of development can inform plesiomorphic conditions and constrain

142 hypotheses of how novel structures originated41. In this study, we analyzed the development and

143 diversity of adipose fins to understand their evolutionary origin. These data show that novel

144 appendages can evolve by individuation of a domain of a plesiomorphic fin fold. Additionally,

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145 adipose fins that develop from the LMFF can evolve skeleton3,32,42, musculature31, and sensory

146 anatomy (here and 43,44). For example, in C. aeneus the domain of the LMFF that contributes to

147 the adipose fin is transformed over ontogeny by the migration and growth of new fin-associated

148 tissues into the territory. Collectively, these observations show the evolutionary trajectory outlined

149 in the MFF hypothesis—partitioning of an existing median fin fold and transformation of such a fin

150 into a structurally and functionally complex appendage—is a viable route by which novel fins can

151 originate.

152 Adipose fins evolved independently in Euteleostei and Otophysii3. Euteleosts uniformly

153 exhibit fold-associated adipose fin development, indicating that adipose fins originated in this

154 clade, at least in part, by the retention of a larval character that would have primitively been

155 reduced over ontogeny. For example, in Polyodon spathula (Acipenseridae) the LMFF reduces

156 fully between the dorsal and caudal fins in development23. In otophysans, adipose fins develop in

157 two ways: fold-associated development in Siluriformes, and de novo outgrowth in Characoidei.

158 These two groups are sister to one another, and data is unavailable for Citharinoidei, the clade

159 sister to that total group that also have adipose fins. Therefore, we cannot currently resolve the

160 primitive pattern of adipose fin development in otophysans. The occurrence of both

161 developmental patterns in Otophysii reveals evolutionary lability between these two states,

162 although the polarity of change is unresolved, and this raises questions of how adipose fins are

163 positioned along the anteroposterior axis in ontogeny.

164 Fin positioning depends upon several processes: genes expressed in the mesoderm

165 (either lateral plate45 or paraxial46,47) establish the fin territory, cells in this territory can migrate

166 and contract asymmetrically to form a mysenchymal bud48, and differential growth of the trunk

167 and fin can affect its eventual adult position26,49,50. Some adipose fins appear to be positioned by

168 an additional mechanism: differential patterns of apoptosis within the LMFF. How this is

169 regulated, or how it relates to positioning in adipose fins that develop de novo as buds is unclear.

170 Future work should characterize adipose fin development in Citharinoidei, to understand whether

171 de novo or fold-associated development is primitive in otophysans. Additionally, characterizing

172 the developmental genetic mechanisms that underlie positioning in Characoidei and Siluriformes

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173 adipose fins would clarify how lineages transition between these two apparently disparate

174 developmental patterns. Developing such a model would allow for detailed hypotheses of how

175 transitions predicted by the MFF hypothesis occurred in early chordates.

176 Adipose fins have evolved anterior dermal spines from midline scutes three times

177 independently (i.e., Sisor spp. (Sisoridae), Amphilidae, and Lorocaricoidea, which includes C.

178 aeneus)3. Scutes can be distinguished from other postcranial dermal plates by the presence of

179 hyaline, a superficial hypermineralized tissue51,52. In C. aeneus, scutes are positioned laterally, on

180 the flank, as well as on the dorsal midline anterior to the adipose fin. Lateral scutes begin

181 developing at the mid-region of a population of mesenchymal cells and not adjacent to an

182 epithelial-mesenchymal boundary52. Similarly, the fin spine of C. aeneus appears to ossify at the

183 core of the LMFF and medial to the actinotrichia and not adjacent to the epithielium. By contrast,

184 lepidotrichia develop from mesenchyme in the space between the basal lamina of the epithelium

185 and actinotrichia; they are, therefore, comprised of paired elements, the hemitrichia18. The

186 medial, unpaired position of the adipose fin spine has consequences for the position of sensory

187 nerves, discussed below.

188 Although both the adipose fin spine and lepidotrichia develop by the migration of

189 osteogenic cells along actinotrichia—bone differentiation and growth proceeding parallel to the

190 orientation of actinotricha53—these two skeletal types differ in the site of initial ossification and

191 direction of skeletal growth. The adipose fin spine of C. aeneus begins ossifying midway along

192 the proximodistal length of the LMFF and extends both proximally and distally. This is in contrast

193 to the general pattern of ossification in vertebrate appendages, where skeleton usually

194 differentiates and grows in a proximal-to-distal direction54. For example, with a single exception

195 (i.e., a pimelodid catfish that has evolved a rayed adipose fin32), lepidotrichia development is

196 initiated proximally and extends distally17,55.

197 Previous descriptions of odontode development in Corydoras observed their

198 differentiation only after the mineralization of associated scutes and fin rays to which they

199 attach36. It was, therefore, suggested that odontode ossification depends upon prior ossification of

200 its attachment site36. However, our data show that ossification of odontodes can precede scute

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201 ossification (Fig. 3 b), indicating the presence of a well-developed stratum compactum in the fin

202 spine mesenchyme at these stages51.

