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1 Monsters with a shortened vertebral column: A population phenomenon in

2 radiating fish Labeobarbus (Cyprinidae)

3

4 Alexander S. Golubtsov1, Nikolai B. Korostelev1, Boris A. Levin2,3*

5 1 Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Moscow, Russia

6 2 Papanin Institute for Biology of Inland Waters, Russian Academy of Sciences, Borok, Russia

7 3 Cherepovets State University, Cherepovets, Russia

8

9 ☯ These authors contributed equally to this work.

10 * [email protected]

11

12

13 Short title: Monstrosity in fish as novelty?

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26 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

27 ABSTRACT

28 The phenomenon of a massive vertebral deformity was recorded in the radiating Labeobarbus

29 assemblage from the middle reaches of the Genale River (south-eastern Ethiopia, East Africa).

30 Within this sympatric assemblage, five trophic morphs – generalized, lipped, piscivorous and two

31 scraping feeders – were reported between 1993 and 2019. In 2009, a new morph with prevalence

32 of ~10% was discovered. The new morph, termed ‘short’, had an abnormally shortened vertebral

33 column and a significantly heightened body. This type of deformity is common in farmed Atlantic

34 salmon and other artificially reared fish, but is rare in nature. In the Genale Labeobarbus

35 assemblage, the deformity was present exclusively within the generalized and lipped morphs. The

36 short morph had between seven and 36 deformed (compressed and/or fused) vertebrae. Their body

37 height was positively correlated with number of deformed vertebrae. In another collection in 2019,

38 the short morph was still present at a frequency of 11%. Various environmental and genetic factors

39 could contribute to the development of this deformity in the Genale Labeobarbus, but based on

40 the available data, it is impossible to confidently identify the key factor(s). Whether the result of

41 genetics, the environment, or both, this high-bodied phenotype is assumed to be an anti-predator

42 adaptation, as there is evidence of its selective advantage in the generalized morph. The Genale

43 “monstrosity” is the first reported case of a massive deformity of the vertebral column in a natural

44 population of African fishes.

45

46

47

48

49

50

51

52

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53 “We have also what are called monstrosities; but they

54 graduate into varieties. By a monstrosity I presume is

55 meant some considerable deviation of structure in one

56 part, either injurious to or not useful to the species,

57 and not generally propagated. If it could be shown

58 that monstrosities were even propagated for a

59 succession of generations in a state of nature,

60 modifications might be effected (with the aid of

61 natural selection) more abruptly than I am inclined to

62 believe they are.”

63 Darwin (1860, pp. 46, 426).

64

65 INTRODUCTION

66 The emergence and establishment of morphological novelties in a population is central to

67 morphological evolution, although this process remains poorly understood. As initially highlighted

68 by Darwin [1], there is no clear discrimination between morphological abnormalities

69 (monstrosities) and regular variation. Morphological abnormalities that are caused by genetic

70 factors – and hence may be shaped by natural selection – are particularly interesting to evolutionary

71 biologists as potential novelties.

72 Ray-finned fishes form one of the major vertebrate groups, comprised of more than 30,000

73 species with extreme variations in morphology and body plans [2]. For example, the ocean

74 sunfishes (Molidae, Tetraodontiformes) exhibit one of the most impressive evolutionary

75 transformations of the axial skeleton among ray-finned fishes [3,4]. It has been suggested that the

76 enigmatic loss of caudal fin and caudal part of the vertebral column is related to “the existence in

77 collections of a number of malformed adult tetraodontiforms without caudal fins” [5,6].

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78 Various body and skeletal deformities in fish have been reviewed in many works [7-13];

79 earlier reports can also be found in Dawson’s bibliographies [13-17]. Many of these deformities

80 apparently can be treated as monstrosities, particularly when the vertebral column is shortened

81 without pronounced curvature, resulting in an altered body form that is extremely short and high.

82 Such deformities are known in at least 26 species of 15 families (S1 Table). Remarkably, all

83 individuals of Aischgrunder Karpfen, the South German carp breed (extinct since 1956), exhibited

84 such deformity [18,19].

85 The riverine adaptive radiations in the large African barbs of the genus Labeobarbus

86 Rüppell 1835 (Cyprinidae) – the dominating fish group in the waters of the Ethiopian Highlands

87 – have been studied by us for almost 30 years. Labeobarbus belongs to the African Torini, a lineage

88 of hexaploids [20-24] that originated in the Middle East via hybridization of tetraploids (maternal

89 Tor lineage) and diploids (paternal Cyprinion lineage), and then dispersed throughout Africa [25].

90 Although the continental-scale phylogeny of Labeobarbus is still poorly resolved [26], the mt-

91 DNA based phylogeny of this group in Ethiopian waters is relatively well-studied [27-32]. It is

92 important to note that the barbs inhabiting the southeastern part of Ethiopia (drained by the Wabi-

93 Shebele and Juba river systems into the Indian Ocean) form a monophyletic group, Labeobarbus

94 gananensis (Vinciguerra, 1895) complex. This is a sister group to Labeobarbus inhabiting

95 enclosed basins of the Ethiopian Rift Valley, as well as all Labeobarbus from the waters of western

96 and northern Ethiopia belonging to the Omo-Turkana and Nile systems, and additional some

97 Kenyan barbs [27,31,32].

98 In addition to the famed Labeobarbus radiation in Lake Tana [33-63], this group has also

99 experienced parallel riverine radiations in four Ethiopian basins. These riverine radiations are

100 similar in terms of their morphological and ecological differentiation, but differ in the ways of

101 genetic divergence [32]. Outside of Ethiopia, the riverine Labeobarbus radiation seems to occur

102 in the Inkisi River basin (Lower Congo) [64].

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103 The Labeobarbus assemblage in the Genale River (the main tributary of the Juba River) is

104 the only radiation occurring in southeastern Ethiopia. It is geographically separated from the other

105 Ethiopian Labeobarbus radiations by the Ethiopian Rift valley. It exhibits the deepest divergence

106 in terms of morphology, ecology and genetics compared to the other riverine radiations [32].

107 During an intensive sampling of the middle Genale River in 2009, we observed a substantial

108 portion of barbs with extremely short and high bodies [31].

