Evolution in Sinocyclocheilus cavefish is marked by rate shifts, reversals and origin of novel traits

Epitomized by the well-studied Astyanax mexicanus, cavefishes provide important model organisms to understand adaptations in response to divergent natural selection. However, the spectacular Sinocyclocheilus diversification of China, the most diverse cavefish clade in the world harboring nearly 75 species, demonstrate evolutionary convergence for many traits, yet remain poorly understood in terms of their morphological evolution. Here, using a broad sample of 49 species representative of this diversification, we analyze patterns of Sinocylocheilus evolution in a phylogenetic context. We categorized species into morphs based on eye-related condition: Blind, Micro-eyed (small-eyed), and Normal-eyed and we also considered three habitat types (Troglodytic – cave-restricted; Troglophilic – cave-associated; Surface – outside of caves). Geometric morphometric analyses show Normal-eyed morphs with fusiform shapes being segregated from Blind/Micro-eyed (Eye-regressed) morphs with deeper bodies along the first principal component (“PC”) axis. The second PC axis accounts for shape complexity related to the presence of horns. Ancestral character reconstructions of morphs suggest at least three independent origins of Blind morphs, each with different levels of modification in relation to the typical morphology of ancestral Normal-eyed morphs. Interestingly, only some Blind or Micro-eyed morphs bear horns and they are restricted to a single clade (Clade B) and arising from a Troglodytic ancestral species. Our geophylogeny shows an east-to-west diversification spanning the Pliocene and the Pleistocene, with Troglodytic species dominating karstic subterranean habitats of the plains whereas predominantly Surface species inhabit streams and pools in hills to the west (perhaps due to the scarcity of caves). Integration of morphology, phylogeny and geography suggests Sinocyclocheilus are pre-adapted for cave dwelling. Analyses of evolutionary rates suggest that lineages leading to Blind morphs were characterized by significant rate shifts, such as a slowdown in body size evolution and a 3.3 to 12.5 fold increase in the evolutionary rate of eye regression. Furthermore, body size and eye size have undergone reversals, but horns have not, a trait that seem to require substantial evolutionary time to form. These results, compared to the Astyanax model system, indicate Sinocyclocheilus fishes demonstrate extraordinary morphological diversity and variation, offering an invaluable model system to explore evolutionary novelty.

2 harboring nearly 75 species, demonstrate evolutionary convergence for many traits, yet remain 24 poorly understood in terms of their morphological evolution. Here, using a broad sample of 49 25 species representative of this diversification, we analyze patterns of Sinocylocheilus evolution in 26 a phylogenetic context. We categorized species into morphs based on eye-related condition: Blind,27 Micro-eyed (small-eyed), and Normal-eyed and we also considered three habitat types 28 (Troglodytic -cave-restricted; Troglophilic -cave-associated; Surface -outside of caves). 29 Geometric morphometric analyses show Normal-eyed morphs with fusiform shapes being 30 segregated from Blind/Micro-eyed (Eye-regressed) morphs with deeper bodies along the first 31 principal component ("PC") axis. The second PC axis accounts for shape complexity related to the 32 presence of horns. Ancestral character reconstructions of morphs suggest at least three independent 33 origins of Blind morphs, each with different levels of modification in relation to the typical 34 morphology of ancestral Normal-eyed morphs. Interestingly, only some Blind or Micro-eyed 35 morphs bear horns and they are restricted to a single clade (Clade B) and arising from a Troglodytic 36 ancestral species. Our geophylogeny shows an east-to-west diversification spanning the Pliocene 37 and the Pleistocene, with Troglodytic species dominating karstic subterranean habitats of the plains 38 whereas predominantly Surface species inhabit streams and pools in hills to the west (perhaps due 39 The specialized traits cavefish bear have led them to be developed as models of evolution 69 especially with respect to adaptations to novel environments and evolutionary convergence 70 (Culver et al., 1995;Dowling et al., 2002;Jeffery, 2001;Li et al., 2008;Strecker et al., 2004;71 Yang et al., 2016). A lion's share of knowledge on evolution and development in cavefishes has 72 come from Astyanax mexicanus (Mexican tetra), a species with both surface-dwelling (pigmented 73 and eyed) and cave-dwelling morphs (depigmented and blind), which can readily interbreed 74 (Borowsky, 2008). In contrast to this well-studied model system, Sinocyclocheilus species not only 75 include blind and normal-eyed morphs (Lan et al., 2013), but demonstrate a continuum from blind 76 to normal-eyed species. Indeed, members of the Sinocyclocheilus genus display remarkable 77 morphological evolution with divergent cave-dwelling, cave-associated, and surface-dwelling 78

