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Integrative ISSN 1540-7063 (PRINT) Integrative ISSN 1557-7023 (ONLINE)

&Comparative Biology & Volume 55 Number 5 November 2015 Comparative Biology

CONTENTS Linking with Crustacea: Comparative Physiology of the Pancrustacea Organized by Sherry L. Tamone and Jon F. Harrison 765 Linking Insects with Crustacea: Physiology of the Pancrustacea: An Introduction to the Symposium Sherry L.Tamone and Jon F. Harrison November 2015 Number 5 Volume 55 771 Exoskeletons across the Pancrustacea: Comparative Morphology, Physiology, Biochemistry and Genetics Robert Roer, Shai Abehsera and Amir Sagi 792 Evolution of Respiratory Proteins across the Pancrustacea Thorsten Burmester 802 Handling and Use of Oxygen by Pancrustaceans: Conserved Patterns and the Evolution of Respiratory Structures Jon F. Harrison 816 Links between Osmoregulation and Nitrogen-Excretion in Insects and Crustaceans Dirk Weihrauch and Michael J. O’Donnell 830 The Dynamic Evolutionary History of Pancrustacean Eyes and Opsins Miriam J. Henze and Todd H. Oakley 843 Integrated Immune and Cardiovascular Function in Pancrustacea: Lessons from the Insects Julián F. Hillyer 856 Respiratory and Metabolic Impacts of Crustacean Immunity: Are there Implications for the Insects? Karen G. Burnett and Louis E. Burnett 869 Morphological, Molecular, and Hormonal Basis of Limb Regeneration across Pancrustacea Sunetra Das 878 Evolution of Ecdysis and Metamorphosis in :The Rise of Regulation of Juvenile Hormone Sam P. S. Cheong, Juan Huang, William G. Bendena, Stephen S. Tobe and Jerome H. L. Hui 891 Neocaridina denticulata: A Decapod Crustacean Model for Functional Genomics Donald L. Mykles and Jerome H. L. Hui Leading Students and Faculty to Quantitative Biology Through Active Learning Organized by Lindsay D. Waldrop and Laura A. Miller 898 Introduction to the Symposium ‘‘Leading Students and Faculty to Quantitative Biology through Active Learning’’ Lindsay D. Waldrop and Laura A. Miller 901 A Mathematical Model and MATLAB Code for Muscle–Fluid–Structure Simulations Nicholas A. Battista, Austin J. Baird and Laura A. Miller 912 Interdisciplinary Laboratory Course Facilitating Knowledge Integration, Mutualistic Teaming, and Original Discovery Robert J. Full, Robert Dudley, M. A. R. Koehl, Thomas Libby and Cheryl Schwab 926 Using Technology to Expand the Classroom in Time, Space, and Diversity Joshua Drew 933 Using Active Learning to Teach Concepts and Methods in Quantitative Biology Lindsay D. Waldrop, Stephen C. Adolph, Cecilia G. Diniz Behn, Emily Braley, Joshua A. Drew, Robert J. Full, Louis J. Gross, John A. Jungck, Brynja Kohler, Jennifer C. Prairie, Blerta Shtylla and Laura A. Miller Integrative &Comparative Biology Volume 55 Number 5 November 2015 www.icb.oxfordjournals.org Integrative and Comparative Biology www.icb.oxfordjournals.org Society for Integrative and Comparative Biology

Editor-in-Chief Executive Officers, Society for Integrative and Harold Heatwole, North Carolina State University, Raleigh, Comparative Biology NC, USA and University of New England, Armidale, NSW, President Australia Peter Wainwright (2017) President-Elect Assistant Editors Louis Burnett (2017) Suzanne C. Miller, North Carolina State University Past President Billie Swalla (2016) Editorial Board Secretary Kathy Dickson (2018) Divisional Representatives Program Officer Behavior (DAB) Sherry Tamone (2016) Matthew Grober (2018), Georgia State University Program Officer Elect Comparative Biomechanics (DCB) Richard Blob (2016) Anna Ahn (2017), Harvey Mudd College Treasurer Comparative Endocrinology (DCE) Karen Martin (2019) Henry John-Alder (2018), Rutgers University Members-At-Large Comparative Physiology and Biochemistry (DCPB) Cheryl Wilga (2016) Dane Crossley (2016), University of North Dakota L. Patricia Hernandez (2017) Ecoimmunology and Disease Ecology (DEDE) Jennifer Bunaford (2018) Ken Field (2020), Bucknell University Divisional Chairs Ecology and Evolution (DEE) DAB Chair: Diana K. Hews (2016) Michael Sears (2017), Clemson University DCB Chair: Melina Hale (2017) Evolutionary Developmental Biology (DEDB) DCE Chair: Mary Mendonca (2016) Robert Zeller (2018), San Diego State University DCPB Chair: Stephen Secor (2016) Invertebrate Zoology (DIZ) DEDB Chair: Sally Leys (2016) Bruno Pernet (2020), California State University, DEDE Chair: Lynn Martin (2017) Long Beach DEE Chair: Michael Sears (2017) Neurobiology (DNB) DIZ Chair: John Zardus (2018) Richard A. Satterlie (2018), University of North DNB Chair: Paul Moore (2018) Carolina at Wilmington DPCB Chair: Michael E. Alfaro (2016) Phylogenetics and Comparative Biology (DPCB) DVM Chair: Callum Ross (2017) Lars Schmitz (2019), W.M. Keck Science Department of Editor-in-Chief, ICB: Harold F. Heatwole (2016) Claremont McKenna, Pitzer, and Scripps Colleges SPDAC Chair: Sean Lema (2016) Vertebrate Morphology (DVM) Ed. Council Chair: Bram Lutton (2018) Lara Ferry (2016), Arizona State University BPC Chair: Michele Nishiguchi (2016) Executive Director: Brett J. Burk Associates Dominique Adriaens (2016), University of Ghent, Ghent, Belgium Christopher R. Bridges (2016), Heinrich-Heine Universita´t, Dusselforf, Germany Berry Pinshow (2016), Ben Gurion University of the Negev, Israel Julia Sigwart (2018), Queen’s University Belfast

Cover image: Mating blue-tailed damselflies (Ischnura elegans). Insects like these damselflies are derived crustaceans, but they have been historically ranked as different taxonomic classes within arthropods and studied separately. In this issue, articles from the symposium ‘‘Linking Insects with Crustacea: Comparative Physiology of the Pancrustacea’’ develop synergies based on our new evolutionary understanding of the group. Photo by Miriam J. Henze. Integrative and Comparative Biology Advance Access published August 28, 2015 Integrative and Comparative Biology Integrative and Comparative Biology, pp. 1–13 doi:10.1093/icb/icv100 Society for Integrative and Comparative Biology

SYMPOSIUM

TheDynamicEvolutionaryHistoryofPancrustaceanEyesandOpsins Miriam J. Henze* and Todd H. Oakley1,† *Department of Biology, Lund University, Lund, Sweden; †Department of Ecology, Evolution and Marine Biology, University of California, Santa Barbara, CA, USA From the symposium ‘‘Linking Insects with Crustacea: Comparative Physiology of the Pancrustacea’’ presented at the Downloaded from annual meeting of the Society for Integrative and Comparative Biology, January 3–7, 2015 at West Palm Beach, Florida. 1E-mail: [email protected]

Synopsis Pancrustacea (Hexapoda plus Crustacea) display an enormous diversity of eye designs, including multiple types http://icb.oxfordjournals.org/ of compound eyes and single-chambered eyes, often with color vision and/or polarization vision. Although the eyes of some pancrustaceans are well-studied, there is still much to learn about the evolutionary paths to this amazing visual diversity. Here, we examine the evolutionary history of eyes and opsins across the principle groups of Pancrustacea. First, we review the distribution of lateral and median eyes, which are found in all major pancrustacean clades (Oligostraca, Multicrustacea, and Allotriocarida). At the same time, each of those three clades has taxa that lack lateral and/or median eyes. We then compile data on the expression of visual r-opsins (rhabdomeric opsins) in lateral and median eyes across

Pancrustacea and find no evidence for ancient opsin clades expressed in only one type of eye. Instead, opsin clades with at University of California, Santa Barbara on August 31, 2015 eye-specific expression are products of recent gene duplications, indicating a dynamic past, during which opsins often changed expression from one type of eye to another. We also investigate the evolutionary history of peropsins and r-opsins, which are both known to be expressed in eyes of arthropods. By searching published transcriptomes, we discover for the first time crustacean peropsins and suggest that previously reported odonate opsins may also be peropsins. Finally, from analyzing a reconciled, phylogenetic tree of r-opsins, we infer that the ancestral pancrustacean had four visual opsin genes, which we call LW2, MW1, MW2, and SW. These are the progenitors of opsin clades that later were variously duplicated or lost during pancrustacean evolution. Together, our results reveal a particularly dynamic history, with losses of eyes, duplication and loss of opsin genes, and changes in opsin expression between types of eyes.

Introduction Here, we review the presence and absence of the With color vision, polarization vision, multiple types two main types of arthropod eyes across major pan- of eyes, and more optical diversity than in any other crustacean groups, we summarize data on gene ex- phylum (Land and Nilsson 2012), pancrustacean eyes pression in those eyes, and we infer phylogenetic are of particular interest to evolutionary biologists. histories of two kinds of pancrustacean opsins, How the diversity and exquisite functionality of visual r-opsins (rhabdomeric opsins) and peropsins. those eyes evolved are enduring questions. While From their last common ancestor, pancrustaceans some , like Drosophila, are well studied inherited two fundamentally different types of ce- (Buschbeck and Friedrich 2008; Wernet et al. phalic visual organs (Paulus 1979, 2000; Bitsch and 2015), we know far less about groups other than Bitsch 2005; Nilsson and Kelber 2007): lateral and winged insects. In recent years, new information on median eyes. Lateral eyes typically are compound the phylogenetic relationships of Pancrustacea (Misof eyes; they consist of up to several thousand omma- et al. 2014; Oakley et al. 2013) and genomic and tidia, individual photoreceptive units, and form a transcriptomic datasets for a wider range of taxa convex retina with an overall erect image. In con- have become available. This offers great potential to trast, median eyes, called nauplius eyes in crustaceans gain a better understanding of the evolutionary his- and median ocelli in insects, are usually single- tory of pancrustacean eyes and their components. chambered eyes, which possess a concave retina

ß The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: [email protected]. 2 M. J. Henze and T. H. Oakley and produce an inverted image (Land and Nilsson (Supplementary Table S3). Unless predicted prote- 2012). omes were available, we used ORF-Finder to detect All animal eyes (with a probable exception in a open reading frames larger than 100 amino acids in sponge; Rivera et al. 2012) express opsins to form length. These sequences were analyzed by phyloge- functional visual pigments sensitive to light. netically informed annotation (PIA; Speiser et al. Arthropods rely on r-opsins for vision, with different 2014), which employs BLASTP to find opsin-like genes specialized for sensitivity to different wave- genes and subsequently applies an evolutionary lengths (Briscoe and Chittka 2001; Arikawa and placement algorithm (Berger and Stamatakis 2011) Stavenga 2014; Cronin and Porter 2014). Previous to determine the most likely placement of a gene research suggests that the ancestral arthropod had on a pre-calculated phylogeny of opsins. To our at least three (Koyanagi et al. 2008) or four dataset from GenBank, we added sequences from ge- (Kashiyama et al. 2009) r-opsins. However, those nomes and transcriptomes that PIA placed within the analyses were based on limited sampling of taxa. arthropod r-opsin clade (Supplementary Table S4). Downloaded from Apart from r-opsins, arthropods have other For all opsins in our list, we revised the available opsins, including c-opsins (ciliary opsins/pteropsins) literature on expression in lateral and/or median expressed in brains (Velarde et al. 2005; Eriksson eyes (Supplementary Tables S2 and S4; et al. 2013), arthropsins of unknown function Supplementary Figs. S1 and S2). (Colbourne et al. 2011), and peropsins/retinal G- http://icb.oxfordjournals.org/ protein-coupled receptors (RGRs) of unknown func- Phylogenetic analyses tion, but known to be expressed in eyes, at least in We conducted phylogenetic maximum-likelihood Limulus, the spiders Hasarius adansoni and analyses in SATe´-II (simultaneous alignment and Cupiennius salei, and possibly in tree estimation; Liu et al. 2012) using onychophoran (Nagata et al. 2010; Eriksson et al. 2013; Battelle opsins (onychopsins) to root the visual r-opsin tree et al. 2015; Futahashi et al. 2015). While searching of arthropods (Hering et al. 2012). Within SATe´-II for r-opsins in transcriptomes, we also found candi- we adopted VT, the best-fit model chosen by at University of California, Santa Barbara on August 31, 2015 date peropsins from multiple pancrustacean groups, ProtTest (Darriba et al. 2011), and applied RAxML indicating that these genes are more common than (Stamatakis 2006) for co-alignment and simulta- previously appreciated. neous topology search. Afterward, we generated 100 The picture that emerges from our analyses is that bootstrap pseudoreplicates using RAxML alone. To visual evolution in Pancrustacea was amazingly dy- infer the evolutionary history of r-opsin genes, we namic. Eyes were lost many times, opsins changed next assumed a phylogeny of arthropod species expression from one type of eye to another, and (Figs. 1 and 2) based on recently published phyloge- opsins of ancient families re-duplicated or were nomic analyses (Oakley et al. 2013; Misof et al. 2014) fully lost in different patterns across the expanse of and reconciled our r-opsin tree (Supplementary Figs. pancrustacean history. S1 and S2) with the species tree in NOTUNG (Durand et al. 2006). Nodes with 590% support in Methods the r-opsin tree were allowed to be rearranged to Occurrence of lateral and median eyes minimize duplications and losses, with duplications being penalized four times more than losses (Fig. 2; We compiled literature on the presence and absence Supplementary Fig. S3). of lateral and median eyes in major pancrustacean taxa (Supplementary Table S1) and displayed the re- Panarthropod peropsin-like genes sults on two recently published arthropod phyloge- We attempted to resolve the phylogenetic placement nies (Oakley et al. 2013; Misof et al. 2014; Fig. 1). of arthropod peropsin/RGR (Battelle et al. 2015) and RGR-like genes (Futahashi et al. 2015) along with Visual r-opsins related sequences we discovered in GenBank and in Sampling of taxa/genes published transcriptomes. Therefore we analyzed Focusing primarily on pancrustacean species with almost 500 opsins, gathered as follows: The characterized opsin expression, we downloaded Uniref90 database (Magrane and Consortium 2011) amino-acid sequences of panarthropod r-opsins was searched twice with BLASTP, first using Limulus from GenBank (Supplementary Table S2). In addi- peropsin (AIT75833) and then human RGR tion, we searched genomes or transcriptomes from (NP_001012738) as queries. For each search, we re- exemplars of major taxonomic groups and key spe- tained the 250 most similar hits. Odonate RGR-like cies such as basal-branching hexapods sequences were not yet incorporated into Uniref90, Pancrustacean eyes and opsins 3 Downloaded from http://icb.oxfordjournals.org/ at University of California, Santa Barbara on August 31, 2015