109 In 1993, an assemblage of the sympatric morphologically distinct morphs of the L.

110 gananensis complex was discovered in the middle reaches of the Genale River [65]. This area was

111 re-sampled twice in the 1990s [66,67], then in 2009 [27,31], and again in 2019. Together with the

112 omnivorous generalized morph of L. gananensis and the scraping-feeder L. jubae (Banister, 1984),

113 three additional morphs specialized in their morphology and feeding habits were always present

114 in these samples from the middle Genale. These include the lipped morph of L. gananensis (having

115 hypertrophied lips), the scraping morph Labeobarbus sp. 1 (provisionally called ‘smiling’) and the

116 large-mouthed morph Labeobarbus sp. 2 (called ‘piscivorous’), as well as a hybrid morph called

117 ‘smiling hybrids’. In 2009, a new morph, ‘short’, was discovered, which has an abnormally short

118 and high body (Fig. 1). It composed approximately 10% of the total barb catch in both 2009 and

119 2019 samplings [31].

120

121 Fig. 1. External appearance (left) and x-ray images (right) of the generalized and short

122 Labeobarbus morphs from the middle Genale assemblage. A: generalized (SL = 242 mm), B:

123 short, with eight deformed vertebrae, marked by red asterisks (SL = 245 mm), C: short, with

124 28 deformed vertebrae (SL = 216 mm), and D: short, with 31 deformed vertebrae (SL = 201 mm).

125

126 Trophic resource partitioning has been demonstrated among most morphs from the middle

127 Genale Labeobarbus assemblage [31]. In contrast to the other morphs, the short morph does not

128 diverge from the generalized morph in its diet [31]. Moreover, based on mtDNA markers, the short

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129 morph shares a common gene pool with the generalized and lipped morphs [31]. Preliminary

130 investigation of the vertebral column revealed that each short individual had a substantial number

131 of deformed (compressed and/or fused) vertebrae (Fig. 1).

132 The objectives of this study were: (1) to investigate the structure of the vertebral column

133 in the short morph compared to other sympatric Labeobarbus morphs from the Genale River

134 assemblage, (2) to analyze morphological differences between the short and related (generalized

135 and lipped) morphs, with clarification of the contribution of the vertebral column shortening to

136 these differences, and (3) to discuss the possible causes of the emergence of the aberrant morph in

137 light of its life history characteristics and decade-long presence in the local Labeobarbus

138 population.

139

140 MATERIALS AND METHODS

141

142 Ethics statement

143 Fish were collected in southeastern Ethiopia under the umbrella of the Joint Ethiopian-Russian

144 Biological Expedition (JERBE), with permission from the National Fishery and Aquatic Life

145 Research Center under the Ethiopian Institute of Agricultural Research and the Ethiopian Ministry

146 of Innovation and Technology. Fish were sacrificed humanely using an anesthetic overdose

147 (American Veterinary Medical Association). The experiments were carried out in accordance with

148 the rules of the Papanin Institute of Biology of Inland Waters (IBIW), Russian Academy of

149 Sciences, and approved by IBIW’s Ethics Committee.

150

151 Study sites and sampling

152 The Ethiopian Highlands are divided by the Rift Valley into the western and eastern

153 plateaus [68]. The southern part of the eastern plateau is drained by the Wabi Shebeli (Webi

154 Shabeelle or Uebi Scebeli) and Juba (Jubba) river systems into the Indian Ocean via Somalia.

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155 Three large tributaries, the Genale (Ganale Doria), the Dawa (Daua) and the Weyb (Gestro), meet

156 near the Ethiopia-Somalia border to form what is known as the Juba River within Somalia. The

157 Genale, Weyb and Wabi Shebeli originate in the Bale Mountains, the highest part of the eastern

158 plateau (Fig. 2).

159 Since 1990, we have sampled a total of 28 sites across the Wabi-Shebele and Juba river

160 systems (dots in Fig. 2). Among these, Labeobarbus were sampled from 15 sites, and not found in

161 the other 13 sites. An additional four sites important for the present work are numbered in Fig. 2:

162 (1) main sampling site, the middle reaches of the Genale River (5º42' N 39º32' E, altitude 1125 m

163 above sea level, asl); (2) the upper reaches the Genale River north of Kibre Mengist (6°02'40" N

164 39°07'19" E, 1324 m asl); (3) the middle reaches of the Welemele River, a northern tributary of

165 the Genale (6°14'30" N 39°47'22" E, altitude c. 1040 m asl); and (4) the upper reaches the Awata

166 River, northern tributary of the Dawa (5º47'07"N 38º55'46"E, 1630 m asl). All sampling sites are

167 listed in S2 Table. Most samples were taken in the different years from the end of January to mid-

168 May during the main rainy season in southeastern Ethiopia [69], but before substantial rise of water

169 level in the rivers.

170

171 Fig. 2. Map of sampling sites in the Juba and Wabi Shebele drainages. Black and red circles

172 indicate sites where Labeobarbus was detected; white circles indicate sites where barbs were not

173 found. Numbered red circles designate the most important sites for the current study – 1: Genale

174 River, middle reaches, 2: Genale River, upper reaches, 3: Welmel River, and 4: Awata River.

175 Geographical coordinates for all sites are given in S2 Table.

176

177 The main sampling site (no. 1, Fig. 2) included two habitats: the continuous pool starting

178 from the shallows around the island, characterized by moderate current and sandy/silty bottom,

179 and the upper part of continuous rapids with small waterfalls, rocky bottom and very fast current

180 (S3 Fig.). Samples were mostly taken from the pool by gill netting (at dawn and overnight), but an

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181 additional sample was obtained from the rapids with cast nets in 2009. The cast net catches differed

182 substantially from the gill net catches, in terms of fish size and frequency of Labeobarbus morphs

183 (S4 Table), therefore these catches were analyzed separately. The remaining sites (nos. 2-4) were

184 sampled by both cast and gill netting during the day, and the catches were analyzed together.

185 The selected fish were maintained for several hours in large barrels submerged into the

186 river, then killed with an overdose of MS-222 and preliminarily investigated for morphology.

187 Tissue samples were taken for genetic and stable isotope analyses because the genetic and trophic

188 specialization studies have been conducted on the same individuals [27,31,32]. Most specimens

189 were preserved in 10% formalin in the field, and subsequently transferred to 70% ethanol in the

190 laboratory. Of the 291 specimens collected in 2009, 132 were preserved with salt to make the dried

191 bone preparations. These specimens were dissected for determination of sex and gonad maturity

192 stages.

193

194 Examination of vertebral column

195 Most formalin-preserved specimens were x-rayed. Based on the film or digital radiograms,

196 the structure of vertebrae were examined. The total vertebral number and numbers of pre-dorsal,

197 pre-anal, trunk, transitional and caudal vertebrae were counted according to Naseka [70].