203 The organization of innervation in the adipose fin of C. aeneus is distinct from what has

204 been described in other actinopterygian fins. In rayed fins, sensory nerves enter the fin medially

205 between the paired hemitrichia, sending branches into the fin membrane through the joints

206 between fin ray segments56,57. By contrast, in the adipose fin of C. aeneus a large nerve branch

207 enters the fin posterior to the adipose fin spine. This nerve bundle maintains an association with

208 the spine, traveling along the length and sending branches both anteriorly through canals in the

209 spine, and also posteriorly into the fin membrane. The posteriorly directed nerve branches run

210 approximately parallel to the actinotrichia in the fin membrane. Additional nerves enter the fin

211 posterior to the adipose fin spine but do not follow a clearly segmented pattern, as is observed in

212 fins with a series of lepidotrichia (e.g., dorsal, anal, caudal fins, Fig 4 and 5). Future work should

213 identify the cues that guide axonal growth into this territory and compare them to the mechanisms

214 by which sensory anatomy is reorganized in other fins.

215 For example, the paired pectoral fins of D. rerio first develop a non-skeletonized fin fold

216 that contains sensory innervation. These nerves are not organized in a strictly proximodistal

217 orientation, but instead form a reticulate network that is reshaped over development; in the adult

218 morphology, sensory nerves are associated with lepidotrichia57. Similarly, the LMFF, which is a

219 passive appendage (i.e., lacking musculature control), contains sensory nerves. These nerves

220 are likely from Rohon Beard cells, primary sensory neurons with peripheral axons that extend

221 along the trunk epithelium, into the caudal fin fold, and likely also extend into the dorsal LMFF58.

222 These cells undergo apoptosis over ontogeny and do not retract during degradation59. In the

223 caudal fin, these are eventually replaced by the dorsal root ganglia.

224 The adipose fin of C. aeneus is innervated by both the recurrent ramus of the facial nerve

225 and by dorsal rami of the spinal cord. We observe both sources projecting nerves dorsally into the

226 adipose fin domain, and other fins have been described with sensory innervation from both

227 cranial and spinal sources60,61. In catfishes, taste buds are distributed across the body and

228 commonly found on both the trunk and fins, including the LMFF and adipose fin38-40. Taste buds

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229 on the trunk have been exclusively described as innervated by the recurrent ramus of the facial

230 nerve40. Superficial neuromasts have been described previously on the caudal fins of various

231 fishes37,62,63, but not on adipose fins. Although we were able to observe nerves entering the

232 adipose fin and extending anteriorly through the fin spine, we were unable to determine whether

233 they terminated upon the superficial neuromasts.

234 The adipose fin of C. aeneus is proprioceptive, able to detect the movement and position

235 of the fin membrane33. Mechanosensation might be achieved through by either the taste buds or

236 free nerve endings in the fin membrane. In the channel catfish, Ictalurus punctatus, the nerves

237 that terminate on extra-oral taste buds on the flank are mechanosensitive64; however, it is

238 unknown how this is achieved. Extra-oral gustatory cells might detect both mechanical and

239 chemical signals, or the nerves that innervate these cells might also terminate on yet unidentified

240 mechanosensory endings.

241 The adipose fin of the brown trout, Salmo trutta (Salmonidae), a euteleost, is also

242 invested with sensory nerve fibers and also hypothesized to detect fin movement43,44,65,66. These

243 nerves terminate upon associated astrocyte-like cells, which are hypothesized to detect the

244 deformation of collagen fibers that span the left-and-right sides of the adipose fin43,44. The

245 organization of collagen is similar to what has been described in the LMFF of D. rerio67, indicating

246 that many structural components of the LMFF are retained over ontogeny and that sensory

247 anatomy is reorganized over ontogeny in this lineage, similar to C. aeneus. Sensory innervation

248 might, therefore, be among the first features to change when a new median fin originates by

249 partitioning of a LMFF.

250 The earliest vertebrate fins lack evidence for muscular attachment5,6 and were likely

251 passive appendages. Typically, discussions of how these fins originated focus on hypotheses of

252 selection for the functions of stabilization, control, and thrust generation11. Given that passive fins

253 (e.g., LMFF and adipose fins) are invested with sensory anatomy41,6 and can be proprioceptive33,

254 we argue that selection for flow detection or other sensory functions could also have shaped the

255 evolution of fins in early vertebrates.

256

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257 The origins of median fins

258 The median fin fold hypothesis is the leading model for how differentiated fins originated

259 in vertebrates. However, there is limited evidence from the paleontological record of a

260 transformational series between a continuous MFF to spatially separated fins by reduction of the

261 MFF in certain positions. Claims that adult fins develop from the LMFF—which, if the MFF is

262 understood as homologous to the LMFF, could provide recapitulist evidence for the MFF

263 hypothesis—are similarly weak (e.g., numerous D. rerio mutants with malformed LMFFs develop

264 normal adult fins25, and most structures that comprise the adult fin are not derived from LMFF

265 tissues20,26,27).

266 The data presented above on the development of adipose fins provide indirect evidence

267 for the MFF hypothesis. Specifically, they demonstrate that a median fin fold, structurally

268 rudimentary and undifferentiated along its anteroposterior length, can be partitioned into an

269 individuated fin. Additionally, they show that fins that develop primitively from LMFF can be

270 transformed by association of new tissues into this territory such that a nascent fin can evolve to

271 become structurally and functionally complex. Indeed, some catfishes have redeployed the

272 developmental program for lepidotrichia into their adipose fins, such that the adipose fin

273 superficially looks like a duplicated first dorsal fin (e.g., Mochokus (Siluriformes))3. Although

274 indirect, adipose fin development and diversity might provide the clearest evidence that the MFF

275 hypothesis describes a viable route by which novel fins can originate.