Fig. 1 Occurrence of lateral (L) and median (M) eyes in Pancrustacea. Median eyes refer to nauplius eyes in Crustacea sensu stricto as defined by Elofsson (2006) and to median ocelli in Insecta. Lateral eyes denote compound eyes or modifications thereof with lateral connections to the protocerebrum. ‘-’ indicates that no eyes of the respective type have been found in any developmental stage, sex, or morph of a living species. Limulus polyphemus (Chelicerata; Battelle 2006) and (; Mu¨ller et al. 2003) are added as representatives of non-pancrustacean arthropods. The phylogeny in (a) and (b) is based on Oakley et al. (2013) and Misof et al. (2014), respectively. For further references see Supplementary Table S1.

so we conducted a third BLAST search looking for analysis (we assumed a WAGþG20 model), matches to a Bombyx peropsin-like gene MAFFT (Katoh and Standley 2013) for alignment (XP_004930922) on GenBank and kept all 18 top and MUSCLE (Edgar 2004) for merging (Fig. 3; ecdysozoan hits. Finally, we added 27 sequences of Supplementary Fig. S4). the RGR/peropsin (¼RPE/peropsin) clade of Hering and Mayer (2014). We combined these datasets and Results removed duplicates found in multiple searches, as well as three long-branch opsins, which were unsta- Occurrence of lateral and median eyes ble in their phylogenetic position in informal, pre- In each of the three major pancrustacean clades pro- liminary analyses. The amino-acid sequences of the posed by Oakley et al. (2013), namely Oligostraca, remaining 494 opsins were analyzed using SATe´-II fast Multicrustacea, and Allotriocarida, entire classes or settings (Liu et al. 2012) with a maximum subproblem subclasses are missing one type of eye or both (Fig. size of 200, blind-mode stopping rule, and iteration 1a). Within Oligostraca, eyes are completely un- limit of 1. SATe´-II ran FastTree (Price et al. 2010) known in Mystacocarida and Pentastomida (Osche for approximate maximum-likelihood phylogenetic 1963; Elofsson 1966; Elofsson and Hessler 2005; 4 4 M.M. J. Henze J. Henze and and T. H.T. H. Oakley Oakley Downloaded from Downloaded from http://icb.oxfordjournals.org/ http://icb.oxfordjournals.org/ at University of California, Santa Barbara on August 31, 2015 at University of California, Santa Barbara on August 31, 2015

Fig.Fig. 2 2EvolutionaryEvolutionary history history of ofvisual visual r-opsins r-opsins in inPancrustacea. Pancrustacea. We We adopted adopted the the illustrated illustrated phylogeny phylogeny of ofarthropod arthropod species species (top (top left) left) from from MisofMisof et et al. al. (2014) (2014)andandOakleyOakley et et al. al. (2013) (2013). For. For each each species, species, amino-acid amino-acid sequences sequences of of r-opsins r-opsins were were downloaded downloaded from from GenBank GenBank (Supplementary(Supplementary Table Table S2), S2 or), or identified identified in in published published genomes genomes or or transcriptomes transcriptomes (Supplementary (Supplementary Tables Tables S3 S3andandS4)S4 and) and subjected subjected to to phylogeneticphylogenetic maximum-likelihood maximum-likelihood analyses analyses using using RAxML RAxML and and SATe SATe´-II´-II (Supplementary (Supplementary Figs. Figs. S1 S1andandS2).S2 We). We reconciled reconciled the the opsin opsin tree tree withwith the the species species tree tree in inNOTUNG NOTUNG (condensed (condensed result result below, below, detailed detailed tree tree in inSupplementarySupplementary Fig. Fig. S3) S3 to) to assess assess the the number number of of opsin opsin (continued)(continued) Pancrustacean eyes and opsins 5

Brenneis and Richter 2010). Lateral eyes are absent in et al. 2004; Fanenbruck and Harzsch 2005; podocopan ostracods, whereas their median (nau- Koenemann et al. 2009). plius) eyes can be highly developed (Tanaka 2005; Below the level of class and subclass, there is con- Elofsson 2006). Within Multicrustacea, copepods siderable variation in the distribution of eyes as well. also lack lateral eyes, but have sophisticated median Within myodocopan ostracods, all halocyprids lack eyes (Elofsson 2006). Cephalocarida and Remipedia, eyes, but myodocopids have both lateral and median two classes within Allotriocarida, are eyeless alto- eyes (Oakley and Cunningham 2002). Thecostraca gether (Elofsson and Hessler 1990; Fanenbruck are quite diverse in whether eyes are present or Downloaded from http://icb.oxfordjournals.org/ at University of California, Santa Barbara on August 31, 2015

Fig. 3 Phylogeny of pancrustacean peropsins. The tree summarizes the results of our SATe´-II analyses of the amino-acid sequences of 494 opsins, focusing on group-4 opsins. Support values mentioned in the text and arthropod taxa are highlighted. For a detailed tree, see Supplementary Fig. S4. (This figure is available in black and white in print and in color at Integrative and Comparative Biology online.)

Fig. 2 Continued genes in the ancestral arthropod (named LW1, LW2, MW1, MW2, SW) and to estimate the timing of duplications (indicated by lowercase letters and numbers) and losses (broken lines). Our main results are summarized next to the species tree (top right), with boxes representing opsin genes and duplications as subdivisions. The unnamed upper and lower compartment of SWc2 in Pterygota illustrates SWc2.1 and SWc2.2, previously known as the UV and blue clade, respectively. Inferred loss of an opsin gene is indicated by ‘-’, blank boxes represent genes that were lost earlier in evolution. The LW2 sequence of Scutigera (*) is very short, and only present in the sequence read archive (SRA), not in the final assembly of the transcriptome. Three r-opsin sequences of Neogonodactylus that belong to the MW1 clade (**) are not yet deposited in GenBank, but reported in a thesis (Bok 2013). B.: Branchinella, E.: Euphilomedes. (This figure is available in black and white in print and in color at Integrative and Comparative Biology online.) 6 M. J. Henze and T. H. Oakley absent (Pe´rez-Losada et al. 2012). Several neopteran and flies arose in and Diptera, respectively insect orders possess only lateral and no median eyes (Supplementary Figs. S1 and S3). The RhLWD opsin (median ocelli) (Fig. 1b; references in Supplementary that is typically expressed in the median eyes of adult Table S1). Even members of the same pancrustacean dragonflies (Futahashi et al. 2015) is nested within family, , or species (age-related, sexual, and/or other odonate LW sequences and is neither known in caste polymorphism) can differ by missing one type Ephemeroptera nor in Neoptera. However, since of eye or both (Kalmus 1945; Parry 1947; Buschbeck RhLWD orthologs are found in Zygoptera as well et al. 2003; Speiser et al. 2013). as in Anisoptera (damselflies like Ischnura and true dragonflies like ), their origin must coin- Expressionofvisualr-opsinsinlateralandmedianeyes cide with or predate the last common ancestor of all living odonate species about 250 mya (Misof et al. The expression of r-opsins has only been investigated 2014). The gene duplication that resulted in the eye- in very few Pancrustacea, even if different techniques Downloaded from specific Rh1-Rh2 paralogs of flies like Musca and to confirm or exclude their existence at the transcrip- Drosophila is probably at least 100 million years tional, translational, and physiological level (proof of younger and happened within the dipteran lineage. mRNA, protein, and function) are taken into ac- Rh1 and Rh2 orthologs have not been identified in count (Supplementary Figs. S1 and S2). All ultravi- non-dipteran insects, and are absent from the olet (UV) opsins identified in median eyes to date http://icb.oxfordjournals.org/ genome of the mosquitoes Anopheles gambiae are expressed in lateral eyes as well (Apis mellifera (Velarde et al. 2005) and Aedes aegypti (Nene et al. UV, Bombus impatiens UV, Triops granarius RhC; 2007), early-branching Diptera. Spaethe and Briscoe 2005; Velarde et al. 2005; Kashiyama et al. 2009) with one possible exception (Gryllus bimaculatus; Henze et al. 2012). Similarly, Phylogeny of visual r-opsins Limulus, a chelicerate with lateral compound and We have analyzed the phylogeny of visual r-opsins median eyes, relies on the same UV opsin in both from representatives of all major pancrustacean at University of California, Santa Barbara on August 31, 2015 types of eyes (Battelle et al. 2014). Opsins that tune clades proposed by Oakley et al. (2013), including the visual pigment to longer wavelengths, however, new data on key taxa such as early-branching hexa- are differentially expressed in Limulus (Battelle et al. pods, for which no opsin sequences were known. 2015) and in most pancrustaceans investigated so far: Our results (Fig. 2; Supplementary Figs. S1–S3) sug- eye-specific opsin paralogs have been found in myo- gest that the ancestral pancrustacean likely had four docopid ostracods (Oakley and Huber 2004), dra- opsins that we call arthropod LW2, MW1, MW2, gonflies (Odonata; Futahashi et al. 2015), crickets and SW (long-, middle-, and short-wavelength), (Orthoptera; Henze et al. 2012), flies (Diptera; based on the maximal spectral sensitivity (where Pollock and Benzer 1988), and bees (Hymenoptera; known) of visual pigments formed by opsins of the Velarde et al. 2005). Exceptions are the branchiopod respective clade in living species. Mandibulata, in- Triops granarius and the hemipteran insect cluding pancrustaceans, may have lost a fifth opsin Nilaparvata lugens, which express all or a subset of (LW1) present in the ancestral arthropod, but known their lateral-eye opsins in median eyes (Kashiyama so far only from Limulus polyphemus. We also infer et al. 2009; Matsumoto et al. 2014). additional r-opsin gene duplications. SW underwent The duplications that gave rise to eye-specific two duplications before the ancestor of Allotriocarida opsin paralogs are phylogenetically much younger plus Multicrustacea, resulting in SWa, SWb, and than are arthropods or pancrustaceans as taxa. A SWc. Before the ancestor of Allotriocarida, MW1 relaxed molecular-clock analysis of lateral- and duplicated, yielding MW1a, MW1b, and MW1c, median-eye opsins of ostracods suggests that they and SWc duplicated, yielding SWc1 and SWc2. originated within Myodocopida about 200 million Finally, LW2 duplicated before the ancestor of years ago (mya) (Oakley et al. 2007). In insects, at Hexapoda, yielding LW2a and LW2b, and SWc2 du- least three evolutionary lines exist, based on the pres- plicated before the ancestor of Pterygota, yielding ence and absence of genes and their phylogeny. SWc2.1 and SWc2.2. While the LW1-LW2 paralogs of Hymenoptera and We failed to detect MW1a, SWa, or SWb in any GreenA-GreenB paralogs of Orthoptera might go hexapod, suggesting that those opsin genes were lost back to an early duplication of a long-wavelength before the hexapod ancestor. While our data show (LW) opsin gene in hexapods (Fig. 2; that MW1c and MW2 are still present in Sminthurus Supplementary Fig. S3) more than 450 mya (Misof viridis (Collembola), a basal-branching hexapod, they et al. 2014), eye-specific opsin paralogs of dragonflies have not been identified in any insect species so far, Pancrustacean eyes and opsins 7 and were thus probably lost before the ancestor of (two sequences) and Stigodyphys mimosarum insects. We found MW1b in Sminthurus and in (Arachnida); Machilis hrabei, Plutella xylostella Machilis hrabei (Archaeognatha), but not in the tran- (three sequences) and Bombyx mori (Insecta). scriptome of Tricholepidion gertschi (Zygentoma). The phylogenetic results of our peropsin/RGR Because genes of the MW1b clade are also unknown analyses are sensitive to which genes are sampled from the comparatively well-investigated Pterygota, and to assumptions of the model. Nevertheless, cer- we assume that MW1b was lost early in insect evo- tain consistencies emerge. We find two reasonably lution, either before the ancestor of Dicondylia supported clades of arthropod ‘peropsins’, one of (Zygentoma and Pterygota) or before the ancestor non-insects, the other of insects (Fig. 3; of Pterygota, implying that none of the MW clades Supplementary Fig. S4). Like previous authors was retained in winged insects. (Hering and Mayer 2014; Battelle et al. 2015), we The amino-acid sequences of the opsins in our obtained consistent and strong support (0.952) for arthropod SW clade (Supplementary Fig. S3) and a clade of peropsins from non-insects, including Downloaded from physiological data (Supplementary Fig. S2; for refer- opsins from Limulus, spiders, and the mite ences see Supplementary Tables S2 and S4) suggest Tetranychus. Our current analyses add to this clade that the SW opsin of the ancestral arthropod prob- another spider opsin (Stegodyphus), two more mite ably formed a UV-sensitive visual pigment. Most of opsins (Metaseiulus), and opsins from a crustacean http://icb.oxfordjournals.org/ the opsins in our SW clade, including Limulus (Argulus), and a myriapod (Scutigera). We also UVOps, have a lysine residue (K) at, or next to, report insect ‘peropsins’. Our new insect opsins the position corresponding to glycine 90 in bovine (from Plutella, Bombyx, and Machilis) form a clade rhodopsin (shifts by one amino acid occurred de- (moderate support of 0.775) with odonate RGR-like pending on the alignment), which was identified as sequences (Futahashi et al. 2015). The arthropod the basis for UV vision in invertebrates (Salcedo non-insect and insect peropsin clades are part of a et al. 2003). larger clade (with very weak support of 0.046) that