198 Examination of vertebral structure and counts of vertebrae mentioned above were made by means

199 of visual inspection of the dried bone preparations. The head magnifier (×4) was used for film and

200 preparation inspection if necessary. In total, the vertebral column structure was studied in 364

201 Genale barbs representing all morphs. This number includes the radiograms of 34 and 45

202 specimens collected before 2009 and in 2019, respectively, as well as 285 radiograms and dried

203 bone preparations of fish collected in 2009.

204 Witten et al. [11] propose a classification of vertebral column deformities that are

205 repetitively observed in salmon (Salmon salar L.) under farming conditions. In our material, we

206 observed all nine types of deformities united by these authors into the category of ‘compressed

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207 and/or fused vertebral bodies’. Taking into account the relatively small size of our samples, we

208 did not consider the different types of such deformities separately. All vertebrae with compressed

209 and/or fused bodies were considered herein as deformed vertebrae. The deformities of other

210 categories [11] were not recorded in our material, except in one specimen of the smiling morph

211 that had vertebral column curvature (kyphosis). Other deformities such as a presence of abnormal

212 additional ribs and neural spines, and splitting of neural spine [71] were recorded in our material,

213 however they were not analyzed because they were not related to the shortening of the vertebral

214 column.

215 Examination of other morphologic characters

216 Prior to preservation, the following measurements were taken (with a ruler, to the nearest

217 0.5 mm) on all specimens: standard length (SL), head length (HL), dorsal spine length (DL),

218 maximum (H) and minimum (h) height of body, and length of caudal peduncle (cpd). For further

219 analysis, the body length (BL) was calculated as SL minus HL.

220 Of all morphs sampled in 2009 (N = 129, SL = 98-308 mm), we randomly selected a sub-

221 set of formalin-preserved specimens and took 23 measurements in the laboratory (with a caliper,

222 to the nearest 0.1 mm): standard length (SL), head length (HL), snout length (R), orbit horizontal

223 diameter (O), interorbital width (IO), postorbital length of head (PO), height of head at mid-orbit

224 (Ch), height of head at occiput (CH), mouth width (MW), dorsal spine length (DL), pre-dorsal

225 distance (PrD), post-dorsal distance (PD), caudal peduncle length (cpd), length of dorsal fin base

226 (lD), maximum (H) and minimum (h) height of body, length of pectoral (lP) and ventral (lV) fins,

227 pectoral-ventral distance (PV), ventral-anal distance (VA), height of anal fin (hA), length of anterior

228 barbel (Ab), and length of posterior barbel (Pb). In the present work, only data on these extended

229 measurements were included for 22 and 28 specimens of the generalized (SL = 108-257 mm) and

230 short (SL = 119-244 mm) morphs, respectively. All measurements were taken by the same person

231 for consistency [72].

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232 In most sampled specimens, the following counts were taken: number of scales in the

233 lateral line, number of scale rows above and below lateral line, number of predorsal scales, number

234 of scales around the caudal peduncle, number of gill rakers on the first gill arch, number of

235 branched rays in the dorsal fin.

236

237 Examination of age and growth

238 In most salt-preserved specimens of the different morphs, age was determined from

239 vertebrae. Annual rings were counted on the anterior and posterior sides of the fifth vertebra after

240 removing the dried intervertebral discs. Counts were made with a binocular microscope at

241 magnification ×16, with a drop of glycerin for contrasting. Age estimates were calibrated with

242 vertebrae from the artificially reared Labeobarbus from Lake Tana [63].

243 Growth rate estimates for the different Labeobarbus morphs were based on comparisons

244 of individual size variation in the different year classes. We did not calculate values of growth rate

245 or parameters of the von-Bertalanffy equation because of the small sample sizes.

246 Statistical analysis

247 Various R packages [73] realized in R-studio v.1.2.5033 were used for statistical analyses

248 and plot construction: summarytools library was used for obtaining basal descriptive statistics,

249 ggpubr library was used to analyze Pearson correlation and plot the result,

250 posthoc.kruskal.dunn.test function was applied for posthoc Dunn’s test, FSA library was applied

251 for Mann-Whitney U test, prcomp function was used for the principal component analysis (PCA)

252 and plotting the results, ggplot2 library was applied for plotting boxplots and histogram bars. The

253 proportions of head and body were used for analyses of single characters, as well as for PCA – all

254 measurements were divided by head length. Data was standardized for PCA.

255

256 Deposition of material

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257 All samples were deposited to the Severtsov Institute of Ecology and Evolution Russian

258 Academy of Sciences and Papanin Institute for Biology of Inland Waters Russian Academy of

259 Sciences under provisional labels of the Joint Ethiopian-Russian Biological Expedition.

260

261 Abbreviation of morph names

262 The names of the morphs from the Genale Labeobarbus assemblage have been assigned

263 the following abbreviations: short SH, generalized GN, lipped LP, L. jubae JB, smiling SM,

264 smiling hybrids HB, and piscivorous PS.

265

266 RESULTS

267 Short definition

268 Initially, in the field, the short morph was identified based on altered body proportions:

269 these individuals had relatively higher and shorter bodies compared to the normal individuals (Fig.

270 1). Preliminary investigation of the vertebral column revealed that each short individual had a

271 substantial number of deformed vertebrae. The data on the structure of the vertebral column for

272 all morphs are presented in Table 1.

273

274 Table 1. Occurrence of individuals with various numbers of deformed vertebrae in the different

275 morphs in the total sample (1993-2019) (N, number of individuals; %dv, percent of individuals

276 with deformed vertebrae in a particular morph).

277

Number of deformed vertebrae per individual

Morph N %dv 0 1 2 3 4 5 6 7 8 ≥ 9

Shorts (SH) 52 100 1 2 49

Generalized 78 16.7 65 1 8 2 2

(GN)

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Lip (LP) 26 11.5 23 2 1

Jubae (JB) 78 12.8 68 1 7 1 1

Smiling (SM) 48 14.6 41 1 4 1 1

Hybrid (HY) 36 5.6 34 2

Piscivorous 46 9.8 41 3 1 1

(PS)

278

279 We defined the SH morph as individuals with seven or more deformed vertebrae. It is

280 important to note that the mouth structure in the SH morph was similar to that in GN and LP

281 morphs that had normal vertebral columns. These morphs – with either shortened or normal

282 vertebral columns – differed in the degree of lip development (S5 Fig.).

283 There were almost no individuals with markedly shortened bodies among the trophically

284 specialized morphs (JB, SM, HY, PS). The only PS individual with eight deformed vertebrae

285 (Table 1) displayed noticeable body shortening (S6 Fig.).

