Genetics: Early Online, published on March 1, 2018 as 10.1534/genetics.118.300793 1 Title: Genetic basis of body color and spotting pattern in redheaded pine sawfly larvae 2 (Neodiprion lecontei) 3 4 Authors: Catherine R. Linnen*, Claire T. O’Quin*, Taylor Shackleford*, Connor R. 5 Sears*†, Carita Lindstedt‡ 6 7 Running title: Genetic basis of larval color 8 9 Keywords: pigmentation, carotenoids, melanin, genetic architecture, convergent 10 evolution, evolutionary genetics 11 12 Corresponding author: Catherine R. Linnen, 204E Thomas Hunt Morgan Building, 13 Lexington, KY 40506, 859-323-3160, [email protected] * Department of Biology, University of Kentucky, Lexington, KY, 40506 † Current affiliation: Department of Biological Sciences, University of Cincinnati, Cincinnati, OH, 45221 ‡ Centre of Excellence in Biological Interactions, Department of Biological and Environmental Sciences, University of Jyväskylä, Jyväskylä, Finland, FI-40014 1 Copyright 2018. 14 ABSTRACT 15 16 Pigmentation has emerged as a premier model for understanding the genetic basis of 17 phenotypic evolution, and a growing catalog of color loci is starting to reveal biases in 18 the mutations, genes, and genetic architectures underlying color variation in the wild. 19 However, existing studies have sampled a limited subset of taxa, color traits, and 20 developmental stages. To expand the existing sample of color loci, we performed 21 quantitative trait locus (QTL) mapping analyses on two types of larval pigmentation traits 22 that vary among populations of the redheaded pine sawfly (Neodiprion lecontei): 23 carotenoid-based yellow body color and melanin-based spotting pattern. For both traits, 24 our QTL models explained a substantial proportion of phenotypic variation and suggested 25 a genetic architecture that is neither monogenic nor highly polygenic. Additionally, we 26 used our linkage map to anchor the current N. lecontei genome assembly. With these 27 data, we identified promising candidate genes underlying: (1) a loss of yellow 28 pigmentation in populations in the Mid-Atlantic/northeastern USA [C locus-associated 29 membrane protein homologous to a mammalian HDL receptor-2 gene (Cameo2) and 30 lipid transfer particle apolipoproteins II and I gene (apoLTP-II/I )], and (2) a pronounced 31 reduction in black spotting in Great-Lakes populations [members of the yellow gene 32 family, tyrosine hydroxylase gene (pale), and dopamine N-acetyltransferase gene (Dat)]. 33 Several of these genes also contribute to color variation in other wild and domesticated 34 taxa. Overall, our findings are consistent with the hypothesis that predictable genes of 35 large-effect contribute to color evolution in nature. 36 37 INTRODUCTION 38 39 Over the last century, color phenotypes have played a central role in efforts to 40 understand how evolutionary processes shape phenotypic variation in natural populations 41 (Gerould 1921; Sumner 1926; Fisher and Ford 1947; Haldane 1948; Cain and Sheppard 42 1954; Kettlewell 1955). More recently, color variation in nature has begun to shed light 43 on the genetic and developmental mechanisms that give rise to phenotypic variation 44 (True 2003; Protas and Patel 2008; Wittkopp and Beldade 2009; Manceau et al. 2010; 45 Nadeau and Jiggins 2010; Kronforst et al. 2012). A growing catalog of color loci is 46 starting to reveal how ecology, evolution, and development interact to bias the genetic 47 architectures, genes, and mutations underlying the remarkable diversity of color 48 phenotypes (Hoekstra and Coyne 2007; Stern and Orgogozo 2008, 2009; Kopp 2009; 49 Manceau et al. 2010; Streisfeld and Rausher 2011; Martin and Orgogozo 2013; Dittmar 50 et al. 2016; Massey and Wittkopp 2016; Martin and Courtier-Orgogozo 2017). To make 51 robust inferences about color evolution, however, genetic data from diverse traits, taxa, 52 developmental stages, and evolutionary timescales are needed. 53 Two long-term goals of research on the genetic underpinnings of pigment 54 variation are: (1) to evaluate the importance of large-effect loci to phenotypic evolution 55 (Orr and Coyne 1992; Mackay et al. 2009; Rockman 2012; Remington 2015; Dittmar et 56 al. 2016), and (2) to determine the extent to which the independent evolution of similar 57 phenotypes (phenotypic convergence) is attributable to the same genes and mutations 58 (genetic convergence) (Arendt and Reznick 2008; Gompel and Prud’homme 2009; 59 Christin et al. 2010; Manceau et al. 2010; Elmer and Meyer 2011; Conte et al. 2012; 2 60 Rosenblum et al. 2014). Addressing these questions will require a large, unbiased sample 61 of pigmentation loci. At present, most of what is known about the genetic basis of color 62 variation in animals comes from a handful of taxa and from melanin-based color 63 variation in adult life stages (True 2003; Protas and Patel 2008; Wittkopp and Beldade 64 2009; Nadeau and Jiggins 2010; Kronforst et al. 2012; Sugumaran and Barek 2016). 