Of the three subclades of UV opsins present in comprises opsins from mollusks, an annelid at University of California, Santa Barbara on August 31, 2015 Multicrustacea and Allotriocarida (SWa, SWb, and (Platynereis), a rotifer (Brachionus), an acorn worm SWc), Hexapoda only retained SWc. SWc1 com- (Saccoglossus), and a crustacean (Calanus). We pro- prises a clade of poorly characterized opsins known visionally refer to these opsins as ‘peropsins’, because as Drosophila Rh7-like sequences, some of which they form a rather well-supported clade (0.848) with might have undergone neo-functionalization (Izutsu chordate peropsins. et al. 2012; Kistenpfennig 2012; Hu et al. 2014). SWc2, in contrast, gave rise to the subclades Discussion SWc2.1 and SWc2.2, which are well-known as the Relation between visual r-opsin and type of eye insect UV- and blue-opsin clades. Interestingly, a Developmental and morphological evidence suggests lysine residue at the spectral tuning site suggests that the median eyes of Pancrustacea and Limulus that some of the odonate sequences in the insect are homologous with onychophoran eyes (Mayer ‘blue’ clade might actually be UV opsins 2006). The last common ancestor of arthropods pre- (Supplementary Fig. S3). It is therefore possible sumably inherited a similar pair of ocellus-like that UV sensitivity was lost several times indepen- organs, which were lost in Myriapoda and modified dently in the SWc2.2 clade of Pterygota. This as- by duplication and fusion in the other euarthropod sumption is supported by different amino-acid lineages (Paulus 2000; Bitsch and Bitsch 2005; residues that substitute lysine in Ephemeroptera (glu- Lehmann et al. 2012). Lateral compound eyes, in tamine), Odonata (histidine), and other insect spe- contrast, appeared in the arthropod stem group cies (asparagine, glutamic acid, or methionine), but more than 500 mya (Paterson et al. 2011) and exist leaves unanswered the question why no such changes today in representatives of Chelicerata (e.g., ever occurred in the SWc1.2 clade. Limulus), Myriapoda (e.g., Scutigera), and Panarthropoda (Paulus 2000; Bitsch and Bitsch Peropsins 2005). Even though compound eyes were trans- From the following arthropod species, we identified formed in many taxa and might have evolved from 11 new sequences that are similar to previously de- a simple progenitor along several independent line- scribed peropsin/RGR-like genes: Argulus siamensis ages (Nilsson and Kelber 2007), the lateral eyes of all and Calanus finmarchicus (Crustacea); Scutigera euarthropods and in particular those of pancrusta- coleoptrata (Myriapoda); Metaseiulus occidentalis ceans are considered homologous, based on 8 M. J. Henze and T. H. Oakley developmental data (Harzsch and Hafner 2006) and mechanisms of opsin co-expression, which is not comparative studies of the nervous system (Harzsch uncommon in photoreceptors of arthropods et al. 2005). (Arikawa et al. 2003; Jackowska et al. 2007; Mazzoni Our phylogeny of r-opsins expressed in arthropod et al. 2008; Rajkumar et al. 2010; Schmelingetal. photoreceptors (Supplementary Figs. S1–S3) does 2014; Battelle et al. 2015), could therefore help to not reflect a separate evolutionary history of opsins understand switches of opsins between median and in median versus lateral eyes from the early lateral eyes. onwards, that is, for the time span The most obvious cause for the co-option of during which the organs presumably diverged. opsins from the other type of eye would be the Opsins in living species often are shared between loss and regain of the whole visual organ, as pro- both types of eyes, which is especially (though not posed for the compound eyes of myodocopid ostra- exclusively) true for UV opsins. Eye-specific opsin cods (Parker 1995; Oakley and Cunningham 2002; paralogs exist, but originate from comparatively Oakley 2003). Myodocopida (subclass Myodocopa) Downloaded from young duplications that occurred independently in are nested phylogenetically within ostracod groups different taxa. that have no eyes or only median eyes. Other pan- Furthermore, there is evidence that opsin expres- crustacean taxa, such as some Rhizocephala sion switched between types of eyes more than (Thecostraca; Pe´rez-Losada et al. 2012) and all in- once. In other words, opsins expressed in median sects (Hexapoda), are in a similar position. http://icb.oxfordjournals.org/ eyes were recruited from opsins expressed in lateral Remipedia, the putative sister group to Hexapoda, eyes and vice versa. All r-opsins identified as part of lack eyes (Fig. 1a; Fanenbruck et al. 2004), and we visual pigments in photoreceptors of arthropods have not found visual r-opsins in the transcriptome (together with the enigmatic ingroup SWc1, which of the remiped Xibalbanus (Speleonectes) tulumensis comprises the Drosophila Rh7-like sequences of (Fig. 2). Protura and Diplura, two of the three basal- unknown function) form a monophyletic sister branching hexapod orders, are also blind (Fig. 1b;

clade to onychophoran visual r-opsins (Hering Bitsch and Bitsch 2000). at University of California, Santa Barbara on August 31, 2015 et al. 2012; Eriksson et al. 2013; Beckmann et al. Since the reduction of visual organs is a recurrent 2015). Thus, the opsins of arthropod lateral eyes feature in the phylogeny of Pancrustacea (Fig. 1), originally must have been co-opted from median and evidence for an independent origin of some lat- eyes. The opposite scenario has also taken place. eral as well as median eyes has been presented RhLWD opsins, for example, are preferentially ex- (Oakley and Cunningham 2002; Elofsson 2006), pressed in the median eyes of adult dragonflies and ingroups and outgroups of those taxa, that lack nested within odonate opsins (Supplementary Figs. one or both types of eyes, are interesting targets S1 and S3) primarily expressed in the larvae or in for investigations of r-opsin expression. Species, in the compound eyes of adults (Futahashi et al. 2015). which a type of eye is missing in part of the popu- In some odonate species, particularly in those that lation, as in many insects with winged and wingless lost the RhLWD ortholog, LW opsins of group E morphs, could also help to gain insight into changes (compound eyes of adults) or C (larvae) became that occur, when eyes are reduced. In the fig wasp specific to the median eyes of the adult. This dem- Ceratosolen solmsi, for example, only females bear onstrates that the involved regulatory mechanisms median eyes, but, surprisingly, the LW2 ortholog, are quite flexible. which is specific for median eyes in bees, is the Is there a general pattern, in which opsin expres- major opsin gene expressed in males (Wang et al. sion switches between types of eyes? Losing the gene 2013). for the major opsin in one type of eye is a plausible The expression pattern of visual r-opsins has been explanation for why a change in the expression pat- studied in just a few pancrustaceans with a bias tern of another opsin gene might be evolutionarily toward pterygote insects (Wernet et al. 2015), nota- advantageous. However, the scenario that both bly butterflies (Wakakuwa et al. 2007; Briscoe 2008). events coincide (loss of gene A and change in expres- Median eyes were often neglected in species that pos- sion pattern of gene B) is less likely than the preced- sess both types of eyes (Supplementary Figs. S1 and ing expansion of gene B expression to photoreceptors S2). In many cases opsin expression was investigated that express gene A. This might be the case in with mRNA only, which leaves the possibility that Ischnura asiatica, in which an LW opsin of group E translation into protein does not take place and is preferentially expressed in the median eyes of adults no functional visual pigment exists. Especially results together with the typical RhLWD (Supplementary Fig. that were obtained by reverse transcription S1; Futahashi et al. 2015). Studying the reasons and polymerase chain recation (RT-PCR) alone have to Pancrustacean eyes and opsins 9 be treated with caution, since opsin sequences can be duplication event back to the last common ancestor amplified from cDNA libraries of tissue, in which of Hexapoda (Fig. 2; Supplementary Fig. S3). they are neither detectable by in situ hybridization MW opsin sequences, which are diverse in non- nor at the protein level (Battelle et al. 2015). More hexapod crustaceans, were not known from detailed data on species from a wider range of taxa Hexapoda (Cronin and Porter 2014), but only hopefully will fill many of the gaps that are left in Pterygota had been investigated. We identified our understanding of the evolutionary history of MW1 and MW2 in Collembola, and MW1 in eyes. Archaeognatha, whereas we did not find any MW sequences in Tricholepidion gertschi (Zygentoma) or Ephemera danica (Ephemeroptera). Thus, opsins of Evolutionary history of visual r-opsins both MW clades were present in the ancestral hexa- The evolutionary history of visual r-opsins in pod and obviously were lost stepwise in the evolu- Pancrustacea is by no means less dynamic than the tion of insects. Downloaded from evolutionary history of the eyes themselves. Our re- The lack of MW opsins in Pterygota coincides sults suggest that the ancestral pancrustacean in- with the exchange of lysine at the position responsi- herited four of five opsins that were present in its ble for UV tuning in opsins of the SWc2.2 clade. arthropod ancestor (Fig. 2; Supplementary Figs. This supports the speculation raised by Bok (2013) http://icb.oxfordjournals.org/ S1–S3). We named these opsins arthropod LW2, that it might have been the loss of MW opsins that MW1, MW2, and SW, following the tradition to caused the evolutionary pressure to tune some insect label visual opsins according to the spectral sensitiv- SW opsins to longer wavelengths. ity of the visual pigments they form. We are aware While some of our clades are distinct in our orig- that the sensitivities may vary in our clades to an inal r-opsin tree (Supplementary Figs. S1 and S2), extent that could exceed the spectral range associated others are only resolved after reconciliation with with LW, MW, or SW, a common problem of this the species phylogeny (Fig. 2; Supplementary Fig. terminology. In addition, some opsins such as mem- S3), which shuffled poorly supported nodes of the at University of California, Santa Barbara on August 31, 2015 bers of the SWc clade might not even form visual gene tree to minimize the implied number of dupli- pigments. Yet, based on the current data, the selected cations and losses. These inferences could be sensi- names represent the best possible guess as to the tive to the addition of new data and may thus spectral sensitivities that the visual pigments change. formed by the different opsins might have had in Apart from r-opsin duplications within higher the ancestral arthropod. taxa, we also inferred many duplications in terminal Presumably starting from a set of four opsins, the branches of our phylogeny, but we do not document opsin repertoire of Pancrustacea soon diversified by those here, because they could be caused by errors in two duplications in the SW clade, before assembling transcriptomes or annotating genomes. Multicrustacea and Allotriocarida split, and by one Similarly, we do not describe terminal losses of more duplication in the SW clade and two duplica- genes, because we and previous investigators may tions in the MW1 clade, before Branchiopoda and have simply failed to detect genes in transcriptomes, Hexapoda split, amounting to at least nine different genomes, or in species that were investigated by opsin clades in pancrustaceans at the time, when the RT-PCR. There is good evidence, though, that last common ancestor of Allotriocarida lived (one visual r-opsins diversified extensively in certain LW, three MW1, one MW2, and four SW opsins). lower pancrustacean taxa, such as ostracods, stoma- This is a surprising number considering that verte- topods, dragonflies, and butterflies (Oakley and brates only possess five visual opsin classes, which Huber 2004; Briscoe 2008; Porter et al. 2009, 2013; did not diversify much except in bony fish Futahashi et al. 2015). Given that only few pancrus- (Bowmaker 2008). tacean species are investigated so far, more examples Unlike in the MW1 and SW clades, we did not are sure to follow in the future. find any evidence for duplications in the LW2 clade early in pancrustacean evolution. This is the most Peropsins diverse visual opsin clade in insects, however, and Arthropods possess genes similar to chordate previous authors have therefore predicted a duplica- peropsins and/or vertebrate RGRs (Eriksson et al. tion of the insect LW opsin gene prior to the emer- 2013; Hering and Mayer 2014; Battelle et al. 2015; gence of Neoptera (Spaethe and Briscoe 2004). Our Futahashi et al. 2015). Our analyses indicate that data support this assumption and move the they may be more closely related to peropsins 10 M. J. Henze and T. H. Oakley than to RGRs (Fig. 3; Supplementary Fig. S4). Even Funding though this conclusion is sensitive to specific choices T.H.O. acknowledges DEB-1146337 from the about phylogenetic analysis, we provisionally refer to National Science Foundation, and M.J.H. Vr 621– these opsins as arthropod peropsins. Arthropod per- 2009–5683 and Vr621–2012–2212 from the Swedish opsins are now known from chelicerates, crustaceans, Research Council. myriapods, and insects, implying that the ancestor of arthropods had at least one peropsin. Presence in a Supplementary data rotifer and several lophotrochozoans suggests that Supplementary Data available at ICB online. peropsin is as old as the common ancestor of pro- tomstomes, and the existence of the gene in deutero- References stomes pushes its origin back to the common Arikawa K, Mizuno S, Kinoshita M, Stavenga DG. 2003. ancestor of bilaterians. Multiple distantly related

Coexpression of two visual pigments in a photoreceptor Downloaded from genes are found in mollusks like Lottia, indicating causes an abnormally broad spectral sensitivity in the eye ancient duplications. However, our current analyses of the butterfly Papilio xuthus. J Neurosci 23:4527–32. are uncertain enough to make specific assignments of Arikawa K, Stavenga DG. 2014. Insect photopigments: photo- orthology difficult. Furthermore, we did not include receptor spectral sensitivities and visual adaptations. In: Hunt DM, Hankins MW, Collin SP, Marshall NJ, editors.