286 Temporal and spatial distribution of the short morph

287 In the samples taken before 2009, SH individuals were not discovered among the 34

288 radiographed individuals of the different morphs, or among the hundreds of superficially examined

289 barbs in catches of 1993, 1997 and 1998. To estimate SH prevalence in the samples of 2009 and

290 2019, we analyzed the total gill net catches. The catch composition appeared to be quite similar in

291 2009 and 2019 (S7 Table). The ratios of morphs remained roughly stable. It is important to note

292 that SH prevalence in the total Labeobarbus catches was almost unchanged: they represented 10%

293 of the 400 barbs sampled in 2009, and 11% of the 179 barbs sampled in 2019.

294 We did not sample the SH morph from the upper reaches of the Genale River (sampling

295 site no. 2, Fig. 2), where most other morphs common to the main sampling site were found.

296 Moreover, the SH morph was not recorded from other sampling sites in the Wabi-Shebele and

297 Juba river systems (Fig. 2, S2 Table) or from other parts of Ethiopia (sampling sites may be found

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298 in [32,74,75]). However, at sampling site no. 3, we found barbs that had shortened bodies but

299 without deformed vertebrae (discussed below).

300 Short vs generalized and lipped morphs: plastic characters

301 As expected, body height of the SH morph was positively correlated (Fig. 3A) with the

302 number of deformed vertebrae, which in turn was negatively correlated with body length (Fig.

303 3B). In other morphs of the Genale Labeobarbus assemblage, no such correlations were found.

304

305 Fig. 3. Pearson correlation of (A) body height and (B) body length with number of

306 deformed vertebrae in the short (SH) morph.

307

308 The changes of body proportions in the SH morph (caused by the deformity of the vertebral

309 column) resulted in a pronounced difference in appearance between this and the other morphs.

310 Based on PCA, the SH and GN morphs were well differentiated from each other in PC1 (Fig. 4).

311 Indices of 21 measurements relative to head length (HL) rather than SL were used in the PCA

312 because of the variable influence of vertebral deformity on SL in the short individuals (Fig. 3B).

313 PC1 explained > 45% of the variance, while PC2 was less than 24%. Eigenvectors of the 10 most

314 loaded characters for PC1 and PC2 are given in S7 Table.

315

316 Fig. 4. PCA of short (SH) and generalized (GN) morphs based on 21 indices of head and

317 body proportions.

318

319 Using the a larger data set (which included the gill net catch of 2009, S5 Table), we

320 compared the characters that had the strongest influence on body form (head length, heights of

321 body and caudal peduncle) between the SH, GN and LP morphs (Fig. 5). The LP and SH morphs

322 were similarly characterized by relatively longer heads (Fig. 5A). In the LP morph, the longer head

323 length arose from elongation of the snout (S5 Fig.), while in the SH morph the head length

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324 appeared longer relative to the shortened body. The LP, SH and lipped SH morphs all had

325 relatively longer heads than the GN; the lipped SH morph had a notably longer head than the SH

326 morph, but the difference was not significant (Fig. 5A). The relative heights of body and caudal

327 peduncle in the SH morph differed significantly from both GN and LP, however there were no

328 such differences between the lipped SH and any of the other morphs (Fig. 5B-C). Notably, lipped

329 SH exhibited the most variation in relative heights of body and caudal peduncle compared to the

330 other morphs. This variation was caused by different degrees of snout elongation and variation

331 caused by body shortening (determined by varying number of deformed vertebrae), which seemed

332 to act additively.

333

334 Fig. 5. Indices of (A) head length, (B) body height, and (C) caudal peduncle height in

335 generalized, lipped, lipped short, and short morphs sampled in 2009. Median is shown as the

336 horizontal black line inside the box. The box represents 1st and 3rd quartiles of variation.

337 Lowercase letters above the boxplots indicate significant differences between morphs (p < 0.05,

338 Dunn’s test with Holm adjustment of p-value).

339

340 The comparison of barbs sampled in 2009 and 2019 yielded unexpected results. Although

341 the later sample was rather small, significant differences in relative head length and height of

342 caudal peduncle, as well as a nearly significant (p = 0.054) difference in body height, were revealed

343 between GN sampled in 2009 and 2019 (Fig. 6). Remarkably, in only 10 years the GN morph

344 became significantly more similar to the SH morph in all three of the indices most important for

345 their discrimination. No differences in SL and absolute values of the corresponding measurements

346 were found between samples of 2009 and 2019 (Mann-Whitney U test).

347

348 Fig. 6. Indices of (A) head length, (B) body height, and (C) caudal peduncle height of

349 generalized and short morphs sampled in 2009 and 2019. Median is shown as the horizontal black

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350 line inside the box. The box represents 1st and 3rd quartiles of variation. Lowercase letters above

351 the boxplots indicate significant differences between morphs (p < 0.05, Mann-Whitney U test; * -

352 nearly significant, p = 0.054).

353

354 Short vs generalized and lipped morphs: structure of vertebral column and other counts

355 Comparison of vertebral counts between the SH morph and the GN and LP morphs did not

356 reveal any differences in total number of vertebrae or in numbers of vertebrae in the three segments

357 of the vertebral column (trunk, transitional and caudal). Moreover, there were no differences in

358 the numbers of pre-dorsal and pre-anal vertebrae (S9 Table).

359 Distribution of the deformed vertebrae along the vertebral column was not random, and

360 was different in the SH morph compared to the other morphs (Fig. 7). When the data on the

361 positions of the deformed vertebra was merged for individuals with the different total number of

362 vertebrae, the frequency estimates for each specific vertebra in the caudal region were obscured.

363 For this reason, only the data for individuals with 41 total vertebrae are presented in Fig. 7. The

364 respective data for individuals with 40, 42 and 43 vertebrae are shown in S10 Figure.

365

366 Fig. 7. Distribution of deformed vertebrae along the vertebral column in (A) short morph

367 (n = 17), and (B) other morphs (n = 47). Only data for individuals with 41 vertebrae are shown

368 here; the deformities were not analyzed for the first four vertebrae (Weberian apparatus) or the 41st

369 (pre-ural 1) vertebra for technical reasons.

370

371 As shown in Fig. 7A , the lowest frequencies of each specific vertebra deformity in the SH

372 morph were found in 5th vertebra, and also in the caudal region, where the minimum frequencies

373 were found in the 36-40th vertebrae. In the other morphs, however, the deformed vertebrae

374 occurred mostly in the posterior half of the vertebral column, with maximum frequencies in the

375 37-38th vertebrae (Fig. 7B).