species. 79
Sinocyclocheilus species are thought to have shared a common ancestor in the late Miocene, 80 undergoing a spectacular diversification spanning the Pliocene and Pleistocene across the 81 southwestern parts of China's 620,000 km 2 of karst habitats (Huang et al., 2008), with nearly 75 82 extant species (Jiang et al., 2019). This resulted in an adaptive diversification into subterranean 83 refugia traversing the intersection of the Guizhou, Guangxi and Yunnan provinces around the time 84 of the uplifting of Tibetan/Guizhou plateau (Li et al., 2008). 85 One of the most striking forms of cave adaptation in Sinocyclocheilus is variation in eye 86 morphology, categorized often into three morphs (Zhao and Zhang, 2009)

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The maximum credibility tree of Sinocyclocheilus is shown in Fig. 2  Normal-eyed species, with two cases of reversal from either Micro-eyed or Blind to Normal-eyed 219 morphs, namely S. zhenfengensis and S. brevibarbatus (Fig. 2). All four clades contain Regressed-220 eyed species and comparatively, clade Blind contains the most cases. Interestingly, clade B 221 originated around the time of the beginning of the aridification process in China in the late Pliocene, 222 whereas the other two transitions to blind species were much more recent (Fig. 2). 223 Interestingly, the evolution of body size and eye diameter seem to have often involved 224 reversals, with little correspondence between body size (Fig. 3A) or eye diameter (Fig. 3B) and 225 their corresponding morphs, except for the case of blind species for which eye diameter is 226 necessarily zero. On the other hand, morphs are clearly distinguished when eye diameter and body 227 11 size are visualized simultaneously (Fig. 3C), which suggests that the evolution of different morphs 228 is achieved by altering the relationships between body size and eye diameter. Habitat associations 229 traced on the phylomorphospace (Fig. 3D) indicates that species with regressed eyes and small-to-230 medium body sizes are obligate cave dwellers. However, normal eyed species can be Troglodytic, 231 Troglophilic or surface dwellers regardless of their body size. Interestingly, all horned species are 232 obligate cave dwellers while all cave species are not horned (Fig. 3E). 233 A more precise description of the overall changes associated with different morphs can be 234 visualized in the projections build from the geometric morphometrics analyses (Fig. 1B). The first 235 PC, which accounted for approximately 32% of the variance in the dataset (see Table S3), tended 236 to distinguish the slender Normal-eyed species on the left and Micro/Blind species on the right, 237 which were characterized by changes in shape and widening of the anterior dorsal area between 238 mouth and beginning of the dorsal fin of their body, resulting in a shift from the fusiform shape of 239 the Normal-eyed forms to a more "boxy" form of the Micro-eyed and Blind forms. The second 240 PC, which explained approximately 17% of the variance in the dataset, emphasized the differences 241 in the type of dorsoventral broadening of the mid-section between morphs, with a shortening of 242 the tail region (Fig. 1B). The variation in this axis is very high among the Micro-eyed and Blind 243 forms when compared to the Normal morphs. 244 The multiple-rate model of evolution provided the best fit to the data for all three quantitative 245 traits (ΔAIC=2.7, 10.7 and 7.9 respectively for ED, sED and SL; Table 1), indicating that the 246 evolution of different Sinocyclocheilus morphs was associated with significant changes in their 247 evolutionary rates. However, there were intriguing differences between traits in their rates (Table  248 2). The rates of evolution of eye diameter and standardized eye diameter were similar between 249 12 Normal-eyed and Micro-Eyed species, but increased between 3.3 to 12.56 times during shifts 250 towards Blind species. 251 Geophylogeny represents the phylogeny overlaid across the geographic location of each 252 species, where phylogenetic clustering is evident across the landscape. Considering the distribution 253 of Sinocyclocheilus, we mainly see a pattern where the basal, Normal-eyed morphs are placed in 254 the east, a substantial portion of Blind/Micro-eyed (Regressed-eyed) species are in the center, and 255 Normal-eyed morphs are predominant towards the western mountains (Fig. 4). 256