related genes from cnidarians (including those called http://icb.oxfordjournals.org/ ‘‘cnidops’’; Plachetzki et al. 2007), because the Evolution of visual and non-visual pigments. New York: Springer. p. 137–62. searches described in the ‘‘Methods’’ section did Battelle BA. 2006. The eyes of Limulus polyphemus not recover any of them, and because they are only (Xiphosura, Chelicerata) and their afferent and efferent distantly related to arthropod opsins, the focus of projections. Arthropod Struct Dev 35:261–74. this study. Nevertheless, they could shed more light Battelle BA, Kempler KE, Harrison A, Dugger DR, Payne R. on the deep history of group-4 opsins. 2014. Opsin expression in Limulus eyes: a UV opsin is ex- The functions of peropsins in pancrustaceans are pressed in each eye type and co-expressed with a visible

light-sensitive opsin in ventral larval eyes. J Exp Biol at University of California, Santa Barbara on August 31, 2015 poorly understood. In dragonflies, a peropsin (RGR- 217:3133–45. like) gene is expressed in the head of larvae and in Battelle BA, Kempler KE, Saraf SR, Marten CE, Dugger DR, the compound eyes of adults, though to a much Jr., Speiser DI, Oakley TH. 2015. Opsins in Limulus eyes: lower extent than genes of most visual r-opsins characterization of three visible light-sensitive opsins (Futahashi et al. 2015). There is a little more data unique to and co-expressed in median eye photoreceptors outside of pancrustaceans, suggesting some connec- and a peropsin/RGR that is expressed in all eyes. J Exp Biol 218:466–79. tion to eyes in chelicerate arthropods. Peropsin was Beckmann H, Hering L, Henze MJ, Kelber A, Stevenson PA, detected in eyes of the wandering spider Cupiennius Mayer G. 2015. Spectral sensitivity in Onychophora (velvet salei (Eriksson et al. 2013), in non-visual cells in the worms) revealed by electroretinograms, phototactic behav- retina of the jumping spider Hasarius adansoni iour and opsin gene expression. J Exp Biol 218:915–22. (Nagata et al. 2010), and in membranes of cells clo- Berger SA, Stamatakis A. 2011. Aligning short reads to refer- sely associated with photoreceptors in Limulus poly- ence alignments and trees. Bioinformatics. 27:2068–75. phemus (Battelle et al. 2015). A functional study of Bitsch C, Bitsch J. 2000. The phylogenetic interrelationships of the higher taxa of apterygote hexapods. Zool Scr 29:131–56. peropsin from Hasarius adansoni indicates molecular Bitsch C, Bitsch J. 2005. Evolution of eye structure and ar- properties of a photoisomerase and a bistable nature thropod phylogeny. In: Koenemann S, Jenner RA, Schram (Nagata et al. 2010). FR, editors. Crustacea and arthropod relationships. New York: CRC Press. p. 185–214. Bok MJ. 2013. The physiological, ecological, and evolutionary basis of polychromatic ultraviolet sensitivity in stomatopod Acknowledgments crustaceans [dissertation]. [Baltimore County]: University of Maryland. We are grateful to Sherry Tamone and Jon Harrison Bowmaker JK. 2008. Evolution of vertebrate visual pigments. for initiating this collaboration by inviting us to the Vision Res 48:2022–41. symposium ‘‘Linking Insects with Crustacea.’’ We Brenneis G, Richter S. 2010. Architecture of the nervous also thank Rolf Elofsson, Michael Bok, Nicolas system in Mystacocarida (Arthropoda, Crustacea) - an Lessios, and Dan Nilsson for advice and providing immunohistochemical study and 3D reconstruction. J Morphol 271:169–89. literature, Almut Kelber for fruitful discussions, and Briscoe A, Chittka L. 2001. The evolution of color vision in the editor and two anonymous reviewers for valu- insects. Annu Rev Entomol 46:471–510. able comments on an earlier version of the Briscoe AD. 2008. Reconstructing the ancestral butterfly eye: manuscript. focus on the opsins. J Exp Biol 211:1805–13. Pancrustacean eyes and opsins 11

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L M mRNAproteinfunction 0.93 M Eoperipatus sp. [AFM43711] onychopsin yes M Principapillatus hitoyensis [AFM75825] onychopsin*** yes 1 1 M Ooperipatus hispidus [AFM43712] onychopsin yes M Euperipatoides rowelli [AFM75824] onychopsin yes M Phallocephale tallagandensis [AFM43710] onychopsin yes M Limulus polyphemus [AIT75830] Ops6 no yes 1 M Limulus polyphemus [AIT75831] Ops7 no yes LW1 0.91 M Limulus polyphemus [AIT75832] Ops8 no yes Limulus polyphemus [AAA02499] Ops2 yes no 1 Limulus polyphemus [AAA02498] Ops1 yes no 0.97 0.94 Argulus siamensis [401512162] rOp1 ? ? Argulus siamensis [401532453] rOp2 ? ? [ ] 1 Argulus siamensis 401513687 rOp3 ? ? Argulus siamensis [401521393] rOp4 ? ? Argulus siamensis [401516392] rOp5 ? ? 0.90 M Heterocypris sp. [contig06320] rOp1§ yes Neogonodactylus oerstedii [ACU00223] rOp4 ? ? 1 Neogonodactylus oerstedii [ABG37008] Rh2 yes ? 1 Neogonodactylus oerstedii [ACU00224] rOp5 ? ? 1 Neogonodactylus oerstedii [ACU00222] rOp6 ? ? Neogonodactylus oerstedii [ABG37007] Rh1 yes ? 0.95 0.95 Neogonodactylus oerstedii [ACU00210] rOp7 ? ? 1 Neogonodactylus oerstedii [ACU00211] rOp8 ? ? 0.96 Neogonodactylus oerstedii [ABG37009] Rh3 yes ? 0.88 Neogonodactylus oerstedii [ACU00218] rOp9 ? ? Neogonodactylus oerstedii [ACU00217] rOp10 ? ? [ ] 1 Neogonodactylus oerstedii ACU00221 rOp11 ? ? 0.73 Neogonodactylus oerstedii [ACU00216] rOp12 ? ? Neogonodactylus oerstedii [ACU00215] rOp13 ? ? 0.71 0.75 1 Neogonodactylus oerstedii [ACU00212] rOp14 ? ? Neogonodactylus oerstedii [ACU00220] rOp15 ? ? 0.78 Neogonodactylus oerstedii [ACU00219] rOp16 ? ? Neogonodactylus oerstedii [ACU00214] rOp17 ? ? 0.73 Neogonodactylus oerstedii [ACU00213] rOp18 ? ? Sminthurus viridis [GATZ01005081] LW1 ? ? Ischnura asiatica [BAQ54909] RhLWA1 ? ? [ ] 1 Sympetrum frequens BAQ54698 RhLWA1 ? ? 0.95 Ischnura asiatica [BAQ54910] RhLWA2 yes ? 0.91 Sympetrum frequens [BAQ54699] RhLWA2 yes ? Sympetrum frequens [BAQ54700] RhLWB1 ? ? Sympetrum frequens [BAQ54701] RhLWC1 ? ? Ischnura asiatica [BAQ54911] RhLWC1 ? ? 0.97 M Ischnura asiatica [BAQ54912] RhLWD1 ? yes Arthropod LW2 M Sympetrum frequens [BAQ54702] RhLWD1 ? yes Ischnura asiatica [BAQ54916] RhLWF3 yes ? 0.71 Ischnura asiatica [BAQ54917] RhLWF4 yes ? Ischnura asiatica [BAQ54914] RhLWF1 yes ? Ischnura asiatica [BAQ54915] RhLWF2 yes ? 0.83 M Ischnura asiatica [BAQ54913] RhLWE1 ? yes Sympetrum frequens [BAQ54703] RhLWE1 yes ? 1 Sympetrum frequens [BAQ54707] RhLWF4 yes ? Sympetrum frequens [BAQ54704] RhLWF1 yes ? 0.80 Sympetrum frequens [BAQ54706] RhLWF3 yes ? Sympetrum frequens [BAQ54705] RhLWF2 yes ? Machilis hrabei [GAUM01017353] LW1 ? ? M Gryllus bimaculatus [AEG78683] GreenA no yes Drosophila melanogaster [CAB06821] Rh6 yes no 1 Musca domestica [XP_005186097] Rh6 yes no Ceratosolen solmsi [AGH15791] LW2 ? ?** 1 Bombus impatiens [AAS55406] LW2 ? ? 0.95 M Apis mellifera [DAA05740] LW2 no yes 0.87 M Drosophila melanogaster [AAA28734] Rh2 no yes 1 M Musca domestica [XP_005191160] Rh2 ? yes 1 Drosophila melanogaster [AAA28733] Rh1 yes no [ _ ] 1 Musca domestica XP 005182995 Rh1a ? no Musca domestica [XP_011291215] Rh1b ? no Musca domestica [XP_005182983] Rh1c ? no Ephemera danica [GAUK01106886] LW1 Oligostraca ? ? Ephemera danica [GAUK01109703] LW2 ? ? Ephemera danica [GAUK01002297] LW3 ? ? 1 Ephemera danica [GAUK01002300] LW4* ? ? 1 Ephemera danica [GAUK01002296] LW4* ? ? 0.78 Ceratosolen solmsi [AGH15790] LW1 ? ?** Apis mellifera [AAA69069] LW1 yes no 1 Bombus impatiens [AAS55402] LW1 ? ? Sminthurus viridis [GATZ01093387] LW2 ? ? Heliconius melpomene [HMEL003344-PA] LW yes Manduca sexta [O02464] Manop1_RhP520 yes ? Sminthurus viridis [GATZ01074098] LW3 ? ? Pediculus humanus [XP_002427337] op1 ? Nephotettix cincticeps [BAO03864] LW ? ? M Nilaparvata lugens [BAO03855] LW yes yes Machilis hrabei [GAUM01184980] LW2 ? ? Schistocerca gregaria [CAA56377] Lo1 yes ? Tribolium castaneum [NP_001155991] LW yes Gryllus bimaculatus [AEG78684] GreenB yes no Tricholepidion gertschi [GASO01008719] LW1 ? ? 0.87 Tricholepidion gertschi [GASO01008718] LW2 ? ? 0.98 Tricholepidion gertschi [GASO01008717] LW3 ? ? Arthropod MW-SW opsin see Supplementary Figure S2

§ one of four, closely related opsins * identical amino-acid sequence, but different nucleotide sequence ** median eyes only present in females *** Principapillatus hitoyensis is referred to as Epiperipatus cf. isthmicola in Hering et al. (2012)

Supplementary Fig. S1 Phylogeny of the amino-acid sequences of pancrustacean LW opsins with onychophoran visual opsins as an outgroup. Branches and branch labels are colored in red, blue, and green for Hexapoda, Multicrustacea, and Oligostraca, respectively. Onychophora and Limulus (Chelicerata) are in black. We show only bootstrap values bigger than 0.70. Opsin expression in lateral (L) and median (M) eyes is indicated as follows: verified (yes), excluded (no), unknown or unclear (?). No entry means that the type of eye is missing. Opsins known to be expressed in median eyes are highlighted and marked by a fat final branch and an M at the end of the branch. The quality of evidence is specified by gray boxes as follows: ‘mRNA’ for transcriptomics, RT-PCR, Northern blot, and in-situ hybridization; ‘protein’ for Western blot and immunohistochemistry; ‘function’ for spectropho- tometry, microspectrophotometry, and electrophysiology. For references see Supplementary Tables S2 and S4. Expression in

L M mRNAproteinfunction M Onychophoran onychopsin yes 1 Arthropod LW opsin see Supplementary Figure S1 0.97 Limulus polyphemus [ACO05013] Ops5 yes no Scutigera coleoptrata [16909] MW yes Machilis hrabei [GAUM01017675] MW ? ? M Triops granarius [BAG80977] RhB yes yes 1 Triops longicaudatus [BAG80982] RhB yes ? 1 M Triops granarius [BAG80980] RhE yes yes 1 Triops longicaudatus [BAG80999] RhE yes ? 0.98 Sminthurus viridis [GATZ01000283] MW1 ? ? Sminthurus viridis [GATZ01005173] MW2 ? ? Sminthurus viridis [GATZ01094783] MW3 ? ? Sminthurus viridis [GATZ01103981] MW7 ? ? 1 Sminthurus viridis [GATZ01010906] MW6 ? ? 1 Sminthurus viridis [GATZ01090057] MW4 ? ? Sminthurus viridis [GATZ01087993] MW5 ? ? Branchinella kugenumaensis [BAG80987] Rhb yes ?

Triops longicaudatus [BAG80998] RhD yes ? Arthropod MW1 1 M Triops granarius [BAG80979] RhD yes yes 0.99 Daphnia pulex [LOPB9] LOpB9 ? ? Daphnia pulex [LOPB1] LOpB1 ? ? 1 Daphnia pulex [LOPB15] LOpB15 ? ? Daphnia pulex [LOPB13] LOpB13 ? ? Daphnia pulex [LOPB14] LOpB14 ? ? Daphnia pulex [LOPB2] LOpB2 ? ? Daphnia pulex [LOPB7] LOpB7 ? ? 1 Daphnia pulex [LOPB8] LOpB8 ? ? 0.90 Daphnia pulex [LOPB4] LOpB4 ? ? 0.97 [ ] 1 Daphnia pulex LOPB5 LOpB5 ? ? Daphnia pulex [LOPB3] LOpB3 ? ? Daphnia pulex [LOPB6] LOpB6 ? ? Hemigrapsus sanguineus [BAA09133] Rh2 yes ? 1 Uca pugilator [ADQ01810] Rh2 yes ? 1 Uca pugilator [ADQ01809] Rh1 yes ? 1 Hemigrapsus sanguineus [BAA09132] Rh1 yes ? 0.73 Daphnia pulex [LOPA3] LOpA3 ? ? [ ] 1 Daphnia pulex LOPA2 LOpA2 ? ? 0.80 Daphnia pulex [LOPA1] LOpA1 ? ? 0.91 Daphnia pulex [LOPA4] LOpA4 ? ? 1 M Triops granarius [BAG80976] RhA yes yes 1 Triops longicaudatus [BAG80981] RhA yes ? Daphnia pulex [LOPA6] LOpA6 ? ? 0.90 0.85 Daphnia pulex [LOPA9] LOpA9 ? ? 1 Daphnia pulex [LOPA10] LOpA10 ? ? Daphnia pulex [LOPA8] LOpA8 ? ?