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376 No differences were found in the counts of scales, gill rakers and fin rays between the GN

377 and LP morphs, on the one hand, and the SH and lipped SH morphs, on the other hand. No changes

378 in the counts for samples 2009 and 2019 were recorded.

379

380 Short vs generalized and lipped morphs: size, age, growth rate, sex ratio and gonad

381 conditions

382 The range of size variation in the SH morph (SL 107-291 mm) was less than that in any

383 other morph from the middle Genale assemblage in 2009 and 2019. The ranges for the GN and LP

384 morphs were 51-375 mm and 106-459 mm, respectively (S4 Table). Most of the salt-preserved

385 barbs were aged from vertebrae. A maximum age of six years was recorded for the SH and HB

386 morphs, seven years for the SM morph, eight years for the GN morph, nine years for the JB morph,

387 12 years for the LP morph and 14 years for the PS morph. There was no correlation between the

388 percentage of deformed vertebrae and age.

389 Analysis of size variation within each year class of the SH and GN morphs revealed that

390 when SL was used as a measure of linear growth, SH did not differ in size from GN during the

391 first four years (S11 Fig.). Modification of the vertebral column in the SH morph, however,

392 apparently resulted in shortening of their body and respective reduction of SL. Hence, it is

393 reasonable to consider the head length as a measure of linear growth in comparisons of the SH

394 morph with the other morphs. In this case, the SH morph demonstrated a markedly fast growth

395 rate during the first four years, which then leveled off during the fifth and sixth years (Fig. 8).

396

397 Fig. 8. Age vs head length in generalized (GN) and short (SH) morphs in 2009. Median is

398 shown as the horizontal black line inside the box. The box represents 1st and 3rd quartiles of

399 variation. Letters above each boxplot designate significant (red S), or non-significant (black NS)

400 differences between morphs within each year class, based on Mann-Whitney U tests (p < 0.05).

401

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402 Gonads were examined in 30 SH individuals: five juveniles, 11 females and 14 males were

403 recorded. There were 16 fish at maturity stage I, 11 fish at stage II, three fish at stages II-III and

404 III. Among the 46 examined individuals of GN and LP morphs, 16 juveniles, 14 females and 16

405 males were recorded. There were 24 fish at maturity stage I, 20 fish at stage II, one fish at stages

406 II-III and one female at stage IV. Thus, neither sex ratio nor gonad condition differed in the SH

407 morph compared to GN and LP morphs.

408

409 Discussion

410 It is impossible to clearly distinguish the SH morph from the GN and LP morphs using

411 only the plastic characters reflecting their body form, which aligns with Darwin’s observation that

412 “monstrosities … graduate into varieties” [1]. Discrimination is possible, however, by using the

413 number of deformed vertebrae per individual: the GN and LP morphs have ≤ 4 such vertebrae,

414 while the SH morph has ≥ 7 (Table 1). Considering the emergence of the SH morph in the

415 Labeobarbus population predominantly composed of GN and LP morphs, one may suggest that it

416 is determined by some factor(s) acting qualitatively or the threshold reaction of the Labeobarbus

417 individuals to some factor(s) with continuously varying parameters.

418 In regards to Darwin’s idea about the propagation of monstrosities “for a succession of

419 generations in a state of nature” [1], we are motivated to estimate the period of SH existence in a

420 population. Taking into account the absence of SH individuals in our sample from 1998 and their

421 maximum age of six years in the sample from 2009, it is reasonable to suggest that the SH morph

422 appeared in the population between 1999 and 2005. The minimum size of SH individuals in the

423 sample from 2019 (SL = 177 mm) corresponds to an age of 2-3 years according to the SH sample

424 from 2009 (S4 Table). Thus, the SH individuals regularly appeared in the population from 2005

425 (or slightly earlier) until at least 2016. This situation could be explained by the influence of some

426 external factor(s) that abruptly appeared and then became permanent in the environment, or by the

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427 reproduction of SH individuals during the ten year period, which obviously includes the life-span

428 of several generations.

429 A central question has arisen from the discovery of the SH morph: does the emergence and

430 10-year persistence of this morph have a genetic background? Based on mtDNA variation, there

431 is no significant genetic difference between the SH, GN and LP morphs [31], but it is still possible

432 that these differences are present but subtle. In the absence of direct genetic evidence, the factors

433 causing vertebral deformities in fish are considered more generally.

434 Factors responsible for abnormal body shortening in fish

435 There is extensive literature devoted to the question of vertebral deformities in fish. The

436 most comprehensive data available pertains to artificially propagated fish species, especially

437 farmed salmonids. Fish with abnormally shortened bodies displaying multiple compressed and/or

438 fused vertebrae are quite frequent in farmed stocks of Atlantic salmon Salmo salar L. 1758 [11,76-

439 86]. Externally (imaged in [76]), they are similar to the short morph from the Genale Labeobarbus

440 assemblage. These salmons, called ‘short tails’ or ‘short-spined’ individuals [78,81], pose a serious

441 problem for the farming industry because of their reduced commercial value [87]. Therefore,

442 special efforts have been undertaken to elucidate the factors determining the appearance of short

443 tails, however the results are vague, at least in terms of the genetic factors. Initially, results

444 suggested that the deformities were heritable [76,79], but this was questioned later [84,85]. As

445 described by Witten et al. [81, p. 244]: “one cannot rule out the possibility that some fish are

446 genetically predisposed and develop vertebral compression as a reaction to external cues”.

447 The factors that increase the risk of vertebral deformities in farmed Atlantic salmon and

448 other salmonids are given by Witten et al. [11]: bacterial and parasitic infections, vitamin C

449 deficiency, phosphorous deficiency, elevated egg incubation temperature, fast growth in under-

450 yearling smolts, inappropriate light regimes, vaccination, inappropriate water current and quality,

451 as well as environmental pollution. All of these factors (except vaccination), along with radiation

452 [71] may be present in the natural population of the middle Genale Labeobarbus. Notably, there

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453 is a relationship between the increased frequency of vertebral deformities and high growth rate in

454 Atlantic salmon smolts [11,82,85]. We also found an increased growth rate in the SH morph

455 compared to the GN and LP morphs at the age of 2-4 years. Thus, the deformations of the vertebral

456 column in the SH morph could be caused by the accelerated early individual growth and

457 mechanical muscle overload of bones, as suggested for Atlantic salmon [11,82,85]. For the

458 Atlantic salmon post-smolts, a retardation of growth is reported in fish with high numbers of

459 deformed vertebrae [86], however the authors used fork length as the measure of linear growth,

460 which may substantially bias the growth estimates.