276

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277 Methods

278 Albino C. aeneus (n=18) were donated for breeding by NBM Aquatics (Chicago, IL) and

279 housed at The University of Chicago in 10 g tanks at 20°C with a standard seasonal light/dark

280 cycle. Fish were conditioned for breeding on a diet of live blackworms, Lumbriculus variegatus,

281 and stimulated to breed with 50% water changes. Females deposited eggs on the aquarium

282 glass, and embryos were transferred by hand to 1 L containers of aerated tank water treated with

283 three drops of the antifungal Methylene blue (VWR International, West Chester, PA, USA; 0.5%

284 diluted in Hank's solution). Water was changed 30% daily until three days post-hatching, at which

285 point larvae were transferred to mesh boxes suspended in the adult tanks and raised on a diet of

286 dried shrimp pellets (OmegaSea, LCC, Painesville, OH).

287 To generate a developmental series, larvae were collected through one month post-

288 hatching. Specimens acquired through the pet trade were used for adult stages. Specimens

289 ranged in size from 0.5–2.7 cm SL. Animals were euthanized with MS222 (Tricaine

290 methanesulfonate, Sigma-Aldrich, St. Louis, MO) at a concentration of 0.5 g/L, fixed in 4%

291 paraformaldehyde at 4°C for 3 days on a rocker, and then stored in 100% methanol at 4°C.

292 Animal care and euthanasia protocols were approved by the University of Chicago’s Institutional

293 Animal Care and Use Committee.

294 Calcein, which fluoresces when bound to calcium, was used to characterize skeletal

295 development (Sigma #: C0875, Sigma Chemical Co., St Louis, MO). Fixed specimens (n=6) were

296 immersed in an aqueous solution of 50% methanol and 1% Calcein for 36 hours at 4°C on a

297 rocker and then rinsed three times in 50% methanol prior to imaging. Photographs of the

298 developmental series and of calcein stained specimens were collected using an Olympus DP72

299 camera mounted to a Leica MZ10 microscope (Leica Microsystems, Wetzlar, Germany) using the

300 software CELLSENS ENTRY v.1.2 (Build 7533) software (Olympus Corporation, Tokyo, Japan).

301 To characterize neuroanatomy, fixed specimens (n=28) were antibody stained using

302 methods adopted from Thorsen and Hale [1]. Nerves were labeled using the primary antibody

303 Mouse monoclonal anti-acetylated tubulin (Sigma-Aldrich), and the secondary antibody goat anti-

304 mouse antibody conjugated with fluorescein (Jackson ImmunoResearch Laboratories, West

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305 Grove, PA). Specimens were stained both undissected and following the removal of skin and

306 myomeres from the left side of the body to expose nerves entering the fin. Antibody stained

307 specimens were imaged with a Zeiss LSM710 confocal microscope (Carl Zeiss Inc., Thornwood,

308 NY, USA). Using the LSM710 software, images with extended depth of field were generated

309 using the ‘maximum intensity projection’ function on a z-stack of images. Also using the LSM710

310 software, large samples were imaged by ‘tiling,’ wherein adjacent z-stacks with 10% overlap were

311 combined into a single image. Brightness and contrast have been uniformly adjusted for images

312 presented in Figures 4 and 5, so that they are consistent across samples and microscope

313 settings.

314

315 Acknowledgements

316 Thank you to M Schauer at NBM Aquatics for guidance on the acquisition, husbandry, and

317 breeding of Corydoras. Thank you to MI Coates, WL Smith, BR Aiello, F Stabile and D Krentzel

318 for feedback on the manuscript. This research was supported by the following: National Science

319 Foundation, IGERT grant no. DGE-0903637 and GRFP to TAS; the Office of Naval Research

320 grant N00014–0910352 to MEH; National Institute of Health grant HD072598 to RKH, the

321 American Society for Ichthyologists and Herpetologists, Raney Award; The University of Chicago,

322 Hinds Fund.

323

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324 Author contributions

325 T.A.S. conceived of the project, collected and analyzed data, and wrote the paper. R.K.H. and

326 M.E.H. contributed to the design of the study, discussed and interpreted results, and helped to

327 write the paper.

328

329 Competing interests

330 The authors declare no competing interests

331

332 Data availability statement

333 The datasets generated during the current study are available from the corresponding author on

334 reasonable request.

