bioRxiv preprint doi: https://doi.org/10.1101/525378; this version posted January 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Title: Microhabitat predicts species differences in exploratory behavior in Lake Malawi 2 cichlids 3 4 Zachary V. Johnson†1, Emily C. Moore†2,3, Ryan Y. Wong4, John R. Godwin2, Jeffrey T. 5 Streelman1, Reade B. Roberts2 6 7 1School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA, USA 8 2 Department of Biological Sciences and W. M. Keck Center for Behavioral Biology, North 9 Carolina State University, Raleigh, NC, USA 10 3 Division of Biological Sciences, University of Montana, Missoula, MT, USA 11 4 Department of Biology and Department of Psychology, University of Nebraska Omaha, 12 Omaha, NE, USA 13 14 † Both authors contributed equally to this work 15 16 Acknowledgements: Preparation of this manuscript was supported by an Arnold and Mabel 17 Beckman Foundation Beckman Young Investigator Award to RBR and by NIH grant 18 R01GM101095 to JTS 19 20 Declarations of interest: none 21 22 23 Address Correspondence to: 24 25 Zachary Johnson 26 E-mail: [email protected] 27 Postal: 950 Atlantic Dr NW 28 Engineered Biosystems Building, 3rd floor 29 Atlanta, GA 30332 30 31 Emily Moore 32 E-mail: [email protected] 33 Postal: 32 Campus Drive 34 Division of Biological Sciences, HS 104 35 Missoula, MT, 59812 36 37 38 39 40 41 bioRxiv preprint doi: https://doi.org/10.1101/525378; this version posted January 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 42 Abstract 43 44 Encountering and adaptively responding to unfamiliar or novel stimuli is a fundamental 45 challenge facing animals and is linked to fitness. Behavioral responses to novel stimuli, or 46 exploratory behavior, can differ strongly between closely related species; however, the 47 ecological and evolutionary factors underlying these differences are not well understood, in 48 part because most comparative investigations have focused on only two species. In this study, 49 we investigate exploratory behavior across 23 species in a previously untested vertebrate 50 system, Lake Malawi cichlid fishes, which comprises hundreds of phenotypically diverse 51 species that have diverged in the past one million years. We investigate behavioral variation 52 across species, across microhabitats, and across environmental contexts. We find strong 53 species differences in behavior that are associated with microhabitat, demonstrate that 54 intermediate microhabitats are associated with higher levels of exploratory behavior, show 55 that patterns of behavioral covariation across contexts are characteristic of modular complex 56 traits, and contrast Malawi cichlid data with behavioral data from selectively bred high- and 57 low-exploratory zebrafish. Taken together, our results tie ecology to species differences in 58 behavior, and highlight Lake Malawi cichlids as a powerful system for understanding the 59 evolution, ecology, and biology of natural behavioral variation. 60 61 Keywords: teleosts, neophobia, neophilia, anxiety-like behavior, bold shy axis, stress 62 response, habitat preference, behavioral syndromes, behavioral modularity, behavioral 63 integration bioRxiv preprint doi: https://doi.org/10.1101/525378; this version posted January 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 64 Highlights 65 66 • Malawi cichlids exhibit high phenotypic variance in exploratory behaviors 67 • Species differences in exploratory behavior are explained by microhabitat 68 • Rock-dwelling species exhibit strong edge preferences across assays 69 • Intermediate habitats are associated with “high exploratory” open field behavior 70 • Patterns of behavioral covariance across contexts are modular in Malawi cichlids 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 bioRxiv preprint doi: https://doi.org/10.1101/525378; this version posted January 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 94 1. Introduction 95 96 Deciding how to respond to unfamiliar or novel stimuli is a fundamental aspect of animal life 97 that has important implications for fitness. For example, how individuals respond to novel 98 conspecifics, heterospecifics, physical environments, food resources, or objects can directly 99 impact survival (N. J. Dingemanse, Both, Drent, & Tinbergen, 2004; Ferrari, McCormick, 100 Meekan, & Chivers, 2015; Lapiedra, Schoener, Leal, Losos, & Kolbe, 2018; Smith & 101 Blumstein, 2008). Behavioral responses to novel stimuli can vary strongly between individuals, 102 populations, and closely-related species; however, the factors underlying this behavioral 103 variation are not well resolved. 