461 As for other salmonids, a short tailed brown trout, Salmo trutta L. 1758 was reported from

462 Scotland over a century ago [89]. Much later, >5% of the brown trout from a fish farm in

463 Hampshire (England) exhibited the short tail phenotype and extensive compression of the vertebral

464 column [90]. The author of the study indicated that inbreeding depression was a probable cause of

465 the abnormalities. However, this explanation is hardly applicable to the Genale barbs for two

466 reasons. First, the generalized morph, which is the ancestor of most individuals of the short morph,

467 is the most common morph among the middle Genale barbs, and hence is unlikely to suffer from

468 inbreeding depression. Second, spatial subdivision of the Genale barb populations can occur only

469 in the spawning grounds; in this case, an increase of inbreeding would only result from almost

470 perfect homing, which is highly unlikely in Labeobarbus.

471 In a study of the vertebral fusion patterns in coho salmon, Oncorhynchus kisutch (Walbaum

472 1792) [91], the author demonstrated that the distribution of vertebral fusions along the spinal

473 column differed significantly among crosses from two hatchery stocks, indicating a genetic basis

474 for this character. Recently, differences in the distribution of vertebral fusions along the spinal

475 column were found between the genetically distinct year-classes of Atlantic salmon and its hybrids

476 with brown trout and Arctic char Salvelinus alpinus (L. 1758) [92]. This result is interesting for

477 understanding the difference in distribution of deformed vertebrae along the spinal column

478 between the SH morph and other Genale Labeobarbus (Fig. 7).

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479 In non-salmonid fishes, it has been reported that body shortening from the compression of

480 the vertebral column was apparently heritable in the south German common carp Aischgrunder

481 Karpfen [18], as well as in laboratory strains of the banded topminnow, Fundulus cingulatus

482 Valenciennes [93] and two poeciliids, blackstripe livebearer Poeciliopsis prolifica Miller 1960

483 [94] and guppy Poecilia reticulata Peters 1859 [95]. In zebrafish, Danio rerio (Hamilton 1822),

484 column shortening and vertebral fusions are exhibited by type I collagen mutants, as well as by

485 individuals with knockout alleles in two genes involved in type I collagen processing [96]. At the

486 same time, the increased frequency of vertebral deformities in wild-type zebrafish is induced by

487 high rearing densities [97]. The stumpbody phenotypes with the shortened vertebral column in

488 channel catfish Ictalurus punctatus (Rafinesque 1818) [98] and blue tilapia Oreochromis aureus

489 (Steindachner 1864) [12,99] are described as not heritable.

490 The situation in the Japanese rice fish, Oryzias latipes (Temminck & Schlegel 1846) is

491 especially informative [100-102]. The shortened vertebral column and vertebral fusions are found

492 in (1) fused mutants obtained from the laboratory strain (‘a simple recessive Mendelian character’

493 with expression modified by temperature [100,102]), (2) ‘wild-fused’ phenotype from ‘a certain

494 region on the eastern outskirts of the City of Nagoya’ that were not heritable, and (3) individuals

495 of the normal strain treated by phenylthiourea at the early embryonic stage [101]. In general, it is

496 obvious that in different species and even in different lineages of the same species, the emergence

497 of fish with shortened columns and fused vertebrae may be determined by various external and

498 genetic cues.

499 Considering the possible role of external cues, the following points must be mentioned.

500 First, among the middle Genale Labeobarbus assemblage, the deformation of the vertebral column

501 is found almost exclusively within the isolated genetic pool including the GN, LP, and SH morphs

502 but excluding the trophically specialized morphs. If the deformation has no genetic background

503 and is triggered by external cues such as mechanic, thermal, chemical or radioactive environmental

504 stress, these impacts are only acting selectively. For example, they would have temporal or spatial

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505 effects on only some of the GN and LP barbs, but not the rest of the assemblage that is possible,

506 in our understanding, mostly on the spawning grounds.

507 Second, environmental stress usually results in multiple malformations of different

508 morphological structures [71,103]. We did not observe such multiple morphological abnormalities

509 in SH. Third, external cues like bacterial [104] or parasitic [105,106] infection, and nutrient

510 deficiency (e.g., vitamin C or phosphorous [107]) are usually manifested as a decrease in the

511 individuals’ conditions, especially in the growth rate, which is often retarded in poor health.

512 However, the growth rate was exceptionally high in the abnormal individuals (SH) observed here.

513 Taking into account all of the above, we suggest that genetic factors (possibly together with some

514 environmental ones) are the most parsimonious explanation of the emergence and 10-year

515 persistence of the SH morph in the middle Genale Labeobarbus assemblage.

516 Nevertheless, to definitively prove this suggestion, we must seek answers to the following

517 questions: do SH individuals reproduce in nature? Would be the progeny of artificial crossing be

518 different for the SH breeders vs. normal GN and LP breeders? Are there any genetic/genomic

519 differences between the SH morph and other morphs? Hopefully, these will be addressed in further

520 studies.

521

522 Shortened vertebral column as a target of natural selection

523 Among several streams in the State of Washington, Patten [108] noted that ‘the presence

524 of many severely deformed fish (some of a mature size) shows that some factor or factors permitted

525 their survival’. Möller [109] also stated that ‘anatomical abnormalities … may become particularly

526 evident in populations that do not suffer from a high selection pressure by predators or food

527 competitors’. Essentially these authors are referring to a relaxation of stabilizing selection; an

528 influence of natural selection is often reasonable to expect, but very difficult to prove

529 unambiguously. There are many records on the increased frequency and severity of skeletal

530 abnormalities in artificially reared fish stocks compared to natural populations [110-117].

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531 Interestingly, the opposite situations are also reported [117,118]. Demonstrating how the intensity

532 of rearing conditions plays a role in increasing the frequency and severity of skeletal abnormalities

533 for several fish species [117,119-122] can lend support to the role of selection relaxation, both in

534 cultivated stocks and natural populations exhibiting the mass abnormalities.

535 Some researchers [123-125] have indicated the relaxation of natural selection as an

536 important mechanism in increasing variation of morphological, ecological and other traits in the

537 course of adaptive radiation. Such an increase often allows the radiating species to occupy new

538 adaptive zones. This phenomenon has been called ‘extralimital specialization’ by Myers [126]. In

539 this perspective, the occurrence of the vertebral deformations at a relatively high frequency only

540 in the radiating Labeobarbus assemblage in the middle Genale River may be evidence for relaxed

541 selection in this particular site.