Sminthurus viridis [GATZ01103643] MW8 ? ? Arthropod MW2 Branchinella kugenumaensis [BAG80986] Rhd yes ? 0.82 1 Branchinella kugenumaensis [BAG80985] Rhc yes ? Calanus finmarchicus [GBFB01009482] rOp2 ? Calanus finmarchicus [GBFB01096267] rOp1 ? M Tigriopus californicus [ADZ45237] opsin yes 0.84 Vargula tsujii [ADZ45236] Vtops ? ? # 0.84 Vargula hilgendorfii [AAL37507] VhCE7 yes no Skogsbergia lerneri [AAL37539] SlCE10# yes no 1 M Skogsbergia lerneri [AAL37521] SlME27# no yes M Vargula hilgendorfii [AAL37513] VhME16# no yes Euphilomedes carcharodonta [UN2129] rOp2 ? ? 0.99 Euphilomedes carcharodonta [UN1394] rOp1 ? ? Uca pugilator [ADQ01811] Rh3 yes ? 0.97 Daphnia pulex [UVOP] UV1 ? ? 1 Neogonodactylus oerstedii [AIF73507] UV1 yes ? 1 Neogonodactylus oerstedii [AIF73509] UV3 yes ? M Limulus polyphemus [AEL29244] UVOps yes yes Daphnia pulex [UNOP2] UnOp2 ? ? 1 Daphnia pulex [UNOP1] UnOp1 ? ? Pediculus humanus [XP_002432663] Op3 ? 1 _ 0.93 Drosophila melanogaster [NP 524035] Rh7 ? ? 1 Musca domestica [XP_005181535] Rh7 ? ? Heliconius melpomene [HMEL022527-PA] Rh7-like ? ? Ischnura asiatica [BAQ54908] Rh7-like ? ? 1 Sympetrum frequens [BAQ54697] Rh7-like ? ? Machilis hrabei [GAUM01184193] UV ? ? Pediculus humanus [XP_002422743] Op2 ? ? Gryllus bimaculatus [AEG78686] UV yes no? 0.84 Schistocerca gregaria [BAP16681] LoUV yes ? 0.90 Ischnura asiatica [BAQ54920] RhUV yes ? 1 Sympetrum frequens [BAQ54713] RhUV yes ? Ephemera danica [GAUK01007309] UV* yes ? 1 Ephemera danica [GAUK01007310] UV* yes ? Nephotettix cincticeps [BAO03866] UV ? ? 0.93 Nilaparvata lugens [BAO03857] UV2 yes no 1 Nilaparvata lugens [BAO03856] UV1 yes no Ceratosolen solmsi [AGH15793] UV ? ?**

Arthropod SW 1 M Apis mellifera [AAC13418] UV yes yes 1 M Bombus impatiens [AAV67326] UV yes yes Drosophila melanogaster [AAA28856] Rh4 yes no _ 1 Musca domestica [XP 005190072] Rh3 yes no 0.94 Drosophila melanogaster [AAA28854] Rh3 yes no Tribolium castaneum [XP_970344] UV yes 1 Manduca sexta [O02465] Manop2_RhP357 yes ? Heliconius melpomene [HMEL022525-PA] UV2 yes 0.99 Heliconius melpomene [HMEL010962-PA] UV1 yes 0.93 Neogonodactylus oerstedii [AIF73508] UV2 yes ? Branchinella kugenumaensis [BAG80984] Rha yes ? Daphnia pulex [BLOP] UV2 ? ? Triops longicaudatus [BAG80983] RhC yes ? 1 Triops granarius [BAG80978] RhC yes yes Argulus siamensis [401535242] rOps6 ? ? 1 Argulus siamensis [401520393] rOps7 ? ? Musca domestica [XP_005175090] Rh5 yes no 1 Drosophila melanogaster [AAC47426] Rh5 yes no 0.98 Heliconius melpomene [HMEL010706-PA] Blue yes 1 Manduca sexta [O96107] Manop3_RhP450 yes no Nephotettix cincticeps [BAO03865] Blue ? ? Gryllus bimaculatus [AEG78685] Blue yes no 0.99 Schistocerca gregaria [CAA56378] Lo2 yes no Ceratosolen solmsi [AGH15792] Blue ? ?** 0.98 1 Apis mellifera [AAC13417] Blue yes no 1 Bombus impatiens [XP_003494923] Blue yes ? Ephemera danica [GAUK01017324] SW1 ? ? [ ] 1 Ephemera danica GAUK01003006 SW2 ? ? 1 Ephemera danica [GAUK01003004] SW4 ? ? 1 Ephemera danica [GAUK01003005] SW3 ? ? 0.97 Sympetrum frequens [BAQ54708] RhSWa1 ? ? # one of multiple opsins Sympetrum frequens [BAQ54709] RhSWb1 yes ? 1 Ischnura asiatica [BAQ54918] RhSWb1 yes ? * identical amino-acid sequence, 0.99 1 Ischnura asiatica [BAQ54919] RhSWb2 yes ? but different nucleotide sequence Sympetrum frequens [BAQ54710] RhSWc1 yes ? 0.94 Sympetrum frequens [BAQ54712] RhSWc3 yes ? ** median eyes only present in females 0.97 Sympetrum frequens [BAQ54711] RhSWc2 yes ?

Supplementary Fig. S2 Phylogeny of the amino-acid sequences of pancrustacean MW-SW opsins with onychophoran visual opsins as an outgroup. Branches and branch labels are colored in red, orange, blue, and green for Hexapoda, Branchiopoda, Multicrustacea, and Oligostraca respectively. Onychophora, Limulus (Chelicerata), and Scutigera (Myriapoda) are in black. Further details are as described for Supplementary Figure S1. Principapillatus hitoyensis [AFM75825] onychopsin 93 Eoperipatus sp. [AFM43711] onychopsin 100 Euperipatoides rowelli [AFM75824] onychopsin 100 Phallocephale tallagandensis [AFM43710] onychopsin Ooperipatus hispidus [AFM43712] onychopsin Mandibulata LOST LW1 [ ] Limulus polyphemus AIT75831 Ops7 100 Limulus polyphemus [AIT75830] Ops6 Limulus polyphemus [AIT75832] Ops8 91 [ ] 97 Limulus polyphemus AAA02498 Ops1 100 Limulus polyphemus [AAA02499] Ops2 Scutigera coleoptrata putative LW (only in SRA) Heterocypris sp. [contig06320] rOp1 90 Myodocopida LOST Argulus siamensis [401512162] rOp1 94 Argulus siamensis [401532453] rOp2 100 Argulus siamensis [401513687] rOp3 Argulus siamensis [401516392] rOp5 Argulus siamensis [401521393] rOp4 Neocopepoda LOST Decapoda LOST Neogonodactylus oerstedii [ACU00218] rOp9 Neogonodactylus oerstedii [ACU00212] rOp14 100 Neogonodactylus oerstedii [ACU00220] rOp15 95 95 Neogonodactylus oerstedii [ACU00219] rOp16 Neogonodactylus oerstedii [ACU00213] rOp18 Neogonodactylus oerstedii [ACU00214] rOp17 Neogonodactylus oerstedii [ACU00217] rOp10 100 Neogonodactylus oerstedii [ACU00221] rOp11 Neogonodactylus oerstedii [ACU00215] rOp13 Neogonodactylus oerstedii [ACU00216] rOp12 Neogonodactylus oerstedii [ABG37008] Rh2 100 Neogonodactylus oerstedii [ACU00223] rOp4 100 Neogonodactylus oerstedii [ACU00222] rOp6 100 Neogonodactylus oerstedii [ACU00224] rOp5 Neogonodactylus oerstedii [ACU00210] rOp7 Neogonodactylus oerstedii [ABG37007] Rh1 100 Neogonodactylus oerstedii [ABG37009] Rh3 96 Neogonodactylus oerstedii [ACU00211] rOp8 Branchiopoda LOST Sminthurus viridis [GATZ01005081] LW1 Machilis hrabei [GAUM01017353] LW1 Tricholepidion gertschi [GASO01008719] LW1 Ephemera danica [GAUK01106886] LW1 Ischnura asiatica [BAQ54909] RhLWA1 Sympetrum frequens LOST 100 Sympetrum frequens [BAQ54699] RhLWA2 91 Ischnura asiatica [BAQ54910] RhLWA2 95 Sympetrum frequens [BAQ54698] RhLWA1 Ischnura asiatica LOST Gryllus bimaculatus [AEG78683] GreenA Schistocerca gregaria [CAA56377] Lo1 Pediculus humanus [XP_002427337] op1 Nephotettix cincticeps [BAO03864] LW Nilaparvata lugens [BAO03855] LW Ceratosolen solmsi [AGH15790] LW1 Bombus impatiens [AAS55402] LW1 100 Apis mellifera [AAA69069] LW1 Tribolium castaneum [NP_001155991] LW Manduca sexta [O02464] Manop1_RhP520 Heliconius melpomene [HMEL003344-PA] LW Musca domestica [XP_005191160] Rh2 100 Drosophila melanogaster [AAA28734] Rh2 100 Drosophila melanogaster [AAA28733] Rh1 100 Musca domestica [XP_005182995] Rh1a LW2a Musca domestica [XP_005182983] Rh1c Musca domestica [XP_011291215] Rh1b Sminthurus viridis [GATZ01074098] LW3 Sminthurus viridis [GATZ01093387] LW2 Machilis hrabei [GAUM01184980] LW2 Tricholepidion gertschi [GASO01008717] LW3 98 Tricholepidion gertschi [GASO01008718] LW2 100 Gryllus bimaculatus [AEG78684] GreenB Schistocerca gregaria LOST Paraneoptera LOST Ceratosolen solmsi [AGH15791] LW2 100 Apis mellifera [DAA05740] LW2 95 Bombus impatiens [AAS55406] LW2 Tribolium castaneum LOST Obtectomera LOST Musca domestica [XP_005186097] Rh6 100 Drosophila melanogaster [CAB06821] Rh6 Ephemera danica [GAUK01109703] LW2 Ephemera danica [GAUK01002297] LW3 100 Ephemera danica [GAUK01002296] LW4* 100 Ephemera danica [GAUK01002300] LW4* Ischnura asiatica [BAQ54911] RhLWC1 Sympetrum frequens [BAQ54700] RhLWB1 Ischnura asiatica [BAQ54912] RhLWD1 97 Sympetrum frequens [BAQ54701] RhLWC1 Ischnura asiatica [BAQ54914] RhLWF1 Sympetrum frequens [BAQ54702] RhLWD1 Sympetrum frequens [BAQ54705] RhLWF2 Sympetrum frequens [BAQ54707] RhLWF4 Ischnura asiatica [BAQ54916] RhLWF3 Sympetrum frequens [BAQ54704] RhLWF1 Ischnura asiatica [BAQ54917] RhLWF4 Sympetrum frequens [BAQ54706] RhLWF3 Ischnura asiatica [BAQ54913] RhLWE1 LW2 LW2b Sympetrum frequens [BAQ54703] RhLWE1 Ischnura asiatica [BAQ54915] RhLWF2 Limulus polyphemus [ACO05013] Ops5 Scutigera coleoptrata [16909] MW Oligostraca LOST Neocopepoda LOST Neogonodactylus oerstedii CRB, CRS and CRV (Bok 2013) Uca pugilator [ADQ01810] Rh2 100 Hemigrapsus sanguineus [BAA09133] Rh2 100 Hemigrapsus sanguineus [BAA09132] Rh1 100 Uca pugilator [ADQ01809] Rh1 Hexapoda LOST Branchinella kugenumaensis LOST Triops longicaudatus [BAG80981] RhA 100 Triops granarius [BAG80976] RhA Daphnia pulex [LOPA3] LOpA3 100 100 Daphnia pulex [LOPA2] LOpA2 Daphnia pulex [LOPA4] LOpA4 91 Daphnia pulex [LOPA1] LOpA1 Daphnia pulex [LOPA10] LOpA10 100 Daphnia pulex [LOPA8] LOpA8 MW1a Daphnia pulex [LOPA9] LOpA9 90 Daphnia pulex [LOPA6] LOpA6 Machilis hrabei [GAUM01017675] MW Dicondylia LOST Sminthurus viridis [GATZ01005173] MW2 98 Sminthurus viridis [GATZ01000283] MW1 Branchinella kugenumaensis LOST Daphnia pulex LOST Triops longicaudatus [BAG80982] RhB 100 Triops granarius [BAG80977] RhB 100 Triops longicaudatus [BAG80999] RhE MW1b 100 100 Triops granarius [BAG80980] RhE Insecta LOST Sminthurus viridis [GATZ01094783] MW3 Sminthurus viridis [GATZ01103981] MW7 97 Sminthurus viridis [GATZ01010906] MW6 Sminthurus viridis [GATZ01087993] MW5 Sminthurus viridis [GATZ01090057] MW4 Branchinella kugenumaensis [BAG80987] Rhb Triops granarius [BAG80979] RhD 100 99 Triops longicaudatus [BAG80998] RhD Daphnia pulex [LOPB9] LOpB9 100 Daphnia pulex [LOPB13] LOpB13 Daphnia pulex [LOPB14] LOpB14 Daphnia pulex [LOPB15] LOpB15 Daphnia pulex [LOPB1] LOpB1 Hemigrapsus ~480 nm Daphnia pulex [LOPB2] LOpB2 90 Daphnia pulex [LOPB8] LOpB8 100 Daphnia pulex [LOPB7] LOpB7 Daphnia pulex [LOPB4] LOpB4 100 Daphnia pulex [LOPB5] LOpB5 MW1 MW1c Daphnia pulex [LOPB6] LOpB6 Daphnia pulex [LOPB3] LOpB3 Limulus polyphemus LOST Scutigera coleoptrata LOST Malacostraca LOST Tigriopus californicus [ADZ45237] opsin Calanus finmarchicus [GBFB01096267] rOp1 Calanus finmarchicus [GBFB01009482] rOp2 Sminthurus viridis [GATZ01103643] MW8 Insecta LOST Phyllopoda LOST Branchinella kugenumaensis [BAG80985] Rhc 100 Branchinella kugenumaensis [BAG80986] Rhd Argulus siamensis LOST Heterocypris sp. LOST 97 Euphilomedes carcharodonta [UN1394] rOp1 Vargula ~460 nm 99 Euphilomedes carcharodonta [UN2129] rOp2 100 Skogsbergia lerneri [AAL37521] SlME27 Vargula hilgendorfii [AAL37513] VhME16 Vargula tsujii LOST Skogsbergia lerneri [AAL37539] SlCE10 MW2 Vargula hilgendorfii [AAL37507] VhCE7 Vargula tsujii [ADZ45236] Vtops Limulus polyphemus [AEL29244] UVOps Scutigera coleoptrata LOST Ostracoda LOST K 93 Argulus siamensis [401520393] rOps7 100 ? Argulus siamensis [401535242] rOps6 Neocopepoda LOST Decapoda LOST Neogonodactylus oerstedii [AIF73509] UV3 100 100 Neogonodactylus oerstedii [AIF73507] UV1 Hexapoda LOST Branchinella kugenumaensis LOST SWa Daphnia pulex [UVOP] UV1 Triops LOST Neocopepoda LOST Neogonodactylus oerstedii [AIF73508] UV2 93 Decapoda LOST Hexapoda LOST Branchinella kugenumaensis [BAG80984] Rha SWb Daphnia pulex [BLOP] UV2 Triops granarius [BAG80978] RhC 100 Triops longicaudatus [BAG80983] RhC Neocopepoda LOST Neogonodactylus oerstedii LOST Uca pugilator [ADQ01811] Rh3 Hemigrapsus sanguineus LOST Branchinella kugenumaensis LOST Triops LOST Daphnia pulex [UNOP1] UnOp1 100 100 Daphnia pulex [UNOP2] UnOp2 Sminthurus viridis LOST Machilis hrabei LOST Tricholepidion gertschi LOST Ephemera danica LOST Sympetrum frequens [BAQ54697] Rh7-like 100 Ischnura asiatica [BAQ54908] Rh7-like Orthoptera LOST Pediculus humanus [XP_002432663] Op3 Euhemiptera LOST Apocrita LOST Tribolium castaneum LOST Heliconius melpomene [HMEL022527-PA] Rh7-like Manduca sexta LOST [ _ ] SWc1 100 Musca domestica XP 005181535 Rh7 Drosophila melanogaster [NP_524035] Rh7 Branchiopoda LOST Sminthurus viridis LOST Machilis hrabei [GAUM01184193] UV Tricholepidion gertschi LOST Sympetrum frequens [BAQ54713] RhUV 100 Ischnura asiatica [BAQ54920] RhUV Ephemera danica [GAUK01007309] UV* 100 Ephemera danica [GAUK01007310] UV* 90 Gryllus bimaculatus [AEG78686] UV Schistocerca gregaria [BAP16681] LoUV Pediculus humanus [XP_002422743] Op2 Nephotettix cincticeps [BAO03866] UV 93 Nilaparvata lugens [BAO03856] UV1 100 Nilaparvata lugens [BAO03857] UV2 Ceratosolen solmsi [AGH15793] UV 100 Bombus impatiens [AAV67326] UV 100 Apis mellifera [AAC13418] UV Tribolium castaneum [XP_970344] UV Manduca sexta [O02465] Manop2_RhP357 100 Heliconius melpomene [HMEL010962-PA] UV1 99 Heliconius melpomene [HMEL022525-PA] UV2 Drosophila melanogaster [AAA28854] Rh3 94 Musca domestica [XP_005190072] Rh3 SWc2.1 100 Drosophila melanogaster [AAA28856] Rh4 Musca domestica LOST ? Ephemera danica [GAUK01017324] SW1 100 Ephemera danica [GAUK01003006] SW2 100 Ephemera danica [GAUK01003005] SW3 100 Ephemera danica [GAUK01003004] SW4 97 Sympetrum frequens [BAQ54708] RhSWa1 Ischnura asiatica LOST 100 Ischnura asiatica [BAQ54919] RhSWb2 100 Ischnura asiatica [BAQ54918] RhSWb1 99 Sympetrum frequens [BAQ54709] RhSWb1 98 Sympetrum frequens [BAQ54710] RhSWc1 94 Sympetrum frequens [BAQ54711] RhSWc2 97 Sympetrum frequens [BAQ54712] RhSWc3 Schistocerca gregaria [CAA56378] Lo2 99 Gryllus bimaculatus [AEG78685] Blue Pediculus humanus LOST 98 Nephotettix cincticeps [BAO03865] Blue Nilaparvata lugens LOST Ceratosolen solmsi [AGH15792] Blue 100 Bombus impatiens [XP_003494923] Blue 100 Apis mellifera [AAC13417] Blue Tribolium castaneum LOST Manduca sexta [O96107] Manop3_RhP450 100 Heliconius melpomene [HMEL010706-PA] Blue SW SWc [ ] Drosophila melanogaster AAC47426 Rh5 SWc2 SWc2.2 100 Musca domestica [XP_005175090] Rh5