104 105 At the interspecies level, large scale comparative studies are a promising strategy for 106 identifying evolutionary and ecological factors contributing to variation in behavioral responses 107 to novel stimuli (Niels J. Dingemanse et al., 2007). For example, a comparative study across 108 61 species of parrots showed that species differences in microhabitat predict species 109 differences in behavioral responses to novel objects: species inhabiting intermediate habitats 110 between the forest and the savannah more readily approached novel objects compared to 111 species inhabiting more uniform savannah habitats (R. Greenberg, 2003; Greenberg & 112 Mettke-hofmann, 2001; Claudia Mettke-Hofmann, Winkler, & Leisler, 2002). These and other 113 findings support hypotheses that ecological plasticity or habitat variability is associated with 114 higher levels of exploratory behavior. However, it is unclear how well this model generalizes 115 across species and vertebrate lineages, in part because many comparative studies of 116 behavioral responses to novel stimuli have compared just two species and have been 117 conducted in birds (Garland & Adolph, 1994; Réale, Reader, Sol, McDougall, & Dingemanse, 118 2007). Furthermore, different behavioral assays and testing parameters have been used 119 across studies, making it difficult to identify organizing principles that explain species 120 differences in behavior. To better elucidate relationships between ecological factors, such as 121 microhabitat, and species differences in exploratory behavior, larger comparative studies 122 employing multiple assays and in new vertebrate systems are needed. 123 bioRxiv preprint doi: https://doi.org/10.1101/525378; this version posted January 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 124 Lake Malawi cichlid fishes are well-suited for comparative investigations of phenotypic 125 variation (R. C. Albertson, Markert, Danley, & Kocher, 1999; Johnson & Young, 2018; Rupp & 126 Hulsey, 2014; Ryan A. York & Fernald, 2017). These fishes have recently (within the past one 127 million years) undergone explosive speciation, radiating into an estimated 500-1000 species 128 that vary in morphology, coloration, diet, habitat preference, and behavior (Brawand et al., 129 2014; Kocher, 2004). Additionally, within Lake Malawi, ecological conditions vary across small 130 spatial scales, resulting in diverse species occupying different microhabitats while living in 131 close geographic proximity. For example, while many species can be grouped into two 132 canonical ecotypes, rock-dwelling and sand-dwelling (Kocher, 2004), a large number of 133 species inhabit intermediate habitats, including the interface between rocky and sandy 134 substrate, and shallow sediment-rich bays near rocks and reed stands. Thus, the Lake Malawi 135 species assemblage is well positioned to test relationships between microhabitat and species 136 differences in behavior. 137 138 Comparative studies in Lake Malawi cichlids have already begun generating insights into the 139 evolution of complex traits (Streelman & Danley, 2003; Sylvester et al., 2010; R. A. York et al., 140 2018). For example, ecological factors have been associated with species differences in 141 aggression and bower-building behavior (Danley, 2011; Ryan A. York et al., 2015). Other 142 studies have investigated the evolution of non-behavioral traits such as oral jaw morphology 143 and color patterning, and have demonstrated modular patterns of phenotypic variation (R. 144 Craig Albertson et al., 2014; Parsons, Cooper, & Albertson, 2011). Briefly, evolutionary 145 modularity and integration refer to patterns of covariation among sets of traits (e.g. dimensions 146 of different oral jaw bones), and they are thought to be related to trait evolvability (Raff & Raff, 147 2000; Wagner, Pavlicev, & Cheverud, 2007). Phenotypic integration refers to more uniform 148 patterns of covariation, while modularity refers to non-uniform patterns of covariation and may 149 reflect increased trait evolvability, although these relationships are complex (Armbruster, 150 Pélabon, Bolstad, & Hansen, 2014). 151 152 Although Lake Malawi cichlids are primely positioned to link ecological and evolutionary 153 factors to species differences