1 Online Supplementary Material 2 CONVERGENT EVOLUTION OF CONSERVED MITOCHONDRIAL 3 PATHWAYS UNDERLIES REPEATED ADAPTATION TO EXTREME 4 ENVIRONMENTS 5 6 Ryan Greenway, Nick Barts, Chathurika Henpita, Anthony P. Brown, Lenin Arias Rodriguez, Carlos 7 M. Rodríguez Peña, Sabine Arndt, Gigi Y. Lau, Michael P. Murphy, Lei Wu, Dingbo Lin, Jennifer H. 8 Shaw, Joanna L. Kelley & Michael Tobler 9 10 Correspondence to: [email protected], [email protected] 11 12 This file includes: 13 • Extended information on materials and methods 14 • Figures S1 to S9 15 • Tables S1 to S14 16 17 Other Supplementary Materials for this manuscript include the following: 18 • Dataset S1 as a separate Excel file 19 • Dataset S2 as a separate Excel file 20 • Dataset S3 as a separate Excel file 21 • Dataset S4 as a separate Excel file 22 1 23 MATERIALS AND METHODS 24 1. Background and hypotheses 25 The overall goals of our study were to test hypotheses about the mechanistic basis of H2S tolerance 26 in evolutionarily replicated populations of Poecilia mexicana and about potential signatures of 27 convergent evolution in sulfide spring fishes across the family Poeciliidae. Poecilia mexicana has been a 28 model for the study of adaptation to H2S-rich environments (1). This species is widely distributed in 29 Mexico and Central America, inhabiting a wide range of freshwater habitats (2, 3). In southern 30 Mexico, this species has also colonized H2S-rich springs in four different river drainages of the Rio 31 Grijalva basin (including the Rios Tacotalpa, Puyacatengo, Ixtapangajoya, and Pichucalco; see Figure 32 S1). Colonization of springs has occurred independently, providing evolutionarily replicated 33 population pairs in sulfidic and nonsulfidic habitats (4, 5). Sulfide spring populations are locally 34 adapted and differ from ancestral populations in adjacent nonsulfidic habitats in a number of 35 physiological, morphological, behavioral, and life-history traits (5-7). Most importantly, sulfide 36 spring populations can tolerate H2S concentrations that are orders of magnitudes higher than what is 37 considered lethal for most metazoans, while ancestral populations are susceptible to the toxic effects 38 of H2S over short periods of time (5, 8), indicating strong survival selection that impacts fitness 39 across small spatial scales. In addition, sulfide spring populations are reproductively isolated from 40 adjacent populations in nonsulfidic water, with low levels of gene flow and high genetic 41 differentiation despite small spatial scales (in some instances <100 m) and an absence of physical 42 barriers that prevent fish movement (8). Reproductive isolation is in part mediated by natural and 43 sexual selection against migrants (8, 9). 44 Comparative genomic and transcriptomic analyses of P. mexicana populations from sulfidic 45 and nonsulfidic habitats have provided insights into mechanisms that potentially underlie tolerance 46 to toxic levels of H2S (10-13). Collectively, these studies have indicated that modification of 47 mitochondrial function may be critical in mediating adaptation to H2S. Mitochondrial modifications 48 may impact H2S tolerance through two mechanisms, resistance and regulation (14). Resistance 49 involves modification of the direct toxicity target (cytochrome c oxidase, COX) of H2S and other 50 components of the oxidative phosphorylation pathway (OxPhos). Regulation involves modification 51 of physiological pathways associated with the maintenance of mitochondrial H2S concentrations; 52 most notably the mitochondrial sulfide:quinone oxidoreductase (SQR) pathway. Past analyses have 53 provided evidence for both resistance and regulation, as multiple components of the OxPhos and 54 SQR pathways exhibit evidence for selection and/or heritable changes in gene regulation (10-13). 55 However, the functional ramifications of amino acid substitutions and gene expression changes 56 remain largely unknown. 57 Here, we hypothesized that the repeated modification of enzymes in the OxPhos and SQR 58 pathways in P. mexicana populations from sulfidic habitats leads to an increased ability to maintain 59 mitochondrial function in the presence of H2S. We tested this hypothesis through multiple, 60 complementary analyses: 61 1. Modification of COX—the primary toxicity target in OxPhos—is predicted to make 62 the enzyme more resistant to the effects of H2S. Previous analyses have already found 63 evidence for this prediction in some P. mexicana populations (15). We followed up on 64 this study and quantified COX activity across a broad range of H2S concentrations in 65 multiple populations of P. mexicana from sulfidic and nonsulfidic habitats (see Section 66 2). 