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a de novo fold

ad.f

b Clupeiformes Alepocephaliformes Gonorhynchiformes Cypriniformes Gymnotiformes Otophysi Citharinoidei Characoidei de novo Siluriformes fold Lepidogalaxidae Euteleostei Argentiniformes Salmonidae fold Esociformes Stomiiformes Osmeriformes fold Retropinnidae Galaxiidae Ateleopodidae Aulopiformes Myctophiformes fold Polymixiidae Percopsiformes Gadiformes Zeiformes Lampriformes Acanthopterygii

Figure 1. Distribution of adipose fin developmental patterns. (a) Adipose fins (ad.f) develop either de novo as a bud following regression of the LMFF or from domain of the LMFF. (b) Phylogenetic distribution of the two developmental patterns. The simplified phylogeny of teleost fishes is based on 68 and modified to show recent phylogenetic hypotheses of Otophysan intra-relationships69,70. Black branches indicate lineages that are observed with adipose fins or where the fin is estimated to have occured3. Species-level data and associated references are provided in Suppl. Table 1. a bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

0.5 mm

Figure 2. Gross anatomy of adipose fin development in C. aeneus. (a) Prior to adipose fin development the LMFF is uniform along its length. (b) The LMFF begins reducing posterior to the dorsal fin and anteri- or to the caudal fin and a condensation is visible where the adipose fin will develop. (c) Ossification is initiated at the anterior portion of the condensation, and the ossification is oriented parallel to the actinori- chia in the LMFF. (d-f) Ossification extends both proximally and distally as the LMFF continues to reduce, and a domain of the LMFF is retained that will become the adipose fin membrane. a dorsal fin caudal fin b bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

anal fin 1 mm 0.5 mm pectoral fin pelvic fin

c d

1 mm 0.5 mm

e f

1 mm 0.5 mm

g h

1 mm 0.5 mm

i j

1 mm 0.5 mm

k l

1 mm 1 mm

Figure 3. Development of C. aeneus adipose fin skeleton. Fixed specimens were stained with calcein to reveal ossification patterns for the adipose fin spine, and odontodes, which are indicated in (b) and (d) by arrow heads. bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

b a dorsal fin larval median fin fold

caudal fin

0.5 mm

c adipose fin adipose fin spine membrane

lateral line larval median fin fold larval median fin fold anal fin 0.5 mm

Figure 4. The larval median fin fold contains sensory innervation. Confocal images of larval C. aeneus immunostained with anti-acetylated tubulin to label nervous tissue show nerves entering the fin fold and terminating as free nerve endings (a) prior to differentiation of the adipose fin spine. (b,c) Nerves in the LMFF are of a similar organization after the fin spine has begun developing and before the spine has grown to reach the epaxial musculature. bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

a b dorsal fin adipose fin nerves

recurrent ramus of the facial nerve spinal nerves

d adipose fin spine e,f anterior scutes j

g,h adipose fin I membrane

lateral scutes 0.1 mm e f c

50 µm 50 µm

g h

25 µm 25 µm

i j

25 µm

0.5 mm 25 µm 25 µm

Figure 5. Adipose fin-associated sensory nerves are observed after the fin spine has grown to reach the epaxial musculature. (a,b) Left side of the specimen has been dissected to expose dorsal rami of the spinal cord and the recurrent ramus of the facial nerve. (c,d) Sensory cells and innervation are observed on the fully developed adipose fin. (e, f) Transmitted light and immonofluorescence images show nerves in the fin membrane run parallel to actinotrichia in the fin membrane. (g, h) Transmitted light and immonofluorescence images showing nerves passing anteriorly through the adipose fin spine and ramifying to extend branches posteriorly into the fin membrane. (i) Superficial neuromasts are observed on the adipose fin spine. (j) Taste buds are observed on the adipose fin membrane. (k) Nerves in the adipose fin membrane also terminate as free nerve endings. Boxes in (d) indicate the approximate position of images in subsequent panels. Panels (i,j) are the same specimen as (c); panels (e-h, k) are of another specimen. bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

335 References

336 1 Coates, M. The origin of vertebrate limbs. Development supplement, 169-180 (1994).

337 2 Janvier, P. Early Vertebrates. Vol. 33 393 (Clarendon Press, 1996).

338 3 Stewart, T. A., Smith, W. L. & Coates, M. I. The origins of adipose fins: an analysis of

339 homoplasy and the serial homology of vertebrate appendages. Proc. Roy. Soc. B. 281,

340 doi:10.1098/rspb.2013.3120 (2014).

341 4 Larouche, O., Zelditch, M. L. & Cloutier, R. Fin modules: an evolutionary perspective on

342 appendage disparity in basal vertebrates. BMC Biology 15, 32, doi:10.1186/s12915-017-

343 0370-x (2017).

344 5 Zhang, X. G. & Hou, X. G. Evidence for a single median fin-fold and tail in the Lower

345 Cambrian vertebrate, Haikouichthys ercaicunensis. Journal of Evolutionary Biology 17,

346 1162-1166, doi:10.1111/j.1420-9101.2004.00741.x (2004).

347 6 Chen, J.-Y., Huang, D.-Y. & Li, C.-W. An early Cambrian craniate-like chordate. Nature

348 402, 518-522 (1999).

349 7 Goodrich, E. S. Studies on the structure & development of vertebrates. (Macmillan,

350 1930).

351 8 Thatcher, J. Median and paired fins, a contribution to the history of vertebrate limbs.

352 Trans. Connecti. Acad. 3, 281-310 (1877).

353 9 Mivart, S. G. Notes on the fins of elasmobranchs. Trans. Zool. Soc. London 10, 439-484

354 (1879).

355 10 Balfour, F. On the development of the skeleton of paired fishes of Elasmobranchii. Proc.