Supplementary Fig. S3 Arthropod r-opsin phylogeny reconciled with the species tree. Gray branch labels denote losses of genes; otherwise branch labels are formatted as in Supp- lementary Figures S1 and S2. Major opsin clades are shaded in green, blue, and purple for long-, middle-, and short-wavelength (LW, MW, and SW), respectively. ‘K’ stands for the gain of a lysine residue at, or next to, the position corresponding to glycine 90 in bovine rhodopsin, ‘X’ indicates the exchange of lysine by another amino acid, and ‘?’ means that the homologous part of the sequence is missing. In addition to the r-opsins in Supplementary Figures S1 and S2, we have included four sequences that were detected by methods different from those described in the main text: The putative LW sequence of Scutigera is very short, and only present in the sequence read archive (SRA). Neogonodactylus CRB, CRS and CRV are reported in a thesis (Bok 2013), but not yet deposited in GenBank. 1 0.96 details. sn ST-I See transcriptomes SATé-II. and using databases from opsins 494 of ces sequen- amino-acid the of analysis Maximum-likelihood S4 Fig. Supplementary 0.95 0.85 0.31 0 0.62 0.81 0.57 Methods „ hlgn o gop4 opsins. group-4 of Phylogeny 0.93 0.96 1 „ eto o mi tx for text main of section 0.62 1 0.8 0.14 0.05 0.78 0.92 0.74 1 1 0.97 0.25 0.6 0.75 0.98 0.96 0.62 0.97 0.97 0.97 1 0.45 0.99 0.1 0.82 0.99 0.84 0.78 0.89 0.92 0.67 0.56 0.9 0.88 0.94 0.78 1 0.94 1 0.99 0.91 0.68 0.83 1 0.9 0.95 1 1 0.3 0.93 0.81 0.87 0.99 0.98 0.63 0.9 0.79 1 0.72 0.93 0.99 0.42 1 0.99 0.75 0.92 0.42 0.21 0.99 0.47 0.27 0.75 0.81 0.97 0.78 1 1 0.89 0.96 1 0.82 1 0.94 0.11 0.97 0.65 1 1 0.91 0.95 0.75 0.83 0.96 0.91 0.82 0.64 1 0.99 0.99 0.87 0.95 0.94 0.81 1 0.98 0.65 0.93 0.02 0.24 0.84 0.99 0.34 0.81 0.71 0.94 0.45 0.87 0.95 0.92 0.9 0.8 0.98 0.32 0.6 0.67 0.55 0.77 0.95 0.96 0.93 0.99 0.85 0.93 0.95 0.98 0.93 0.5 0.55 0.74 0.77 0.92 0.6 0.93 0.95 0.93 0.85 0.93 0.75 0.98 1 0.88 0.86 0.8 0.6 1 0.84 0.42 0.95 0.85 0.75 0.48 0.72 0.97 0.93 0.63 1 0.69 0.2 0.55 0.81 0.82 0.64 1 0.88 0.82 0.9 0.57 0.83 0.67 0.93 0.95 1 0.96 1 0.94 0.85 0.95 0.35 0.98 0.69 0.91 0.94 0.9 0.97 1 0.83 0.82 0.82 0.89 1 0.44 1 1 0.88 0.94 0.96 0.99 0.74 0.99 1 0.93 0.77 0.72 0.56 0.74 0.73 0.58 0.84 0.96 0.18 0.95 0.94 1 1 0.97 0.99 0.21 0.96 0.81 0.95 0.56 0.9 0.25 0.23 1 0.98 0.25 0.93 0.91 0.47 0.98 0.89 1 0.85 0.81 0.47 0.43 0.93 0.94 0.93 0.13 0.9 1 1 0.85 0.99 0.93 1 0.97 0.88 0.74 0.52 0.95 0.99 0.35 1 1 0.88 1 1 1 0.24 0.82 0.83 0.92 0.85 0.3 0.64 0.83 0.99 0.97 0.77 0.98 0.58 0.99 0.74 0.88 0.87 0.25 1 1 0.9 0.78 1 0.72 1 0.98 0.85 0.86 0.72 0.99 0.91 1 0.99 0.93 0.88 0.73 1 0.8 0.17 0.95 0.98 1 0.96 0.92 0.47 0.99 0.91 0.93 0.68 0.94 1 0.97 1 0.84 0.12 1 1 0.26 0.79 1 0.99 1 0.29 0.83 0.85 1 1 0.98 0.04 1 0.95 0.85 0.85 0.51 1 1 0.99 0.75 1 1 0.97 0.7 0.9 0.84 0.47 0.96 0.22 0.86 0.98 1 0.83 0.94 0.99 1 1 0.87 1 1 0.99 1 0.99 0.48 0.96 1 0.99 0.99 0.82 1 1 1 1 1 1 0.1 0.75 0 0.91 1 1 1 0.66 1 0.74 0.92 0.96 0.98 1 1 0.99 0.99 0.93 1 0.99 0.97 0.79 0.8 0.98 0.97 1 0.19 0.46 1 0.93 0.72 0.91 0.95 0.98 1 0.91 1 1 0.78 0.41 0.94 1 1 0.98 0.94 0.85 1 1 1 0.86 0.88 0 0.75 0.84 0.88 0.76 1 0.98 0.84 1 1 1 0.85 1 0.99 1 0.44 1 1 1 1 0.99 0.29 Homo sapiens Danio rerio Tetraodon nigroviridis Hasarius adansoni Takifugu rubripes Heterocephalus glaber Nannospalax galili Lottia gigantea Branchiostoma floridae Schistosoma mansoni Pan troglodytes Eurypyga helias Papio anubis Cupiennius salei Larimichthys crocea Todarodes pacificus Catarrhini P47804-3 Oncorhynchus mykiss Chelonia mydas Notothenia coriiceps Poecilia Sympetrum frequens Rattus norvegicus Perissodactyla Glires I3MWT1 Esox lucius Esox lucius Tetraodon nigroviridis Anax parthenope Elephantulus edwardii Oncorhynchus mykiss Manacus vitellinus Otolemur garnettii Lepisosteus oculatus Manacus vitellinus Mesocricetus auratus Branchiostoma belcheri Gasterosteus aculeatus Lepisosteus oculatus Chrysemys pictabellii Argulus siamensis Tetranychus urticae Fukomys damarensis Oncorhynchus mykiss Esox lucius Rattus norvegicus Lottia gigantea Aplysia californica Asiagomphus melaenops Simiiformes F6VQB0 Lottia gigantea Strongylocentrotus purpuratus Poecilia formosaUPI0004438247 Xenopus tropicalis Mizuhopecten yessoensis Callithrix jacchus Gallus gallus Callorhynchus milii Sarcophilus harrisii Poeciliinae M4A1Y4 Notothenia coriiceps Xenopus tropicalis Chrysochloris asiatica Branchiostoma belcheri Scutigera coleoptrata Canis familiaris Equus caballus Capitella teleta Boreoeutheria OPSX Dicentrarchus labrax Phasianidae A1IHF8 Stegastes partitusUPI000495C2A6 Latimeria chalumnae Limulus polyphemus Spermophilus tridecemlineatus Bubalus bubalis Homo sapiens Alligator mississippiensis Platynereis dumerilii Callorhynchus milii Pseudocrenilabrinae I3KGH7 Bos taurusNP Neognathae A0A093CF33 Capitella teleta Canis familiaris Brachionus Branchiostoma floridae Anolis carolinensis Tetraodon nigroviridis Astyanax mexicanus Oryzias latipes Cynoglossus semilaevis Orycteropus aferUPI00045E3454 Danio rerio Pan troglodytes Loliginidae OPSD Aves H0Z6G8 Branchiostoma floridae Spermophilus tridecemlineatus Takifugu rubripes Lepisosteus oculatus Takifugu rubripes Poecilia formosa Takifugu rubripes Plutella xylostella Platynereis dumerilii Takifugu rubripes Anas platyrhynchos Chlorocebus sabaeus Canis familiaris Cynoglossus semilaevis Takifugu rubripes Acanthisitta chloris Latimeria chalumnae Myotis lucifugus Haplorrhini RGR Danio rerio Percomorphaceae M4AB16 Anolis carolinensis Pan troglodytes Alligator sinensis Latimeria chalumnae Crassostrea gigas Branchiostoma floridae Lottia gigantea Poecilia Galeopterus variegatus Gallus gallus Chelonia mydas Indolestes peregrinus Plutella xylostella Otolemur garnettii Pan troglodytes Echinops telfairi Dasypus novemcinctus Fulmarus glacialis Esox lucius Takifugu rubripes Microtus ochrogaster Jaculus jaculus Myotis G1NYV5 Percomorphaceae I3K9S0 Astyanax mexicanus Emberiza bruniceps Takifugu rubripes Danio rerio Todarodes pacificus Monodelphis domestica Taeniopygia guttata Xiphophorus maculatus Astyanax mexicanus Callorhynchus milii Cavia porcellus Asiagomphus melaenops Trichechus manatus Tetraodon nigroviridis Loxodonta africana Crassostrea gigas Pteropus L5KFC5 Python bivittatusUPI000441C2D1 Eutheria OPN5 Neognathae U3J749 Tetraodon nigroviridis Canis lupus Strongylocentrotus purpuratus Condylura cristata Terebratalia transversa Lottia gigantea Cupiennius salei Tupaia chinensis Lottia gigantea Branchiostoma belcheri Chrysochloris asiatica Sorex araneus Protacanthopterygii A0A060VYB3 Anax parthenope Cricetidae G3HLQ9 Alligator Aves B3XZF6 Stegastes partitusUPI0004954BEF Neognathae H0ZVE9 Oncorhynchus mykiss Crassostrea gigas Nannospalax galili Strongylocentrotus purpuratus Schistosoma japonicum Homo sapiens Cynoglossus semilaevis Tetraodon nigroviridis Phasianidae F1NEY8 Carnivora OPN4 Neognathae H0Z174 Crassostrea gigas uchidai Limulus polyphemus Murinae RGR Mus musculus Homo sapiens Cynoglossus semilaevis Ictalurus punctatus Ornithorhynchus anatinus Ovalentaria H2MLK7 Xenopus tropicalis Crassostrea gigas Tarsius syrichtaUPI00046B5C4A Enteroctopus dofleini Capitella teleta Mus musculus Aves D9N3D0 Xenopus tropicalis Plutella xylostella Ictalurus punctatus Apaloderma vittatum Loxodonta africana Takifugu rubripes Erinaceus europaeus Pseudocrenilabrinae I3JZA7 Percomorphaceae H2UBK9 Neognathae A0A091PXY2 Oryzias latipes Xenopus laevis Simiiformes OPN4 Takifugu rubripes Struthio camelusaustralis Canis familiaris Mustela putoriusfuro Canis lupus Branchiostoma floridae Lagomorpha G1SQD6 Erinaceus europaeus Lottia gigantea Tetraodontidae H3CNY8 Equus F7C7B5 Pteropus vampyrus Ochotona princeps Tupaia chinensis Takifugu rubripes Larimichthys crocea Cynoglossus semilaevis Astyanax mexicanus Anas platyrhynchos Takifugu rubripes Takifugu rubripes Poecilia A0A087Y593 Orthetrum albistylum Anolis carolinensis Xiphophorus maculatus Danio rerio Trichechus manatuslatirostris Cryptodira K7G2U1 Tetraodon nigroviridis Larimichthys crocea Bos taurusNP Macaca mulatta Perissodactyla F7AC99 Ursus maritimus Esox lucius Pelodiscus sinensis Takifugu rubripes Oncorhynchus mykiss Oryzias latipesR9R6L1 Echinops telfairi Rattus norvegicus Ladona fulva Metaseiulus occidentalis Tupaia chinensis Galeopterus variegatus Chrysochloris asiatica Larimichthys crocea Tetraodon nigroviridis Crassostrea gigas Oryzias melastigma Myotis brandtii Saccoglossus kowalevskii Oreochromis niloticus Euperipatoides kanangrensis Notothenia coriiceps Esox lucius Percomorphaceae E6ZHK6 Macaca mulatta Loxodonta africana Taeniopygia guttata Neognathae A0A094NLK3 Cetartiodactyla W5NPY9 Callorhynchus milii Branchiostoma floridae Fukomys damarensis Branchiostoma floridae Oryctolagus cuniculus Latimeria chalumnae Lepisosteus oculatus Takifugu rubripes Lottia gigantea Platynereis dumerilii Vespertilionidae Ailuropoda melanoleuca Afrotheria UPI0002C4630A Takifugu rubripes Cryptodira K7F7F1 Neognathae UPI000549943A Pseudocrenilabrinae I3JPV7 Erinaceus europaeus Bos taurusNP Poecilia Xenopus tropicalis Lottia gigantea Vespertilionidae G1PVT8 Danio rerio Danio rerio Stegodyphus mimosarum Neognathae UPI00039467BD Branchiostoma floridae Danio rerio Anolis carolinensis Danio rerio Lottia gigantea Poecilia Danio rerio Ailuropoda melanoleuca Crassostrea gigas Pelodiscus sinensis Asiagomphus melaenops Metaseiulus occidentalis Astyanax mexicanus Oryzias latipes Takifugu rubripes Megaselia scalaris Elephantulus edwardii Archelosauria