Habitat utilization in context of eye-morphology 259
Integrating evolution of eye size and habitat manifests interesting and previously 260 unrecognized evolutionary patterns in the evolution of Sinocyclocheilus. The Eye-size based 261 ancestral reconstruction suggests the base of the phylogeny is most likely an Eyed species (i.e. 262 Normal-or Micro-), but habitat reconstructions places, with high probability, Troglodytic species 263 at the base (Fig. S1). This suggests an ancestral Eyed-species evolved a Troglodytic habit before 264 they became blind. This may be an example of preadaptation in Sinocyclocheilus, i.e., the 265 advancement of a functional change with little or no evolutionary modification (Ardila, 2016). In 266 Astyanax cavefish, surface-dwelling forms are scotophilic, they prefer to remain away from direct 267 light suggesting that scotophilia may be preadaptive for colonizing the dark, cave environment 268 (Espinasa et al., 2001). In Sinocyclocheilus, since a basal (eyed) species demonstrated preference 269 for the cave habitat, this preadaptation to darkness may hint towards why certain species tend to 270 become cave-dwellers while others do not. This pattern is supported by two principal lines of 271 evidence. First, most of the basal species are eyed, and Troglodytic (except for one species with 272 13 an unusual eye-related polymorphic condition that we discuss below). Second, the most westward 273 group (Clade D; Normal-eyed Surface fish), re-colonized caves whenever cave habitats were 274 available within that area, suggesting a strong predisposition for cave-dwelling across all 275 Sinocyclocheilus. In other words, when caves were present, members of Sinocylocheilus, 276 irrespective of eye-related condition, preferred the cave habitat. 277 The preference for caves may not be a preference for darkness, but in fact a preference for 278 depth, in search of water for survival. In a karstic environment where drying of surface running 279 water is common, a preference for such deeper habitats may have provided an evolutionary 280 advantage. In the presence of an array of subterranean waterways, such a predisposition would 281 have given rise to the eye-regressed forms living close to, or associated with, caves that are 282 characteristic of the genus. 283 Furthermore, apart from the Troglodytic and Troglophilic species of Clade D, some of the 284 putative Surface species of Clade D are often observed at the entrances of caves or at windows to 285 subterranean rivers (Zhao and Zhang, 2009). Hence, with more intensive ecological studies, some 286 species recognized as Surface species may indeed be Troglophilic species, bolstering the notion 287 that Sinocyclocheilus are predisposed to seek deeper waters of the karstic caves. 288 Resource utilization plays a key survival role in harsh environments (Culver and Pipan, 2009). 289 Some of the Troglophilic, eyed-species are nocturnal, emerging from submerged caves, 290 presumably to feed at night to reduce competition from other non-cave inhabiting fish species 291 (personal observations). Some species like S. altishoulderus, S. donglanensis ), 292 S. bamaensis (Su et al., 2003, S. malacopterus (Chen et al., 2017) and S. longibarbatus (personal 293 observation, video evidence as Supplementary information); are known to come out of caves 294 during the high water season, presumably to feed and breed. This explains dependence on the cave 295 14 as a diurnal refugium, from where these species can exploit the surface habitats at night. Strategies 296 such as this, where multiple resources are utilized simultaneously, points to the adaptability of 297 some Sinocyclocheilus species, resulting in their evolutionary success in a harsh and changing 298 environment. Hence, the cave entrances are possibly an important ecotone that is important in 299 Sinocyclochelius diversification and conservation. 300 Season and time of day seem to be important factors in determining habitat utilization patterns, 301 but this level of resolution in habitat data is not currently available for a majority of the species to 302 carry out a comprehensive habitat analysis across the diversification -indeed, many species are 303 known only from one or a few specimens (Zhao and Zhang, 2009). 304 305

Adaptations in the light of geophylogeny 306