356 Zool. Soc. London, 662-671 (1881).

357 11 Weihs, D. & Webb, P. W. in Fish Biomechanics (ed P. W. Webb and D. Weihs) 339–

358 371 (Praeger Publishers, 1983).

359 12 Freitas, R., Gómez-Skarmeta, J. L. & Rodrigues, P. N. New frontiers in the evolution of

360 fin development. Journal of Experimental Zoology Part B: Molecular and Developmental

361 Evolution 322, 540-552, doi:10.1002/jez.b.22563 (2014).

15 bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

362 13 Coates, M. I. The evolution of paired fins. Theory in Biosciences 122, 266-287,

363 doi:10.1007/s12064-003-0057-4 (2003).

364 14 Coates, M. Hox genes, fin folds and symmetry. Nature 364, 195, doi:10.1038/364195b0

365 (1993).

366 15 Abe, G., Ide, H. & Tamura, K. Function of FGF signaling in the developmental process of

367 the median fin fold in zebrafish. Developmental Biology 304, 355-366,

368 doi:http://dx.doi.org/10.1016/j.ydbio.2006.12.040 (2007).

369 16 van den Boogaart, J. G. M., Muller, M. & Osse, J. W. M. Structure and function of the

370 median finfold in larval teleosts. The Journal of Experimental Biology 215, 2359-2368,

371 doi:10.1242/jeb.065615 (2012).

372 17 Parichy, D., Elizondo, M., Mills, M., Gordon, T. & Engeszer, R. Normal table of

373 postembryonic zebrafish development: staging by externally visible anatomy of the living

374 fish. . Dev. Dyn. 238, 2975–3015 (2009).

375 18 Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. & Schilling, T. F. Stages of

376 embryonic development of the zebrafish. Developmental Dynamics 203, 253-310,

377 doi:10.1002/aja.1002030302 (1995).

378 19 Cole, L. K. & Ross, L. S. Apoptosis in the Developing Zebrafish Embryo. Developmental

379 Biology 240, 123-142, doi:http://dx.doi.org/10.1006/dbio.2001.0432 (2001).

380 20 Freitas, R., Zhang, G. & Cohn, M. J. Evidence that mechanisms of fin development

381 evolved in the midline of early vertebrates. Nature 442, 1033-1037,

382 doi:http://www.nature.com/nature/journal/v442/n7106/suppinfo/nature04984_S1.html

383 (2006).

384 21 Akimenko, M. A., Johnson, S. L., Westerfield, M. & Ekker, M. Differential induction of four

385 msx homeobox genes during fin development and regeneration in zebrafish.

386 Development 121, 347-357 (1995).

387 22 Yonei-Tamura, S. et al. Competent stripes for diverse positions of limbs/fins in

388 gnathostome embryos. Evolution & Development 10, 737-745, doi:10.1111/j.1525-

389 142X.2008.00288.x (2008).

16 bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

390 23 Bemis, W. E. & Grande, L. in Mesozoic Fishes 2 - Systematics and Fossil Record (eds

391 G. Arratia & H.-P. Schultze) 41-68 (Verlag Dr. Friedrich Pfeil, 1999).

392 24 Lalonde, R. L. & Akimenko, M.-A. Effects of fin fold mesenchyme ablation on fin

393 development in zebrafish. PLOS ONE 13, e0192500, doi:10.1371/journal.pone.0192500

394 (2018).

395 25 van Eeden, F. et al. Genetic analysis of fin formation in the zebrafish, Danio rerio.

396 Development 123, 255-262 (1996).

397 26 Goodrich, E. S. Memoirs: Notes on the Development, Structure, and Origin of the Median

398 and Paired Fins of Fish. Journal of Cell Science s2-50, 333-376 (1906).

399 27 Lee, R. T. H., Thiery, J. P. & Carney, T. J. Dermal fin rays and scales derive from

400 mesoderm, not neural crest. Current Biology 23, R336-R337,

401 doi:http://dx.doi.org/10.1016/j.cub.2013.02.055 (2013).

402 28 Durán, I., Marí-Beffa, M., Santamaría, J. A., Becerra, J. & Santos-Ruiz, L. Actinotrichia

403 collagens and their role in fin formation. Developmental Biology 354, 160-172,

404 doi:http://dx.doi.org/10.1016/j.ydbio.2011.03.014 (2011).

405 29 Wood, A. & Thorogood, P. An analysis of in vivo cell migration during teleost fin

406 morphogenesis. Journal of Cell Science 66, 205-222 (1984).

407 30 Haeckel, E. Generelle morphologie der organismen. Allgemeine grundzüge der

408 organischen formen-wissenschaft, mechanisch begründet durch die von Charles Darwin

409 reformirte descendenztheorie. Vol. Bd.2 (G. Reimer, 1866).

410 31 Stewart, T. A. & Hale, M. E. First description of a musculoskeletal linkage in an adipose

411 fin: innovations for active control in a primitively passive appendage. Proc. Roy. Soc. B.

412 280, doi:10.1098/rspb.2012.2159 (2013).

413 32 Stewart, T. A. The origin of a new fin skeleton through tinkering. Biol. Letts 11,

414 doi:10.1098/rsbl.2015.0415 (2015).

415 33 Aiello, B. R., Stewart, T. A. & Hale, M. E. Mechanosensation in an adipose fin.