F1NEL9 Saccoglossus kowalevskii Mnais costalis Stegodyphus mimosarum Octodon degus Cryptodira K7FFW2 Takifugu rubripes Tetraodon nigroviridis Callorhynchus milii Heterocephalus glaber Xenopus Ornithorhynchus anatinus Tinamus guttatusA0A099ZRE5 Capitella teleta Capitella teleta Poecilia formosa Astyanax mexicanus Neognathae A0A091K6W3 Crassostrea gigas Pteropus alecto Oryzias latipesH2M2R3 Lottia gigantea Platynereis dumerilii Macromia amphigena Caniformia UPI0003EE08EA Gasterosteus aculeatus Anolis carolinensis Cynoglossus semilaevis Metaseiulus occidentalis Ovalentaria I3KQV6 Pseudocrenilabrinae I3IXF4 Lepisosteus oculatus Gasterosteus aculeatus Strongylocentrotus purpuratus Gasterosteus aculeatus Mustelinae M3YB07 Gallus gallus Larimichthys crocea Mizuhopecten yessoensis Chinchilla lanigera Monodelphis domestica Canis lupus Tupaia chinensis Xenopus Lottia gigantea Sorex araneus Aplysia californica Euprymna Chrysemys pictabellii Percomorphaceae M4ALW3 Ischnura asiatica Cuculus canorus Schistosoma haematobium Cynoglossus semilaevis Branchiostoma floridae Metatheria G3W284 Crassostrea gigas Tetraodon nigroviridis Bombyx moriXP Bos taurusUPI000572B4AA Astyanax mexicanus Cebidae F6RXG7 Latimeria chalumnae Ornithorhynchus anatinus Tetraodon nigroviridis Mustela putoriusfuro Takifugu rubripes Oryzias latipes Gasterosteus aculeatus Danio rerio Lepisosteus oculatus Neognathae A0A091W7K7 Condylura cristata Crassostrea gigas Chinchilla lanigera Cetartiodactyla UPI0003AE9F7D Daphnia pulex Xenopus Passeriformes H0Z5K8 Hystricognathi H0V2M9 Spermophilus tridecemlineatus Hymenochirus curtipes Astyanax mexicanus Danio rerio Merops nubicus Machilis hrabei Lottia gigantea Orycteropus aferUPI00045DE648 Cynoglossus semilaevis Astyanax mexicanus Branchiostoma floridae Takifugu rubripes Arvicanthis ansorgei Pteropus L5KG58 Pteropus alecto Cetartiodactyla Rattus norvegicus Peromyscus maniculatusbairdii Pseudocrenilabrinae I3JZL2 Latimeria chalumnae Lottia gigantea Gasterosteus aculeatus Lepisosteus oculatus Python bivittatusUPI0004423AE1 Lepisosteus oculatus Dasypus novemcinctus Lepisosteus oculatus Decapodiformes OPSD Chinchilla lanigera Cetartiodactyla RGR Lepisosteus oculatus Macaca mulatta Ictalurus punctatusC3V151 Heterocephalus glaber Bathyergidae A0A091DSC5 Ochotona princeps Condylura cristata Ornithorhynchus anatinus Lepisosteus oculatus Percomorphaceae G3NB50 Bos taurusNP Callorhynchus milii Capitella teleta Pseudocrenilabrinae I3JH16 Branchiostoma floridae Oryzias latipes Helobdella robusta Callorhynchus milii Metaseiulus occidentalis Poecilia Poecilia reticulata Percomorphaceae I3KQ82 Oncorhynchus mykiss Glires UPI00022B50CF Branchiostoma floridae Mus musculus Xenopus tropicalis Tetraodon nigroviridis Salmo salar Tetraodontidae H2V568 Callorhynchus milii Xenopus tropicalis Mus musculus Carnivora M3VX16 Takifugu rubripes Aves A0A099Z381 Calanus finmarchicus Sus scrofaF1S135 Ornithorhynchus anatinus Limulus polyphemus Laurasiatheria G3MZW2 Oryzias latipes Tetranychus urticae Poeciliinae M4AG56 Pseudocrenilabrinae I3KSN8 Poecilia Hystricognathi UPI000334031A Vespertilionidae G1P3M8 Epiophlebia superstes Pan troglodytes Ophiophagus hannah Mus musculus Calypte anna Tanypteryx pryeri Cricetulus griseus A0A087XC66 A0A087Y377 A0A087X5H8 A0A087XB82 A0A087XBP4 UPI0003C264A7 UPI000444424A Q28EK0 (Silurana F6UZB2 B8Q2W3 OPN4A NP F1QCL1 F1QM45 F1QPE1 Q6DH22 Q567Y2 NP Q66I55 NP R9R6C7 B3DK10 sp.AIT99330 UPI000576A105 UPI0005768E1C UPI000576A9F4 UPI000576E77F UPI00057726F2 UPI0005772C91 XP I1W5U7 XP XP NP AAR02098 AAR02099 A0A096N043 A0A091I4U0 FAA01168 _ _ BAQ54923 _ _ UPI0003D70503 OPSX Q80XL3 NP EDL23393 NP UPI0005D028F3 NP NP EFX84680 _ _ _ _ F7AFM0 UPI000331659D UPI0001580F58 R9R6H4 UPI0005CBFE3F _ _ _ H2LRK0 R9R6D2 H2M0A1 H2LBK8 XP XP XP XP V4CNF1 XP XP XP XP XP V3ZDT4LOTGI MOUSE ELT97785 ELU02401 ELT87076 ELT87227 ELU09017 ELU11093 _ GAUM01022346 CHICK F1MR98 S7PSS6 _ UPI00005A077C E2R3W5 E2R8U6 J9JH94 UPI000332FFE0 TAEGU XP _ Q0QFY9 UPI0003336952 H0W479 COTJA _ _ _ V3ZSU7 V4AUU9 XP 001014890 786969 001193009 001179153 XP UPI00051238AD UPI0003F0FF0D UPI000512496C 001017877 001186975 001004654 H9F008 UPI0001D55C84 UPI0004F056BE F7HTU3 UPI00042CC902 _ A0A093IHD7 SPETR M7B015 M7CIJ5 UPI0004DFCFBB MOUSE XP UPI0005122CA3 UPI0003F183B6 HORSE _ _ UPI000333E099 UPI000333D1E7 864762 003639476 853139 T2I4T4 A0A096MAM0 G1PJY3 MYOLU _ A0A087XRU1 UPI0003C4487A _ A0A091GF67 F7DJ88 UPI0003C2A4E3 L9KXS8 UPI0003C92126 UPI0003C8C65D BAQ54907 MYOLU CCP46949 XP XP XP BAQ54813 H2U9V4 H2THH8 H2RU69 H2RWN0 UPI000298F6C6 H2RWN1 H2V570 H2THH6 UPI000298C154 H2UBK4 H2SBY2 H2TZX8 H2TZA5 H2SBY1 H2SBV8 UPI000298A95A H2U9V5 H2TVH5 L8YFB3 HUMAN FELCA UPI0004A27321 001124215 _ _ A0A0C6G2W9 BAQ54848 004930922 A0A061ILW0 H0XGM2 H0X693 _ _ _ UPI00051B2F75 UPI0003599B0D XP EDM18681 H2TVH4 H2UBL0 H2RU68 UPI0000DA2166 NP _ _ EKC25796 K1PPZ4 UPI0003343C70 UPI0005C356B2 EKC42752 EKC29416 XP _ _ _ K1QCZ3 XP K1R077 UPI0003342FC7 EKC40616 UPI000334322B A0A093QH01 A0A093PPB7 F7A4M9 F7BYN3 UPI00034FCCB8 UPI00038F039A UPI00038F1995 UPI00038F11CA UPI0001DE7084 F7BWP4 ______) tropicalis UPI0004E47BE5 H9GQ69 H9GM86 G1KNV3 G1KWC5 V9L506 UPI00045763FA UPI00045757C2 UPI0004572E50 UPI0004574B0B A0A091MXY0 _ T1G602 401527168 UPI0004ED1174 CALJA UPI0004ED048E W5UEE6 W5UHC8 UPI00032AEB69 _ ALLSU UPI00032AE234 UPI000273AD3B _ _ F7EFF0 F6T3T2 _ PTEAL XENTR PTEAL T1GU18 _ G3SXB6 G3TDN1 XENTR 033128 _ 002912 859528 006574 G1KAU7 E1CFG1 _ _ UPI0004574CCF UPI0004573F1E UPI000457602E UPI0005B771C9 009051341 009048325 009051446 009045392 009058695 009058696 009053517 009049184 009065058 009060307 HUMAN _ _ U3IU26 R0L581 UPI0003C454C0 K7FPC1 I1SRN3 tetur04g04260 XP G3T3L1 _ A0A068EPX1 _ TINGU UPI00054BCBBC UPI00054BB946 UPI00054B7829 UPI00054B736F _ _ _ 001502825 R4S0M6 _ PELSI OPSD _ P31356 FELCA _ _ DANRE latirostrisUPI0002C43D61 _ _ PIG _ _ T1K297 DANRE _ UPI000552603D UPI0005557FC0 001154882 001146167 UPI0002B43977 AIT75833 A0A097J9F3 1Lpperopsin E6ZJD5 _ _ UPI00054B7493 W5KY57 W5KA16 W5LC48 W5LE41 UPI000440D61D W5LL00 W5LI72 W5KL95 W5KS89 _ _ BAQ54716 R4RY94 AHI16247 Q5SBP8 CRIGR PELSI BAQ54696 _ _ _ _ M1JJ88 UPI0003C192E0 H3AHR5 H3B1A3 H3AZS5 UPI0003C157B4 H2ZYF5 BAQ54791 _ _ PELSI UPI00038C4F36 UPI000555F89E UPI000344467E W5N199 _ DANRE UPI0003EAE1AC W5MQT2 W5MEJ9 W5MAS1 W5NIT5 W5NDA1 W5NFQ1 EUPSC ORENI DANRE _ DANRE UPI0005219769 SARHA 4779 MUSPF XIPMA UPI0004444F2F UPI000443E3D2 UPI000443C1D3 A0A091DUP0 A0A091D6F6 UPI0003596710 DANRE DANRE _ DANRE DANRE GBFB01041842 XIPMA _ BAQ54938 V8P4Z0 _ H3CBM4 H3CR14 H3DQ07 H3DFD6 H3DHG5 H3CWD0 Q4RK24 Q4RRX8 BAQ54765 H3CKG3 M3YAY1 H3BXN0 W5MJC7 W5NID2 BAQ54740 OPSD W5MJ35 UPI000388B256 UPI00046C1A8C SALSA BOVIN _ _ _ UPI00045DD6A9 CHICK _ _ ANAPL UPI0003F1550E UPI0003F15D07 UPI0003F101B6 A0A060Y2K3 A0A060VV44 A0A060X8T0 A0A060WQS9 G1TDZ2 ORYLA 011552121 011552092 011552091 UPI0003F06522 UPI0003F0EA8F _ TAEGU CALJA HUMAN _ _ BAQ54883 UPI0003946B9D Q4SH06 Q4RE69 _ MOUSE _ TAEGU A0A060YPF6 A0A060XP54 Q9NJC9 _ G5AQM3 CHICK G5BDP8 G5APG0 005094221 G5DZT2 001101196 011435061 011438181 POEFO POEFO EEN65038 C3XR28 C3ZBH0 XP XP XP C3ZQG5 XP XP UPI0003291F46 AGJ70278 UPI00019566C8 POEFO _ _ UPI0004D0597F UPI0004D0700F POEFO POEFO _ _ POEFO _ _ BAC76023 Q868G2 RABIT M4AFJ5 M3ZMH3 _ _ _ C3YCQ8 XP XP G3QAZ6 F6R2C0 G3NJT0 G3PFC6 G3PVJ9 F6SMD5 G3QAZ5 G3ND31 _ CHICK ______UPI0004964505 UPI00049685FA UPI000494E016 UPI00049681A1 UPI000497B269 UPI0004967FDA UPI000497ED93 _ _ Q868G0 G1LTN8 _ _ _ _ _ TAEGU C1LEE2 MOUSE UPI000265855B XP UPI00026586FF _ _ LOLFO _ 002189255 CANFA UPI000496F9DE HORSE _ _ ORYLA CAVPO MYOBR _ G1LD74 _ UPI0003D0C028 _ ORYLA _ HORSE ORYLA XP LOTGI TAKRU ORYLA ORYLA _ LOTGI ORYLA _ BAQ54831 A0A0C6G6R3 A0A0C6FPS8 LOTGI _ ORYLA BOVIN CUPSA CANFA A0A087TK86 KFM65525 _ BOVIN CHEMY MACMU _ F7FTA8 UPI000454579E UPI000155CB9E F6XNY7 F7E735 OPSD1 TETNG O15974 _ XP CAVPO XENLA CANFA SHEEP MACMU _ CHEMY UPI00051E3FF9 _ _ MYOLU _ _ CALJA UPI0003F06E32 ______MYOLU _ _ _ _ _ TUPCH _ _ TUPCH ______TAKRU _ _ _ TAKRU TAKRU _ _ _ _ ORENI 9AVES _ TAKRU TAKRU TAKRU _ _ _ 9AVES _ TAKRU TAKRU A0A095A2W7 TAKRU TAKRU _ OTOGA TAKRU 002594196 002613137 TAKRU TAKRU 002587828 002609116 002609117 002604146 002605849 _ ORENI CALAN LEPDC TAKRU TAKRU TAKRU ______XENTR _ CRAGI _ _ _ _ _ OPIHO COLST PAPAN _ _ _ XENTR TODPA CRAGI _ CALMI _ _ _ _ TAKRU TAKRU CRAGI XENTR DICLA XENTR _ HELRO _ MEGSC _ _ _ XIPMA GASAC NP ICTPU OTOGA TAKRU _ ORENI _ _ ANAPL T2I6P9 FUKDA LOXAF XENTR 003744578 9ARAC 003744590 ENTDO _ ORENI _ ANOCA ANOCA ANOCA ANAPL ORYME _ ANOCA XIPMA TETUR ORENI ORENI _ _ _ ORENI _ LOXAF _ LOXAF UPI0002C43A13 ANOCA _ _ _ _ PELSI ARVAN ICTPU _ _ _ ORENI ICTPU _ _ _ _ ASTMX XP XP ORENI _ PLADU _ _ DICLA _ _ PLADU PLADU 002732329 UPI00038BF4F8 _ I3M253 ASTMX XP XP _ _ _ _ _ EURHL _ _ _ _ LATCH _ LEPOC ASTMX ASTMX _ LATCH ASTMX _ LATCH _ _ _ _ _ ASTMX ASTMX ASTMX _ LEPOC _ _ I3MFC3 OPHHA LATCH LEPOC UPI00042A9825 MUSPF _ LEPOC _ _ _ LEPOC TETNG LEPOC TETNG TETNG TETNG _ TETNG TETNG LEPOC TETNG _ LEPOC 9AVES _ _ LEPOC _ RABIT SCHMA TETNG LEPOC TETNG POEFO TETNG TETNG ______TINGU _ TETNG POEFO CRIGR ______9PIPI BRAFL _ HETGA _ BRAFL HETGA _ 001016013 XIPMA ANAPR BRAFL GASAC BRAFL MONDO _ _ _ _ GASAC BRABE HETGA BRABE _ SCHJA 9PASS _ 9PASS GASAC GASAC _ _ MONDO GASAC GASAC _ AILME _ AILME XIPMA 9PASS _ 001199309 003723700 783329 784266 ONCMY MIZYE LIMPO ORNAN 9EMBE _ ORNAN ORNAN ______FUKDA _ FUKDA ONCMY _ ONCMY ONCMY ONCMY ONCMY 9BILA ONCMY _ _ _ _ SPETR _ SPETR 9ARAC 9ODON 9ODON _ SCHHA

