bioRxiv preprint doi: https://doi.org/10.1101/2020.09.09.288886; this version posted September 9, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC 4.0 International license. 1 From scales to armour: scale losses and trunk bony plate gains in ray- 2 finned fishes 3 Alexandre Lemopoulos1, Juan I. Montoya-Burgos1,2,3 4 1. Department of Genetics and Evolution. University of Geneva, Geneva, Switzerland 5 2. iGE3 institute of Genetics and Genomics of Geneva 6 3. E-mail: [email protected] ORCID: https://orcid.org/0000-0001-9080-9820 7 Article type : Letter 8 Running title: Tegument cover transitions along actinopterygian evolution 9 Keywords: Tegument, actinopterygians, gene network, skeleton evolution, functional 10 innovation, ancestral state, phylogeny 11 12 Abstract 13 Actinopterygians (ray-finned fishes) are the most diversified group of vertebrates and are 14 characterized by a variety of protective structures covering their tegument, the evolution of 15 which has intrigued biologists for decades. Paleontological records showed that the first 16 mineralized vertebrate skeleton was composed of dermal bony plates covering the body, 17 including odontogenic and skeletogenic components. Later in evolution, the exoskeleton of 18 actinopterygian's trunk was composed of scale structures. Although scales are nowadays a 19 widespread tegument cover, some contemporary lineages do not have scales but bony 20 plates covering their trunk, whereas other lineages are devoid of any such structures. To 21 understand the evolution of the tegument coverage and particularly the transition between 22 different structures, we investigated the pattern of scale loss events along actinopterygian 23 evolution and addressed the functional relationship between the scaleless phenotype and 24 the ecology of fishes. Furthermore, we examined whether the emergence of trunk bony 25 plates was dependent over the presence or absence of scales. To this aim, we used two 26 recently published actinopterygian phylogenies, one including > 11,000 species, and by using 27 stochastic mapping and Bayesian methods, we inferred scale loss events and trunk bony 28 plate acquisitions. Our results reveal that a scaled tegument is the most frequent state in 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.09.288886; this version posted September 9, 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-NC 4.0 International license. 29 actinopterygians, but multiple independent scale loss events occurred along their phylogeny 30 with essentially no scale re-acquisition. Based on linear mixed models, we found evidence 31 supporting that after a scale loss event, fishes tend to change their ecology and adopt a 32 benthic lifestyle. Furthermore, we show that trunk bony plates appeared independently 33 multiple times along the phylogeny. By using fitted likelihood models for character 34 evolution, we show that trunk bony plate acquisitions were dependent over a previous scale 35 loss event. Overall, our findings support the hypothesis that tegument cover is a key 36 evolutionary trait underlying actinopterygian radiation. 37 38 Impact Summary 39 Ray-finned fishes (actinopterygians) are the most diverse vertebrate group in the world. The 40 majority of these fishes possess scales as a protective shield covering their trunk. However, 41 several lineages display a body armour composed of trunk bony plates or are devoid of any 42 protective structures. The diversity and the transitions between different tegument 43 coverage types have not been previously studied in an evolutionary framework. Here, we 44 investigate which structure was present at the origin of ray-finned fishes and how the 45 different phenotypes emerged through time. 46 We show that a scaled tegument was the most widespread sate along ray-finned fish 47 evolution, yet scale losses occurred multiple independent times, while acquiring scales again 48 almost never happened. Moreover, we reveal that scaleless teguments most probably led 49 species to change their ecology and colonise the floors of oceans and water bodies. The 50 functional advantages of a scaleless tegument in a benthic environment are yet to be 51 demonstrated, but the increased cutaneous respiration could be an explanation. We show 52 that trunk bony plates also emerged independently multiple times along the evolution of 53 ray-finned fishes but these armours protecting the trunk can only appear after a scale loss 54 event. Therefore, while the acquisitions of trunk bony plates are phylogenetically 55 independent, they need a “common ground” to emerge. All together, our findings provide 56 evidence that the various tegument covers have contributed to the outstanding 57 diversification of ray-finned fishes. 