416 Proceedings of the Royal Society of London B: Biological Sciences 283,

417 doi:10.1098/rspb.2015.2794 (2016).

17 bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

418 34 Bender, A. & Moritz, T. Developmental residue and developmental novelty – different

419 modes of adipose-fin formation during ontogeny. Zoosystematics and Evolution 89, 209-

420 214, doi:10.1002/zoos.201300007 (2013).

421 35 Fuiman, L. in Ontogeny and systematics of fishes: based on an international symposium

422 dedicated to the memory of Elbert Halvor Ahlstrom / sponsored by the National Marine

423 Fisheries Service, National Oceanic and Atmospheric Administration, United States Dept.

424 of Commerce 126-138 (American Society of Ichthyologists and Herpetologists, 1985).

425 36 Sire, J.-Y. & Huysseune, A. Structure and Development of the Odontodes in an

426 Armoured Catfish, Corydoras aeneus (Siluriformes, Callichthyidae). Acta Zoologica 77,

427 51-72 (1996).

428 37 Schmitz, A., Bleckmann, H. & Mogdans, J. Organization of the superficial neuromast

429 system in goldfish, Carassius auratus. Journal of Morphology 269, 751-761,

430 doi:10.1002/jmor.10621 (2008).

431 38 Herrick, C. J. The cranial nerves and cutaneous sense organs of the north american

432 siluroid fishes. Journal of Comparative Neurology 11, 177-249,

433 doi:10.1002/cne.910110302 (1901).

434 39 Atema, J. Structures and Functions of the Sense of Taste in the Catfish (Ictalurus

435 natalis). Brain, Behavior and Evolution 4, 273-294 (1971).

436 40 Northcutt, R. G. Taste bud development in the channel catfish. Journal of Comparative

437 Neurology 428, 1-16 (2005).

438 41 Wagner, G. P. Homology, Genes and Evolutionary Innovation. (University Press, 2014).

439 42 Matsuoka, M. & Iwai, T. Adipose fin cartilage found in some teleostean fishes. Jpn. J.

440 Ichthyol 30, 37-46 (1983).

441 43 Buckland-Nicks, J. A. New details of the neural architecture of the salmonid adipose fin.

442 Journal of Fish Biology, n/a-n/a, doi:10.1111/jfb.13098 (2016).

443 44 Buckland-Nicks, J. A., Gillis, M. & Reimchen, T. E. Neural network detected in a

444 presumed vestigial trait: ultrastructure of the salmonid adipose fin. Proc. Roy. Soc. B.

445 279, 553-563, doi:10.1098/rspb.2011.1009 (2012).

18 bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

446 45 Ahn, D.-g., Kourakis, M. J., Rohde, L. A., Silver, L. M. & Ho, R. K. T-box gene tbx5 is

447 essential for formation of the pectoral limb bud. Nature 417, 754,

448 doi:10.1038/nature00814

449 https://www.nature.com/articles/nature00814 - supplementary-information (2002).

450 46 Chen, J., Liu, X., Yao, X., Gao, F. & Bao, B. Dorsal fin development in flounder,

451 Paralichthys olivaceus: Bud formation and its cellular origin. Gene Expression Patterns

452 25-26, 22-28, doi:https://doi.org/10.1016/j.gep.2017.04.003 (2017).

453 47 Freitas, R., Zhang, G. & Cohn, M. J. Evidence that mechanisms of fin development

454 evolved in the midline of early vertebrates. Nature. 442, doi:10.1038/nature04984 (2006).

455 48 Mao, Q., Stinnett, H. K. & Ho, R. K. Asymmetric cell convergence-driven fin bud initiation

456 and pre-pattern requires Tbx5a control of a mesenchymal Fgf signal. Development,

457 doi:10.1242/dev.124750 (2015).

458 49 Tanaka, M., Yu, R. & Kurokawa, D. Anterior migration of lateral plate mesodermal cells

459 during embryogenesis of the pufferfishTakifugu niphobles: insight into the rostral

460 positioning of pelvic fins. Journal of Anatomy 227, 81-88, doi:10.1111/joa.12324 (2015).

461 50 Murata, Y. et al. Allometric growth of the trunk leads to the rostral shift of the pelvic fin in

462 teleost fishes. Developmental Biology 347, 236-245,

463 doi:https://doi.org/10.1016/j.ydbio.2010.07.034 (2010).

464 51 Sire, J.-Y. & Huysseune, A. N. N. Formation of dermal skeletal and dental tissues in fish:

465 a comparative and evolutionary approach. Biological Reviews 78, 219-249,

466 doi:10.1017/S1464793102006073 (2003).

467 52 Sire, J.-Y. Development and fine structure of the bony scutes in Corydoras arcuatus

468 (Siluriformes, callichthyidae). Journal of Morphology 215, 225-244,

469 doi:10.1002/jmor.1052150305 (1993).

470 53 Géraudie, J. & Landis, W. J. The fine structure of the developing pelvic fin dermal

471 skeleton in the trout Salmo gairdneri. American Journal of Anatomy 163, 141-156,

472 doi:10.1002/aja.1001630204 (1982).