542 Concerning the possibility of directional selection – positive or negative – in shaping the

543 abnormal barbs (SH morph) in the middle Genale River, the following points must be mentioned.

544 First, some retardation of growth in the 5-6 year classes of the abnormal barbs (Fig. 8), along with

545 their relatively short life span (< 7 years), could be treated as evidence for negative selection at

546 later stages of ontogenesis. For Atlantic cod, Gadus morhua L. 1758 from the German Wadden

547 Sea, a few individuals with spinal compression were found in the larger size classes [127]. This

548 reduced frequency in later life stages was explained by negative selection for this phenotype. At

549 the same time, seasonal variation of spinal compression frequencies in the smaller size classes was

550 explained by the migratory activity that differed between the abnormal and normal individuals

551 [127], where the former are considered to be less active.

552 Second, one may expect that the barbs with deformed vertebral columns should exhibit

553 reduction in swimming performance, but this is not straightforward. Studying the triploid lines of

554 Atlantic salmon, Powell et al. [128] did not find a difference in swimming performance between

555 normal fish and those with visible spinal shortening. They did note a poor recovery from

556 exhaustive swimming exercises in the deformed fish compared to the normal fish. Based on this

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557 result, we cannot exclude the possibility of variable swimming performances between the

558 deformed and normal Genale barbs. At the same time, it is clear that the deformed barbs can avoid

559 habitats with fast water current in order to reduce the energy cost of their abnormality.

560 Third, there could be various reasons for the difference in distribution of the deformed

561 vertebrae along the vertebral column between barbs with few and many compressed and/or fused

562 vertebrae (Fig. 7). As described earlier, most of these vertebrae in the individuals with slightly

563 deformed vertebral columns occur in the posterior end of the fish [9,83,86,97,121,129,130]. This

564 phenomenon is explained by the morphogenetic influence of the caudal complex, whose normal

565 development includes several vertebral fusions [130]. The increased frequency of abnormalities in

566 the caudal region was also detected as a result of thyroid hormone disruption [131,132]. However,

567 in the deformed (SH) barbs, the frequency of deformed vertebrae was lower in the pre-caudal

568 region (Fig. 7). This is unlike the farmed stock of Atlantic salmon [86]. In the wild Genale

569 Labeobarbus, this frequency distribution of deformed vertebrae may be caused by the genetic

570 distinctiveness [91,92] of these deformed individuals, or by selection against the individuals with

571 deformed vertebrae in the caudal region, for example because of their potentially decreased

572 swimming capability.

573 The data obtained from individuals with normal vertebral columns allows us to shed light

574 on the susceptibility of column deformations to directional natural selection. First, comparison of

575 the samples from 2009 and 2019 unexpectedly revealed changes in the body form of the GN morph

576 (Fig. 6). The GN individuals became more similar to the SH morph, particularly in the relative

577 body height. At the same time, the SH morph also became more high-bodied than in 2009. The

578 most parsimonious explanation for this phenomenon is a selective pressure for the increase in

579 relative body height in the population studied. Second, in our small sample from the Welemele

580 River (the northern tributary of the Genale; Fig. 2, site no. 3), together with the normal generalized

581 Labeobarbus morph, we found a few individuals with body proportions very similar to that in the

582 SH morph from the middle Genale assemblage. However, their vertebral columns were not

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583 deformed (Fig. 9). This finding might provide evidence for a selective pressure for an increase in

584 the relative body height in another Labeobarbus population.

585 The increased body height is considered as an anti-predator adaptation in the crucian carp

586 Carassius carassius (L. 1758) [133,134]. Indeed, the differentiation among Labeobarbus in the

587 Chamo-Abaya lake basin (Ethiopian Rift valley) corroborates this idea. The lakes are inhabited by

588 the high-bodied barbs classified as L. bynni (Fabricius 1775) [135], while the tributaries are

589 inhabited by the low-bodied barbs classified as L. intermedius (Rüppell 1835) [135]. However,

590 phylogeny based on mt-DNA [30] demonstrates a close relationship between these two forms and

591 their distant relation to L. bynni from the Nile basin. Thus, high-bodied forms evolved

592 independently in the Chamo-Abaya and Nile basins most probably as an anti-predator adaptation,

593 as the faunal data [74,136,137] indicate the greater predator diversity in these basins compared to

594 the Chamo-Abaya tributaries and most other rivers of the Ethiopian Highlands that are populated

595 by the low-bodied Labeobarbus. In our understanding, the importance of the predator-induced

596 high-bodied phenotype is evident in some Labeobarbus populations. In these cases, natural

597 selection could favor the Genale deformed barbs (SH morph) because of their anti-predator high-

598 bodied phenotype.

599

600 Fig. 9. X-ray images of two Labeobarbus from the Welemele River with (A) short-like

601 exterior (SL = 160 mm) and (B) normal exterior (SL = 177 mm). No deformed vertebrae were

602 detected in either individual.

603

604 Shortened vertebral column as a population phenomenon

605 In natural fish populations, the frequency of individuals with few compressed and/or fused

606 vertebrae usually ranges from less than one percent to several percents (usually <10%) [109-112,

607 114,118,138-143], but sometimes it appears substantially higher (up to or over 50%)

608 [115,117,143-145]. Considering these facts, the frequencies of individuals with 1 to 6 compressed

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609 and/or fused vertebrae (ranging from 5% to 15% in the morphs other than SH; Table 1) do not

610 look outstanding. However, among the 34 individuals sampled from the Awata River (Fig. 2, site

611 no. 4), such deformed vertebrae were not found at all. Hence, in the absence of data for other

612 Labeobarbus populations, we may consider the frequency of individuals with deformed vertebrae

613 in the middle Genale populations somewhat increased. Unfortunately, we cannot say with certainty

614 how this is related to an emergence of the SH morph there.

615 There are many reports of individuals with the markedly shortened bodies and deformed

616 vertebral columns in natural populations of different fish groups (S1 Table). Among wild

617 cyprinids, there are reports of a Mesopotamian barb, Mesopotamichthys sharpeyi (Günther 1874),

618 with nine deformed vertebrae, and a yellowfin barbell, Luciobarbus xanthopterus Heckel 1843 ,

619 with 22 deformed vertebrae [146,147]. The situations, as in the Genale Labeobarbus, where the

620 abnormality becomes a population phenomenon occurring with substantial frequency among

621 several successive generations are rare. It is important to note that in the Genale barb assemblage,

622 only two morphs (GN and JB) were always present in catches with frequencies higher than 10%

623 recorded for the abnormal (SH) morph (S7 Table).