65 Another important source of bias stems from a tendency to focus on candidate genes and 66 discrete pigmentation phenotypes (Kopp 2009; Manceau et al. 2010; Rockman 2012; but 67 see Greenwood et al. 2011; O’Quin et al. 2013; Albertson et al. 2014; Signor et al. 2016; 68 Yassin et al. 2016). Here, we describe an unbiased, genome-wide analysis of multiple, 69 continuously varying color traits in larvae from the order Hymenoptera, a diverse group 70 of insects that is absent from our current catalog of color loci (Martin and Orgogozo 71 2013). 72 More specifically, our study focuses on pine sawflies in the genus Neodiprion. 73 Several factors make Neodiprion an especially promising system for investigating the 74 genetic basis of color variation. First, there is intra- and interspecific variation in many 75 different types of color traits (Figures 1-2). Second, previous phylogenetic and 76 demographic studies (Linnen and Farrell 2007, 2008a; b; Bagley et al. 2017) enable us to 77 infer directions of trait change and identify instances of phenotypic convergence in this 78 genus. Third, because many different species can be reared and crossed in the lab (Knerer 79 and Atwood 1972, 1973; Kraemer and Coppel 1983; Bendall et al. 2017), unbiased 80 genetic mapping approaches are feasible in Neodiprion. Fourth, a growing list of genomic 81 resources for Neodiprion—including an annotated genome and a methylome for N. 82 lecontei (Vertacnik et al. 2016; Glastad et al. 2017)— facilitate identification of causal 83 genes and mutations. And finally, an extensive natural history literature (Coppel and 84 Benjamin 1965; Knerer and Atwood 1973) provides insights into the ecological functions 85 of color variation in pine sawflies, which we review briefly for context. 86 Under natural conditions, pine sawfly larvae are attacked by a diverse assemblage 87 of arthropod and vertebrate predators, by a large community of parasitoid wasps and 88 flies, and by fungal, bacterial, and viral pathogens (Coppel and Benjamin 1965; Wilson et 89 al. 1992; Codella and Raffa 1993). When threatened, larvae adopt a characteristic “U- 90 bend” posture and regurgitate a resinous defensive fluid (Figure 1), which is an effective 91 repellant against many different predators and parasitoids (Eisner et al. 1974; Codella and 92 Raffa 1995; Lindstedt et al. 2006, 2011). Although most Neodiprion species are 93 chemically defended, larvae vary from a green striped morph that is cryptic against a 94 background of pine foliage to highly conspicuous aposematic morphs with dark spots or 95 stripes overlaid on a bright yellow or white background (Figure 1). Thus, larval color is 96 likely to confer protection against predators either via preventing detection (crypsis) or 97 advertising unpalatability (aposematism) (Ruxton et al. 2004; Lindstedt et al. 2011). 98 Beyond selection for crypsis or aposematism, there are many abiotic and biotic 99 selection pressures that could act on Neodiprion larval color. For example, insect color 100 can contribute to thermoregulation, protection against UV damage, desiccation tolerance, 101 and resistance to abrasion (True 2003; Lindstedt et al. 2009; Wittkopp and Beldade 102 2009). Color alleles may also have pleiotropic effects on other traits, such as behavior, 103 immune function, diapause/photoperiodism, fertility, and developmental timing (True 104 2003; Wittkopp and Beldade 2009; Heath et al. 2013; Lindstedt et al. 2016). Temporal 3 105 and spatial variation in these diverse selection pressures likely contribute to the abundant 106 color variation in the genus Neodiprion. 107 As a first step to understanding the proximate mechanisms that generate color 108 variation in pine sawflies, we conducted a quantitative trait locus (QTL) mapping study 109 of larval body color and larval spotting pattern in the redheaded pine sawfly, Neodiprion 110 lecontei. This species is widespread across eastern North America, where it feeds on 111 multiple pine species. Throughout most of this range, larvae have several rows of dark 112 black spots overlaid on a bright, yellow body (e.g., center image in Figure 1). However, 113 previous field surveys and demographic analyses indicate that there has been a loss of 114 yellow pigmentation in some populations in the Mid-Atlantic/northeastern United States 115 and a pronounced reduction in spotting in populations living in the Great-Lakes region of 116 the U.S. and Canada (Bagley et al. 2017; Figure 2). To describe genetic architectures and 117 identify candidate genes underlying these two reduced-pigmentation