bioRxiv preprint doi: https://doi.org/10.1101/2020.08.03.232421; this version posted August 3, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license. 1 Genomics of a killifish from the Seychelles islands supports transoceanic island colonization and reveals 2 relaxed selection of developmental genes 3 4 Rongfeng Cui1, Alexandra M Tyers1, Zahabiya Juzar Malubhoy1, Sadie Wisotsky2, Stefano Valdesalici3, Elvina 5 Henriette4, Sergei L Kosakovsky Pond2, Dario Riccardo Valenzano*1,5 6 1 Max Planck Institute for Biology of Ageing, Cologne, Germany 7 2 Department of Biology, Institute for Genomics and Evolutionary Medicine, Temple University, Temple, U.S.A. 8 3 Associazione Italiana Killifish, Italy 9 4 Island Biodiversity Conservation centre, University of Seychelles, Anse Royale, Mahe, Seychelles 10 5 University of Cologne, Cologne, Germany 11 12 *corresponding author: [email protected] 13 14 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.03.232421; this version posted August 3, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-ND 4.0 International license. 15 Abstract 16 How freshwater fish colonize remote islands remains an evolutionary puzzle. Tectonic drift and trans-oceanic 17 dispersal models have been proposed as possible alternative mechanisms. Integrating dating of known tectonic events 18 with population genetics and experimental test of salinity tolerance in the Seychelles islands golden panchax 19 (Pachypanchax playfairii), we found support for trans-oceanic dispersal being the most likely scenario. At the 20 macroevolutionary scale, the non-annual killifish golden panchax shows stronger genome-wide purifying selection 21 compared to annual killifishes from continental Africa. Reconstructing past demographies in isolated golden panchax 22 populations provides support for decline in effective population size, which could have allowed slightly deleterious 23 mutations to segregate in the population. Unlike annual killifishes, where relaxed selection preferentially targets 24 aging-related genes, relaxation of purifying selection in golden panchax affects genes involved in developmental 25 processes, including fgf10. 26 27 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.03.232421; this version posted August 3, 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-ND 4.0 International license. 28 Introduction 29 Taxa with limited dispersal capabilities, such as flightless mammals, amphibians and freshwater fishes are thought to 30 speciate largely through vicariance, i.e. population isolation caused by the presence of physical barriers, such as rivers 31 and mountain ranges (Wiley 1988). Whether species with limited dispersal capacity, such as freshwater fishes, 32 colonize remote islands through tectonic drift or trans-oceanic events is still debated (Briggs 2003; Sparks and Smith 33 2005). 34 Aplocheiloid killifish (Aplocheiloidei, Cyprinotondiformes) are small fresh-water and brackish water fishes 35 distributed in South-east Asia, America, Africa, Madagascar, Seychelles and India. Killifishes are often adapted to 36 survive in extreme environments, such as those characterized by seasonal drought. Some killifishes evolved an annual 37 life cycle (Figure 1), which allows fertilized eggs to remain in diapause and survive through the dry season (Cellerino, 38 et al. 2016). The main mechanism explaining the current distribution of killifishes across the planet is the mesozoic 39 divergence of the major killifish clades, which followed the tectonic drift order of Gondwana (Murphy and Collier 40 1997; Costa 2013). Hence, if taxa mainly speciated as a consequence of geographical isolation due to tectonic events, 41 the phylogenetic branching order and timings are expected to agree with those of continental drift. 