624 To the best of our knowledge, apart from the Genale Labeobarbus, the similar situations

625 are described for only two other fish species, Atlantic cod Gadus morhua from the Elbe estuary

626 and German Wadden Sea [127,148-150] and Arctic char Salvelinus alpinus from Transbaikalian

627 Lake Dzhelo [151]. The high prevalence of vertebral column deformities was recently reported in

628 a wild population of lumpfish Cyclopterus lumpus L. 1758 from Masfjorden, Norway [117], but

629 changes of the body form and temporal stability of the abnormal phenotypes are not evident in the

630 latter species.

631 In 1971, Wunder [148] reported the high prevalence (10-15%, sometimes up to 20%) of

632 cod individuals with shortened bodies and deformed vertebrae in catches from the Elbe estuary

633 and adjacent regions of the Northern Sea. In each deformed individual, 40-80% of vertebrae were

634 compressed and/or fused. This abnormality was most frequent in the young cods (length < 20 cm).

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635 The situation persisted at least until the late 1980s [127,149,150]. The geographical range of this

636 cod deformity expanded to some regions of the Wadden Sea; the mean frequency of the deformed

637 individuals in the catches of all inshore regions of the German Wadden Sea was 9.2% in the size

638 class of 11-23 cm [127].

639 Dzhelo is a small lake (1.4×0.3 km, maximum depth 25 m) in the upper reaches of the Lena

640 River system in Transbaikalia. According to Alekseyev [151], the lake is inhabited by dwarf char

641 (maximum length 24 cm). There are numerous deformed individuals with the shortened body,

642 which also show multiple fusions of vertebrae. Their prevalence is 12%, and a change of the body

643 form in the individuals with ≤ 6 deformed vertebrae is not noticeable. The lake was sampled once

644 in 2003 [151].

645 The main question about these deformities in cod and char is whether they still persist with

646 the substantial frequencies. Comparative etiology of vertebral deformities in cyprinid, salmonid

647 and gadid species could be of great interest. Moreover, deeper understanding of the action of

648 natural selection on these abnormalities can shed the light on how morphological novelties are

649 established in a population, especially if the abnormalities in question have the genetic

650 background.

651

652 Conclusions

653 The striking emergence of individuals with deformed (shortened) vertebral columns

654 resulting in a high-bodied phenotype is a phenomenon rarely detected among wild fish

655 populations. For the particular case of the radiating Labeobarbus assemblage from the middle

656 Genale River, the following circumstances must be highlighted: i) the recent emergence (~ 15

657 years ago) of the high-bodied phenotype with the shortened vertebral column, ii) the persistence

658 of this phenotype with a substantial frequency (~ 10%) in several generations (>10 years) in a

659 population, iii) the fact that this is the only type of morphological deformity; virtually all other

660 morphological abnormalities are absent, iv) the presence of the deformity in the isolated gene pool

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661 within the radiating Labeobarbus assemblage, and v) the rapid growth of the abnormal individuals

662 at 2-4 years of life compared to other sympatric morphs. Taking into account all the above, as well

663 as the evidence for a genetic contribution to such abnormality in some other fish species,

664 particularly the common carp, we assume that this phenomenon in this Labeobarbus population

665 most likely has a genetic background. Moreover, the high-bodied phenotype in Labeobarbus may

666 have a selective advantage as an anti-predator defense.

667

668

669 Acknowledgments

670

671 Material for this study was collected within the scope of the Joint Ethiopian-Russian Biological

672 Expedition (JERBE). We gratefully acknowledge the JERBE coordinators A.A. Darkov

673 (Severtsov Institute of Ecology and Evolution, Moscow, Russia - IEE) and Simenew Keskes

674 Melaku (Ministry of Innovation & Technology, Addis Ababa, Ethiopia) for administrative

675 assistance. We express our gratitude to S.E. Cherenkov and Yu.Yu. Dgebuadze (both from IEE),

676 Fekadu Tefera and Genanaw Tesfaye (both from National Fishery and Other Aquatic Life

677 Research Center of the Ethiopian Institute of Agricultural Research - EIAR, Sebeta), M.V. Mina

678 (Institute of Developmental Biology, Moscow, Russia) for sharing field operations and assistance

679 in collecting material. We thank S.E. Cherenkov for his help with photography, F.N. Skil (IEE)

680 for aging materials from experimentally reared barbs, O.N. Artaev for creating the map, M.V.

681 Mina for critical comments on the manuscript, and Jacquelin DeFaveri for linguistic corrections.

682 This work was financially supported by the Russian Science Foundation, project no. 19-14-00218.

683

684

685

686

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687 References

688 1. Darwin C. On the origin of species by means of natural selection, or the preservation of

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1148 isser.pdf

1149

1150

1151

1152

1153

1154

1155

1156

1157

1158

1159

1160

1161 Supporting information

1162

1163 S1 Table. Occurrence of aberrant individuals or stocks with shortened vertebral column and

1164 abnormally short and high body in the different groups of fish.

1165 (DOCX)

1166 S2 Table. Complete list of sampling sites.

46 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1167 (XLSL)

1168 S3 Figure. Photographs of two habitats at the main sampling (no. 1) of the middle Genale River

1169 – (A) the continuous pool and (B) the upper section of rapids downstream of the pool.

1170 (DOCX)

1171 S4 Table. Size characteristics of the cast and gill net catches for different Labeobarbus forms

1172 from the middle Genale assemblage sampled in 2009 and 2019. Size characteristics of the aged

1173 individuals from the gill net catches of 2019.

1174 (DOCX)

1175 S5 Figure. Variation in lower lip development in short individuals.

1176 (DOCX)

1177 S6 Figure. Two piscivorous barbs with (A) normal and (B) deformed vertebral column.

1178 (DOCX)

1179 S7 Table. Gill net catch composition and percentage of different Labeobarbus morphs in the

1180 middle Genale assemblage in 2009 and 2019.

1181 (DOCX)

1182 S8 Table. Eigenvectors of the 10 most loaded characters from Fig. 4.

1183 (DOCX)

1184 S9 Table. Data on vertebral counts.

1185 (DOCX)

1186 S10 Figure. Supplement to Fig. 7. Distribution of deformed vertebrae along the vertebral column

1187 in individuals with 40 and 42 total vertebrae.

1188 (DOCX)

1189 S11 Figure. Growth of short (SH), generalized (GN) and lipped (LP) forms in the middle Genale

1190 Labeobarbus assemblage sampled in 2009, estimated with standard length (SL).

1191 (DOCX)

1192

47 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.11.292763; this version posted September 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.