67 2. Modification of SQR—the first step in enzymatic H2S oxidation—is predicted to 68 increase the rate of H2S detoxification. We tested this prediction by conducting enzyme 69 activity assays, quantifying the rate at which H2S is oxidized by SQR in multiple 70 populations of P. mexicana from sulfidic and nonsulfidic habitats (see Section 2). 2 71 3. If regulation of mitochondrial H2S is a primary mechanism of H2S tolerance, P. mexicana 72 from sulfidic habitats should be able to maintain low endogenous H2S concentrations 73 upon environmental exposure, whereas endogenous concentrations should increase in 74 individuals from nonsulfidic habitats. We tested this prediction by experimentally 75 exposing laboratory-reared individuals of P. mexicana to environmental H2S and 76 quantifying endogenous H2S where it matters—in the mitochondria—using a novel 77 mitochondria-targeted probe (MitoA) that can be deployed to measure H2S in vivo (see 78 Section 3). 79 4. If regulation of endogenous H2S is a primary mechanism of H2S tolerance, 80 mitochondrial function should be maintained in the presence of H2S even in sulfide 81 spring populations that do not exhibit an H2S-resistant COX. We tested this prediction 82 by quantifying mitochondrial respiration across multiple P. mexicana populations in 83 presence and absence of H2S in vitro (see Section 4). 84 5. We also asked whether adaptive alleles in P. mexicana from different sulfidic populations 85 rose to high frequency through selection on standing genetic variation, or whether 86 adaptive alleles arose independently in different sulfide spring populations through de 87 novo mutations. To do so, we sequenced whole genomes of multiple P. mexicana 88 populations to conduct local ancestry analyses (see Section 5). 89 Overall, our analyses in P. mexicana revealed that some sulfide spring populations have 90 adaptive modifications to COX, an increased SQR activity, a higher capacity in maintaining low 91 endogenous H2S concentration upon environmental exposure, and an ability to maintain 92 mitochondrial respiration in presence of H2S irrespective of the presence of an H2S-resistant toxicity 93 target. Adaptive alleles at some loci (e.g., COX) arose independently in different populations, while 94 there is also evidence for selection on standing genetic variation (e.g., genes associated with 95 detoxification). Based on these findings, we asked whether other species that successfully colonized 96 H2S-rich habitats exhibit convergent modifications of genes associated with H2S toxicity and 97 detoxification. This was possible because sulfide springs are widely distributed across the globe (16), 98 and species of the family Poeciliidae are among the few metazoans that have colonized and adapted 99 to these extreme environments (1). Collecting species from multiple genera allowed for the 100 investigation of mechanisms underlying the evolution of H2S tolerance across broad phylogenetic 101 scales (over 40 million years of divergence among lineages) and in different ecological and 102 biogeographical contexts (Figure S1). 103 We hypothesized that convergent molecular modifications underlie the convergent evolution 104 of H2S tolerance across different lineages of poeciliid fishes. We sequenced transcriptomes of five to 105 six individuals from each of 10 independent lineages of sulfide spring fishes—as well as closely 106 related lineages from nonsulfidic habitats—to test the following predictions (see Section 6): 107 6. Genes associated with H2S toxicity and detoxification should exhibit repeated changes 108 of gene expression upon colonization of H2S-rich habitats. We used phylogenetic 109 comparative analyses to identify genes with convergent expression shifts in sulfide 110 spring fishes. 111 7. Genes associated with H2S toxicity and detoxification should exhibit evidence for 112 positive selection upon colonization of H2S-rich habitats. We used analyses of 113 molecular evolution to identify convergent targets of selection in sulfide spring fishes. 114 Note that all procedures used were approved by the Institutional Animal Care and Use 115 Committees at Kansas State University (protocol #3418, 3473, 3581, 3886, 3992) and Oklahoma 116 State University (protocol #1015, 1102). All statistical analyses were conducted in R version 3.6.1 117 (17) unless otherwise stated. 118 3 119 2. Enzyme activity assays 120 2.1. Sample procurement 121 We collected fish from three population pairs of Poecilia mexicana from the Tacotalpa (Tac), 122 Puyacatengo (Puy), and Pichucalco (Pich) drainages in Mexico, each including an H2S-tolerant and 123 ancestral, intolerant population (Table S1). All fish were collected using a seine (2 ´ 5 m, 3 mm mesh 124 size), immediately euthanized, and livers were extracted. Liver tissues were immediately frozen in 125 liquid nitrogen