19 bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

473 54 Hall, B. K. Fins into limbs: Evolution, Development and Transformation. (University of

474 Chicago Press, 2007).

475 55 Hass, H. Studies on mechanisms of joint and bone formation in the skeleton rays of fish

476 fins. Dev. Biol 5, 1-34 (1962).

477 56 Williams Iv, R., Neubarth, N. & Hale, M. E. The function of fin rays as proprioceptive

478 sensors in fish. Nat Commun 4, 1729, doi:10.1038/ncomms2751 (2013).

479 57 Thorsen, D. H. & Hale, M. E. Neural development of the zebrafish (Danio rerio) pectoral

480 fin. J. Comp. Neurol. 504, 168-184 (2007).

481 58 Williams, J. A. et al. Programmed Cell Death in Zebrafish Rohon Beard Neurons Is

482 Influenced by TrkC1/NT-3 Signaling. Developmental Biology 226, 220-230,

483 doi:https://doi.org/10.1006/dbio.2000.9860 (2000).

484 59 Reyes, R., Haendel, M., Grant, D., Melancon, E. & Eisen, J. S. Slow degeneration of

485 zebrafish Rohon-Beard neurons during programmed cell death. Developmental

486 Dynamics 229, 30-41, doi:10.1002/dvdy.10488 (2004).

487 60 Kotrschal, K., Whitear, M. & Finger, T. E. Spinal and facial innervation of the skin in the

488 gadid fish Ciliata mustela (Teleostei). The Journal of Comparative Neurology 331, 407-

489 417, doi:10.1002/cne.903310310 (1993).

490 61 Codina, E. et al. The barbel-like specialization of the pelvic fins in Ophidion rochei

491 (Ophidiidae). Journal of Morphology 273, 1367-1376, doi:10.1002/jmor.20066 (2012).

492 62 Bensouilah, M. & Denizot, J. P. Taste Buds and Neuromasts of Astyanax jordani:

493 Distribution and Immunochemical Demonstration of Co-localized Substance P and

494 Enkephalins. European Journal of Neuroscience 3, 407-414, doi:10.1111/j.1460-

495 9568.1991.tb00828.x (1991).

496 63 Asaoka, R., Nakae, M. & Sasaki, K. The innervation and adaptive significance of

497 extensively distributed neuromasts in Glossogobius olivaceus (Perciformes: Gobiidae).

498 Ichthyol. Res. 59, 143-150, doi:10.1007/s10228-011-0263-x (2012).

499 64 Davenport, C. & Caprio, J. Taste and tactile recordings from the ramus recurrens facialis

500 innervating flank taste buds in the catfish. J. Comp. Physiol. 142, 217-229 (1982).

20 bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

501 65 Temple, N. F. & Reimchen, T. E. Adipose fin condition and flow regime in catfish.

502 Canadian Journal of Zoology 86, 1079-1082, doi:10.1139/Z08-086 (2008).

503 66 Reimchen, T. E. & Temple, N. F. Hydrodynamic and phylogenetic aspects of the adipose

504 fin in fishes. Canadian Journal of Zoology 82, 910-916, doi:10.1139/z04-069 (2004).

505 67 Feitosa, N. M. et al. Hemicentin 2 and Fibulin 1 are required for epidermal–dermal

506 junction formation and fin mesenchymal cell migration during zebrafish development.

507 Developmental Biology 369, 235-248, doi:https://doi.org/10.1016/j.ydbio.2012.06.023

508 (2012).

509 68 Near, T. J. et al. Resolution of ray-finned fish phylogeny and timing of diversification.

510 Proceedings of the National Academy of Sciences 109, 13698-13703,

511 doi:10.1073/pnas.1206625109 (2012).

512 69 Chen, W.-J., Lavoué, S. & Mayden, R. L. Evolutionary origin and early biogeography of

513 Otophysan fishes (Ostariophysi: Teleostei). Evolution 67, 2218-2239 (2013).

514 70 Nakatani, M., Miya, M., Mabuchi, K., Saitoh, K. & Nishida, M. Evolutionary history of

515 Otophysi (Teleostei), a major clade of the modern freshwater fishes: Pangaean origin and

516 Mesozoic radiation. BMC Evolutionary Biology 11, 1-25 (2011).

517 71 Huysentruyt, F., Moerkerke, B., Devaere, S. & Adriaens, D. Early development and

518 allometric growth in the armoured catfish Corydoras aeneus (Gill, 1858). Hydrobiologia

519 627, 45-54, doi:10.1007/s10750-009-9714-z (2009).

520 72 Rodríguez-Ithurralde, D., del Puerto, G. & Fernández-Bornia, F. Morphological

521 development of Corydoras aff. paleatus (Siluriformes, Callichthyidae) and correlation with

522 the emergence of motor and social behaviors. Iheringia. Série Zoologia 104, 189-199

523 (2014).

524 73 Ramnarine, I. W. Larval culture, development and growth of the cascadu, Hoplosternum

525 littorale (Hancock 1828; Callichthyidae). Aquculture 126, 291-298 (1994).

526 74 Jones, P. W., Martin, F. D. & Hardy, J. D. Development of Fishes on the Mid-Atlantic: An

527 Atlas of Egg, Larvae, and Juvenile Stages. . (Fish and Wildlife Service, U.S. Department

528 of the Interior, 1978).