In combination with the data analyzed, basal Sinocyclocheilus (Clade A) are Normal-Eyed, 307 predominantly cave dwelling and non-horned species from the Eastern region of their distribution. 308 As pointed out, this suggests that the earliest ancestors of Sinocyclocheilus species where Normal-309 eyed and but still lived in close association with caves. The ancestral reconstructions suggest that 310 the affinity to caves would have evolved early and is present in most Sinocyclocheilus. The clade 311 comprising basal species are from the east of the Sinocyclocheilus distribution, i.e. the He Jiang 312 and Gui Jiang river basin in northeastern Guangxi, and hence, it seems that the diversification of 313 these fish occurred from East to West (Fig. 4). 314 Within this predominantly Normal-eyed clade (Clade A), there is an exception, 315 Sinocyclocheilus guanyangensis, a species that we coded as Micro-eyed, has Normal-Eyed, Micro-316 eyed and effectively Blind morphs within the same population -polymorphic for this trait. But 317 these blind morphs have their eyes completely covered by skin and the Micro-eye is not itself 318 degenerate. This kind of condition has been noted in several other taxa also (S. xunlensis and S. 319 flexuodorsalis -not available for our analysis), but is uncommon. This suggests a degree of 320 polymorphism for this trait, suggesting that the earliest ancestors of Sinocyclocheilus may have 321 been able to lose or gain eyes relatively easily as an adaptation to local conditions, this ability 322 appears several times within this cave-driven diversification. 323 The major adaptation for cave dwelling evolves predominantly in the expansive karstic area 324 in northwestern Guangxi (associated with the Liu Jiang basin and Hongshui river basin joining the 325 main Xijiang River system from the North), in Clade B, the southeastern corner of Guizhou 326 province (upper reaches of Hongshui River) and the northeastern plateau of Yunnan province. This 327 region can be considered the center for novel adaptations for Sinocyclocheilus, where these fishes 328 express their full morphological diversity, blindness, micro-eyedness, and their remarkable horns. 329 In the shape-related analyses, these species cluster on the right of morphospace (Fig. 1B). The 330 deeper caves and extensive subterranean river system associated with the Guangxi plains (Zhao 331 and Zhang, 2009) would have facilitated this extensive adaptive diversification (Fig. 4). 332 The karstic northwestern region that the Guangxi-dominated Clade (Clade B) experiences 333 drought conditions during much of the year, and one of the major sources of rain for the region is 334 through storms sweeping from the southeast that are strong enough to persist through the vast 335 plains of Guangxi, mainly from April to August (Zhao and Zhan, 2009). During unfavorable 336 periods, these fishes seem to have found refuge in the subterranean caves. The morphologically 337 most diverse clade being present in the region where the climatic conditions are most unfavorable 338 for surface fish, reinforces the notion that Sinocyclocheilus species adapted to life in caves as 339 climatic refuges (Zhao and Zhang, 2009).

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The distribution of Clade C, characterized by mostly Normal-eyed but Troglodytic species 341 largely overlaps Clade B. In Clade C, the single Blind species (S. xunlensis) and the two Micro-342 Eyed species (S. cyphotergous and S. multipunctatus) are shown within a narrow geographic area 343 on the Liu Jiang and Hongshui river system (Fig. 4). 344 Species in the clade that is found in the west (Clade D), predominantly on the hilly terrain of 345 Yunnan plateau, are predominantly Normal-eyed Surface species lacking horns (Fig. 1, Fig. 4). 346 However, wherever there are cave habitats and subterranean river systems, some of these putative 347 surface species have become facultative or obligate cave species. The obligate cave species found 348 within the region, S. anophthalmus, is blind. However, this Blind species stands clustered with the 349 Normal-eyed morphs in the morphospace, signifying that the shape of the species has not 350 extensively changed, possibly due to recent (Pleistocene) invasion of the cave habitat from a 351 Normal-eyed ancestor (Fig. 2) -time since becoming blind is not been long enough for change 352 into the box-like shape of the Blind species of Clade B. 353 Horn distributions show several peculiar trends. In most Sinocyclocheilus species, a prominent 354 hump is present (He et al., 2013). However, this hump is markedly low in the Normal-eyed surface 355 inhabiting species of the Yunnan clade (Fig. 4). For the species that bears a horn, the horn 356 represents the region in which the dorso-frontal hump is present, and always occurs before the 357 hump begins, at the boundary of the edge of the dorsal skull. The exception to this is S. 358 cyphotergous (Clade C), where a horn like structure is placed on top of the hump. S. cyphotergous, 359 a species found in clade C, is phylogenetically separate from other horned species, suggesting that 360 the origins of the "horn" for this species is evolutionarily different from the other horned species 361 (Fig. S2). Though the function of the horn remains unknown (protection of head, anchoring in 362 strong current and protection of head has been suggested; Zhao and Zhang, 2009), functionally 363 this structure may be similar to other species, if it is actually anchoring in strong current is the 364 main function. 365 The rates of evolution of various traits show some incongruent (non-allometric), but 366 interesting patterns that can be explained in the context to adaptations to a Troglodytic condition. 367 The rates of evolution in eye diameter are similar between Normal-and Micro-Eyed species but, 368 increases dramatically (3.3-12.56 times) with shifts towards blind forms. However, body size 369 evolution for these morpho groups shows a reversed pattern, with a 0.03 decrease in body size 370 evolution in the Blind morphs compared to the eyed-morphs. These patterns in rate variation 371 suggest that the evolution of Blind morphs to a Troglodytic habitat were simultaneously associated 372 with an increase in the rate of evolution of the eye-size degeneration and a decrease in the rate of 373 body size evolution. The smaller body size resulting from a sluggish rate of change will facilitate 374 both navigation within constricted spaces and sustenance on a limited supply of resources expected 375 to be experienced in subterranean habitats. 376 Much of our collective knowledge of the patterns and mechanisms of regressive evolution 377 come from studies of animals that have colonized the subterranean biome. Within this group, a 378 several studies have focused on the Mexican tetra, Astyanax mexicanus (Jeffery, 2009). This 379 natural animal model system comprises multiple cave-adapted morphs and a surface-dwelling 380 morph that resides in near the caves themselves (Gross, 2012). Since the discovery of Astyanax 381 cavefish in 1936, countless studies have provided insight to the developmental and genetic bases 382 for cave-associated traits (Hubbs and Innes, 1936). Indeed, much of this insight has emerged from 383 the interbreeding studies of conspecific cave and surface morphs (reviewed in Wilkens, 2016). 384 However, several aspects of regressive evolution and troglomorphic adaptation remain unresolved. 385 Owing to several of the differences with Astyanax, we argue that Sinocyclocheilus is well- Sinocyclocheilus species allows keener resolution for understanding broad phylogenetic processes, 396 such as trait reversals and directions of diversification. Although a reversal from an eyeless to an 397 eyed form has been reported for one cave population in Astyanax (Caballo Moro; Krishnan and 398 Rohner, 2017), this phenomenon appears to be much less common than in Sinocyclocheilus. 399 Additionally, a clear polarity of diversification is lacking in Astyanax cavefish, rather ancient 400 stocks of surface-dwelling forms appear to have recurrently invaded caves to the east (i.e., Sierra 401 de El Abra caves), with more recent invasions having occurred in the northern (Sierra de Colmena) 402 and the western caves (Sierra de Guatemala; Bradic et al., 2012). However, the well-characterized 403 gene flow between the cave and surface waters obscures the ability to understand clear boundaries 404 between different cave groups. Further, most Astyanax cave populations are believed to have 405 diverged over the course of the last ~200 -500 Ky (Herman et al., 2018). By contrast, 406 Sinocyclocheilus species are much older, and therefore one can determine how longer-term 407 processes unfold in these cave-dwelling animals. Thus, despite clear phylogenetic differences 408 between Astyanax and Sinocyclocheilus, both genera have the ability to provide complementary 409 and critical insights to the processes underlying cave evolution and diversification. 410 The integration of morphology, phylogeny, rate analyses, dating and distribution show not 411 only several remarkable patterns of evolution, but also interesting exceptions to these patterns that 412 signifies the diversification of Sinocyclocheilus as a unique model system to study evolutionary 413 novelty.