19The discus fish (Symphysodon spp.) is an endemic species of the Amazon that is 20 among the most popular ornamental fish around the world, and is usually used as the 21 model animal for studying the diversification of Amazon fish. Here, a comparative 22 analysis of two species of discus fish, i.e., S. haraldi and S. aequifasciatus, based on 23 several antioxidant indexes was conducted, to test the hypothesis that cold resistance 24 might correlate with the diversification of discus fish. We set up a continuous 25 sequence of three temperature programs, namely cooling (28 °C to 14 °C; -1 °C/h), 26 cold maintenance (14 °C for 12 h) and recovery (14 °C to 28 °C; +1 °C/h).
27Subordinate function (SF) combined with principal component analysis (PCA) 28 showed that the cold hardiness of S. haraldi during cold treatment was in the order of 29 cooling > cold maintenance ≈ recovery, but the cold hardiness of S. aequifasciatus 30 during cold treatment was in the order of cold maintenance > cooling > recovery. 31 Specifically, the lowest cold hardiness was observed in S. aequifasciatus during 32 recovery, indicating that cold stress resulted in more seriously oxidative stress in S. 33 aequifasciatus than in S. haraldi. Overall, these results show a significant interspecific 34 variation, indicating the correlation between environmental adaptation and the 35 diversification of discus fish.36 37 38The discus fish (Symphysodon spp.) is an important ornamental tropical fish all over 40 the world, originating from the Amazon River (Wen et al., 2018(Wen et al., , 2017. In addition to 41 their Amazon basin-wide distribution, the 3 currently recognized species of the genus 42 Symphysodon (S. aequifasciatus, S. discus, and S. haraldi, Cichlidae, Perciformes) 43 (Bleher et al., 2007; Gross et al., 2009) exhibit a large amount of morphological 44 variation (different color and color patter) and genetic variability associated with 45 different types of biotopes (Farias and Hrbek, 2008; Koh et al., 1999). For example, S. 46 haraldi, the 'bule' discus, is found in the central portion of the Amazon basin (type 47 locality Manacapuru river), S. aequifasciatus, the 'green' discus, is found in the west 48 portion of the Amazon basin (type locality Tef´e River), and S. discus, the. Heckel 49 discus, is found in the Negro River basin (Farias and Hrbek, 2008; Gross et al., 2010). 50 Fish as an ectotherm, ambient temperature which constrains whole-organism 51 performance is one of the most important factors affecting the biogeographic 52 distribution and abundance (Troia and Gido, 2017). Recently, several studies have 53 shown that distinct responses of antioxidant defense systems (ADS) would occur 54 between different locations of fish species toward temperature stress (e.g., Bryant et 55 al., 2018; Chung et al., 2017; Johnston et al., 1998; Rudneva-Titova et al., 1994 56 Shaliutinakolešová et al., 2013). Yet surprisingly few studies have compared thermal 57 performance among closely related warm-water species (Troia and Gido, 2015).58...