42 However, the genus Pachypanchax challenges this model. Pachypanchax are non-annual killifishes restricted to 43 Madagascar and the neighboring Seychelles, with the sister genus Aplocheilus found in India and South-east Asia. 44 Only a single species, Pachypanchax playfairii (the golden panchax) is found on the Seychelles islands. Despite the 45 current geographical proximity to Madagascar, the Seychelles islands have a closer geological relationship with the 46 Indian subcontinent (Plummer and Belle 1995; Seton, et al. 2012). Hence, based on the continental drift hypothesis of 47 taxa distribution, species from Seychelles with limited dispersal capacity, like freshwater fish, are expected to have 48 closer phylogenetic proximity to India than to Madagascar. However, Seychelles and Madagascar are home to fish of 49 the same genus (Pachypanchax), suggesting the possibility for transoceanic dispersal following tectonic drift as a 50 possible mechanism for species dispersal. Evidences for transoceanic dispersal have been found in other groups of 51 animals. For example, palaeontological and molecular clock analyses place the divergence time of cichlid fish in the 52 Palaeocene (65-57 Myr ago), much later than Mesozoic tectonic rifts of Gondwana (Friedman, et al. 2013). Based on 53 molecular clock and nucleotide substitution saturation analysis in mitochondrial genes, Malagasy amphibian, reptilian 54 and non-flying mammals are too closely related to continental sister groups to fit the ancient tectonic events (Vences 55 2004). The Malagasy chameleon Archaius tigris was shown to be sister to a continental African chameleon genus, 56 which diverged not until the Eocene–Oligocene, again supporting the possibility for transoceanic dispersal 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.03.232421; this version posted August 3, 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-ND 4.0 International license. 57 (Townsend, et al. 2011). Molecular dating of the now extinct Malagasy Elephant Bird reveals its relatively recent 58 divergence with the sister group New Zealand Kiwis, suggesting transoceanic dispersal by their flying ancestors 59 (Grealy, et al. 2017). 60 To directly test whether tectonic or transoceanic dispersal can explain the evolution of the golden panchax 61 (Pachypanchax playfairii), we used phylogenetic and population genomic methods, as well as a lab experiment. 62 The golden panchax has a long evolutionary history of isolation on the Seychelles islands lasting several million years, 63 which combined with its limited dispersal capacity, represents a paradigmatic example of island evolution. Evolution 64 through island colonization has been used as an idealized model for understanding speciation (MacArthur and Wilson 65 2001). Endemic species are known to be more susceptible to extinction attributed to inbreeding (Frankham 1998), 66 despite some taxa evolving mechanisms for its mitigation (Wheelwright and Mauck 1998). Reduction of effective 67 population normally lowers the efficacy of selection, leading to the accumulation of deleterious mutations 68 (Charlesworth, et al. 1993). On the other hand, shifts in the ecological niche can also lead to relaxed selection by 69 removing niche-specific selective constraints (Wertheim, et al. 2014). Colonizing new island habitats is likely 70 accompanied by both a reduction of effective population size and by a change of the ecological niche, which may 71 affect a suite of phenotypes. For instance, relaxation of pre-existing selective pressures, such as a lack of predators in 72 novel environments, can explain the loss of anti-predator behaviors in oceanic tortoises (Austin and Nicholas Arnold 73 2001) and the loss of flight in the Kakapo (Livezey 1992) and Dodo (Livezey 1993). Positive selection has also been 74 invoked to explain island-specific phenotypes. For example, gigantism in oceanic tortoises is proposed to confer a 75 selective advantage during the unpredictable long dry seasons on islands (Jaffe, et al. 2011). However, still little is 76 known about the genomic patterns of species undergoing long-term island isolation. 77 Previous studies conducted in continental African killifishes have shown that relaxation of selection dominates 78 genome evolution in short-lived annual African killifishes, preferentially targeting aging-related pathways, likely 79 resulting from population fragmentation, population size reduction and a shift in the selective regime (Cui, et al. 2019; 80 Willemsen, et al. 2019). Here, to assess the effects of island isolation in the golden panchax, we inferred the historical 81 changes in effective population size in wild populations from different islands of the Seychelles archipelago and 82 conducted an unbiased characterization of the relative contribution of relaxation of selection, intensified selection and 83 positive selection in shaping the golden panchax genome. 84 Our results show that golden panchax likely colonized the Seychelles via transoceanic dispersal and indicate that 85 prolonged insularism led to progressive