58 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.09.288886; this version posted September 9, 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-NC 4.0 International license. 59 Introduction 60 Ray-finned fishes (Actinopterygii) represent the most diversified vertebrate lineage in the 61 world, with more than 33 thousand described species (Alfaro 2018). One of the most 62 prominent features among Actinopterygii representatives is the presence of scales in their 63 trunk tegument forming a protective layer. Scales can display various shapes and structures, 64 as they can contain different compounds and differ in histological characteristics (Moyle and 65 Cech 2004). The diversity of scales has created some confusion in the scientific community, 66 because different skeletal elements have been referred as scales despite being of different 67 origin (Schultze 2018). Yet, given the great diversity and the complexity of these structures, a 68 consensus over their nomenclature and classification still needs to be established based on a 69 comprehensive understanding of their evolutionary origin (Sire et al. 2009; Vickaryous and 70 Sire 2009). In this study, we primarily focus on two categories of mineralized structures 71 developing within the tegument of actinopterygians, micromeric scales and macromeric 72 trunk bony plates. 73 Scales, as differentiated micromeric dermal skeletal elements (sensu Sire 2003 and Sire et al. 74 2009) were present in the ancestral lineage that gave rise to Actinopterygii and Sarcopterygii 75 (Sire et al. 2009). Therefore, scales are considered a plesiomorphic trait for ray-finned fishes 76 and today the majority of them possess some type of scales (Gemballa and Bartsch 2002; 77 Sire et al. 2009). Based on different histological and morphological properties, scales have 78 been classified in two main groups: ganoid scales (in Protopteridae (bichirs) and 79 Lepisosteiformes (gars) [Meunier and Brito 2004; Ichiro et al. 2013]) and elasmoid scales (in 80 the majority of actinopterygian lineages; e.g. Sire et al., 1997; Mongera and Nüsslein- 81 Volhard, 2013). All scales possess a bony layer (e.g. bony-ridge, lammellar bone) in their 82 structure (Benthon 2004; Moyle and Cech 2004; Zhu et al. 2012). Thus, scales are a bony 83 structure covered with a scale-specific odontogenic-like tissue, in general. The nature of the 84 odontogenic-like cover and the scale organization then define the type of scale (e.g. ganoin 85 in ganoid scales; Ichiro et al. 2013). Therefore, two components are in general necessary for 86 the formation of a scale: a) a bone micromeric structure; and b) an odontogenic-like cover 87 tissue that is scale-specific (but this tissue is sometimes reduced or even absent). 88 Trunk bony plates (TBP) represent another type of tegument protection, which is present in 89 some extant actinopterygians. The origin of TBP can be traced back to the first vertebrate 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.09.288886; this version posted September 9, 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-NC 4.0 International license. 90 mineralized skeleton, which was composed of TBP covered with an odontogenic tissue 91 (Keating and Donoghue 2016). Independently of their evolutionary history, TBP sensu lato, 92 can be differentiated from scales as macromeric tegumental elements composed of bone 93 only (i.e. lacking the odontogenic-like cover). TBP, as macromeric tegument structures, 94 reappeared in specific actinoptetygian lineages. For instance, the iconic seahorse 95 (Syngnathidae) exoskeleton is made of dermal bony plates covering the entire body (Lees et 96 al., 2012; Porter et al., 2013). Other examples are the Callichthyidae and the Loricariidae, 97 two species-rich families of Neotropical catfishes, that have their trunks covered with TBP 98 (Sire 1993; Covain et al. 2016; Rivera-Rivera and Montoya-Burgos 2017). Interestingly, 99 micromeric scales and macromeric TBP seem to be mutually exclusive as no extant fish 100 displays both exoskeletal structures in the trunk. 101 Despite the widespread occurrence of protective elements in the