21 bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

529 75 Hinaux, H. et al. A developmental stating table for Astyanax mexicanus surface fish and

530 Pachón cavefish. Zebrafish 8, 155-165 (2011).

531 76 Gorodilov, Y. Description of the early ontogeny of the Atlantic salmon, Salmo salar, with a

532 novel system of interval (state) identification. Environmental Biology of Fishes 47, 109-

533 127 (1996).

534 77 Ré, P. & Meneses, I. Early stages of marine fishes occuring in the Iberian Peninsula.,

535 (IPIMAR/IMAR, 2008).

536 78 Sassa, C., Kawaguchi, K. & Loeb, V. Early development of Diaphus garmani

537 (Myctophidae) in the transition region of the western North Pacific. Ichthyol. Res. 50, 94-

538 97. (2003).

539 79 Moser, H. & Watson, W. in Early Stages of Atlantic Fishes: an identification guide for the

540 Western Central North Atlantic (ed WJ Richards) (Taylor and Frances Group, 2004).

541

542

22 bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

543 Suppl. Table 1: Data aggregated from the literature on adipose fin developmental diversity. 544 Information on adipose fin development was found in one study of the fin’s development 34, the 545 taxonomic keys of larval fishes, and in developmental staging papers. The information is 546 summarized in Fig. 1.

order family genus species dev. mode reference

Siluriformes Callichthyidae Corydoras aeneus fold this study, 71

Siluriformes Callichthyidae Corydoras paleatus fold 72

Siluriformes Callichthyidae Hoplosternum littorale fold 73

Siluriformes Ictaluridae Ictalurus catus fold 74

Siluriformes Ictaluridae Ictalurus punctatus fold 74

Siluriformes Ictaluridae Noturus insignis fold 74

Characiformes Alestidae Brycinus longipinnis de novo 34

Characiformes Alestidae Phenacogrammus interruptus de novo 34

Characiformes Characidae Astyanax Mexicanus de novo 75

Characiformes Characidae Hemigrammus erythrozonus de novo 34

Characiformes Characidae Hyphessobrycon herbertaxelrodi de novo 34

Characiformes Characidae Pironobrama filigera de novo 34

Characiformes Serrasalmidae Pygocentrus nattereri de novo 34

Characiformes Ctenoluciidae Ctenoluccius hujeta de novo 34

Salmoniformes Salmonidae Salmo salmar fold 76

Salmoniformes Salmonidae Salmo trutta fold 34

Salmoniformes Coregonidae Coregonus maraena fold 34

Myctophiformes Myctophidae Benthosema glaciale fold 77

Myctophiformes Myctophidae Ceratoscopelus maderensis. fold 77

Myctophiformes Myctophidae Electrona risso fold 77

Myctophiformes Myctophidae Diaphus garmani fold 78

Myctophiformes Myctophidae Benthosema suborbitale fold 79

Myctophiformes Myctophidae Centobranchus nigroocellatus fold 79

Osmeriformes Osmeridae Osmerus eperlanus fold 34

23 bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Supplementary Fig. 1. Photographs of adult C. aeneus by Yen-Chyi Liu. bioRxiv preprint doi: https://doi.org/10.1101/283432; this version posted March 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

a b c d

50 µm 25 µm e f g h

i j k l

Supplementary Fig. 2. Superficial neuromasts on the adipose fin spine and lateral scutes of C. aeneus. (a) Maximum-intensity projection of a superficial neuromast on the adipose fin spine. Panels (b-l) are of a neuromast on a lateral scute, which is oriented perpendicular to the image plane. Panels are from apex (b) to base (l). Depth between panels is 2.7 μm. Figure 2. Gross anatomy of adipose fin development in C. aeneus. (a) Prior to adipose fin development the LMFF is uniform along its length. (b) The LMFF begins reducing posterior to the dorsal fin and anteri- or to the caudal fin and a condensation is visible where the adipose fin will develop. (c) Ossification is initiated at the anterior portion of the condensation, and the ossification is oriented parallel to the actinori- chia in the LMFF. (d-f) Ossification extends both proximally and distally as the LMFF continues to reduce, and a domain of the LMFF is retained that will become the adipose fin membrane.

a b c d

50 µm

e f g h

i j k l

Supplementary Fig. 3. Anatomy of a taste buds on the adipose fin of C. aeneus. (a) Maximum intensity projection showing three taste buds, indicated by arrowheads, and associated nerves. The taste buds are oriented perpendicular to the image plane, and panels are from apex (b) to base (l). Adipose fin afferents, which run parallel to actinotrichia in the membrane, terminate upon taste buds. Depth between panels is 3 μm. Figure 3. Development of C. aeneus adipose fin skeleton. Fixed specimens were stained with calcein to reveal ossification patterns for the adipose fin spine, and odontodes, which are indicated in (b) and (d) by arrow heads.

25 µm f g h i j

Supplementary Fig. 4. Anatomy of a lateral line cell of C. aeneus. Panels (a-j) are of a lateral line cell positioned ventral to the adipose fin on the left side of the body; anterior is left. The lateral line cell is oriented perpendicular to the image plane, and panels are organized from apex (a) to base (j). The apex of the lateral line cell is characterized by a filamentous tip. Depth between panels is 5.5 μm.