PEROPSIN see main Fig 2 RGR NEUROPSIN Go CILIARY RHABDOMERIC Supplementary Tables

The Dynamic Evolutionary History of Pancrustacean Eyes and Opsins

Miriam J. Henze and Todd H. Oakley

CONTENTS

Table S1 Literature on the occurrence of lateral and median eyes in Pancrustacea as illustrated in Figure 1. …………………………………………………………………………………………………………………..………………..….. page 2

Table S2 Visual r-opsins from GenBank used for phylogenetic analyses, including references on the identification and expression of opsins. ………………………….………………..…………………………………….…….. page 3

Table S3 Genomes and transcriptomes searched for visual r-opsins and peropsins. .……….….…….. page 12

Table S4 Visual r-opsins identified in the genomes and transcriptomes listed in Supplementary Table S3, and references on the expression of opsins. .………………………………………………………..…………………….. page 14

References …………..……………………………………………………….…..……………..…………....………………………….. page 17

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Table S1 Literature on the occurrence of lateral and median eyes in Pancrustacea as illustrated in Figure 1.

______Taxon References ______OLIGOSTRACA Mystacocarida (Elofsson 1966; Elofsson and Hessler 2005; Brenneis and Richter 2010) Branchiura (Elofsson 1966; Hallberg 1982; Cronin 1986; Elofsson 2006) Pentastomida (Osche 1963) Podocopa (Elofsson 1966; Oakley and Cunningham 2002; Oakley 2005; Tanaka 2005; Elofsson 2006; Tsukagoshi et al. 2006) Myodocopa (Elofsson 1966; Land and Nilsson 1990; Parker 1995; Oakley and Cunningham 2002; Oakley 2005; Elofsson 2006) MULTICRUSTACEA Malacostraca (Elofsson 1963, 1965; Paulus 1979; Fincham 1980; Cronin 1986; Elofsson 2006; Cronin and Porter 2008) Copepoda (Vaissière 1961; Elofsson 1966, 1992, 2006) Thecostraca (Elofsson 1966; Hallberg and Elofsson 1983; Hallberg et al. 1985; Grygier 1987; Elofsson 2006; Glenner et al. 2008; Pérez-Losada et al. 2012) ALLOTRICARIDA Cephalocarida (Elofsson 1966; Elofsson and Hessler 1990) Branchiopoda (Elofsson 1966; Güldner and Wolff 1970; Elofsson and Odselius 1975; Paulus 1979; Cronin 1986; Diersch et al. 1999; Elofsson 2006; Reimann and Richter 2007) Remipedia (Fanenbruck et al. 2004; Fanenbruck and Harzsch 2005; Koenemann et al. 2009) Hexapoda Protura (Tuxen 1931; Bedini and Tongiorgi 1971; Haupt 1972; Bitsch and Bitsch 2000) Collembola (Paulus 1972a, 1972b, 1974, 1977, 1979; Bitsch and Bitsch 2000, 2004; Meyer-Rochow et al. 2005) Diplura (Bitsch and Bitsch 2000, 2004) Archaeognatha (Paulus 1974, 1979; Bitsch and Bitsch 2000) Zygentoma (Elofsson 1970; Paulus 1974; Bitsch and Bitsch 2000; Blanke et al. 2014) Pterygota (Wigglesworth 1941; Kalmus 1945; Parry 1947; Goodman 1970; To et al. 1971; Dickens and Eaton 1973; Weber and Renner 1976; Wilson 1978; Paulus 1979; Goodman 1981; Hallberg and Hagberg 1986; Mizunami 1994; Grünewald and Wunderer 1996; Insausti and Lazzari 2002; Klass et al. 2002; Buschbeck et al. 2003; Leschen and Beutel 2004; Beutel and Weide 2005; Grimaldi and Engel 2005; Taylor et al. 2005; Warrant et al. 2006; Berry et al. 2007; Nilsson and Kelber 2007; Beutel et al. 2010; Wei and Hua 2011; Wipfler et al. 2011; Gullan and Cranston 2014)

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Table S2 Visual r-opsins from GenBank used for phylogenetic analyses, including references on the identification and expression of opsins.

Expression in Species Accession No. Opsin lateral median References name eyes eyes ONYCHOPHORA (Outgroup) Euonychophora Peripatidae Principapillatus AFM75825 onychopsin not yes (Hering et al. 2012; hitoyensis applicable Beckmann et al. 2015) (Epiperipatus cf. isthmicola) Eoperipatus sp. AFM43711 onychopsin not yes (Hering et al. 2012) applicable Peripatopsidae Euperipatoides AFM75824 onychopsin not yes (Hering et al. 2012; rowelli applicable Beckmann et al. 2015) Phallocephale AFM43710 onychopsin not yes (Hering et al. 2012) tallagandensis applicable Ooperipatus AFM43712 onychopsin not yes (Hering et al. 2012) hispidus applicable CHELICERATA (Outgroup) Xiphosura Xiphosurida Limulus AAA02498 Ops1 yes no (Nolte and Brown 1970; polyphemus Smith et al. 1993; Dalal et al. 2003; Katti et al. 2010; Battelle et al. 2014) Limulus AAA02499 Ops2 yes no (Nolte and Brown 1970; polyphemus Smith et al. 1993; Dalal et al. 2003; Katti et al. 2010; Battelle et al. 2014) Limulus ACO05013 Ops5 yes no (Nolte and Brown 1970; polyphemus Katti et al. 2010; Battelle et al. 2014) Limulus AIT75830 Ops6 no yes (Nolte and Brown 1970; polyphemus Battelle et al. 2015) Limulus AIT75831 Ops7 no yes (Nolte and Brown 1970; polyphemus Battelle et al. 2015) Limulus AIT75832 Ops8 no yes (Nolte and Brown 1970; polyphemus Battelle et al. 2015) Limulus AEL29244 UVOps yes yes (Nolte and Brown 1970; polyphemus Battelle et al. 2014, 2015)

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OLIGOSTRACA Ostracoda, Myodocopa Myodocopida Skogsbergia lerneri AAL37539 SlCE10# yes no (Huvard 1993; Oakley and Huber 2004) Skogsbergia lerneri AAL37521 SlME27# no yes (Huvard 1993; Oakley and Huber 2004) Vargula AAL37507 VhCE7# yes no (Oakley and Huber 2004) hilgendorfii Vargula AAL37513 VhME16# no yes (Oakley and Huber 2004) hilgendorfii Vargula tsujii ADZ45236 Vtops unknown unknown (Huvard 1993; Colbourne et al. 2011) MULTICRUSTACEA Malacostraca Decapoda Hemigrapsus BAA09132 Rh1 yes unknown (Sakamoto et al. 1996) sanguineus Hemigrapsus BAA09133 Rh2 yes unknown (Sakamoto et al. 1996) sanguineus Uca pugilator ADQ01809 Rh1 yes unknown (Jordão et al. 2007; Rajkumar et al. 2010) Uca pugilator ADQ01810 Rh2 yes unknown (Jordão et al. 2007; Rajkumar et al. 2010) Uca pugilator ADQ01811 Rh3 yes unknown (Rajkumar et al. 2010) Stomatopoda Neogonodactylus ACU00223 rOp4 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00224 rOp5 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00222 rOp6 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00210 rOp7 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00211 rOp8 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00218 rOp9 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00217 rOp10 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00221 rOp11 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00216 rOp12 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00215 rOp13 unknown unknown (Porter et al. 2009) oerstedii

#one of multiple candidates [4 of 25]

Neogonodactylus ACU00212 rOp14 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00220 rOp15 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00219 rOp16 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00214 rOp17 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ACU00213 rOp18 unknown unknown (Porter et al. 2009) oerstedii Neogonodactylus ABG37007 Rh1 yes unknown (Cronin and Marshall oerstedii 1989; Brown 1996; Cronin et al. 1996; Porter et al. 2007, 2009; Cronin et al. 2010) Neogonodactylus ABG37008 Rh2 yes unknown (Cronin and Marshall oerstedii 1989; Brown 1996; Cronin et al. 1996; Porter et al. 2007, 2009; Cronin et al. 2010) Neogonodactylus ABG37009 Rh3 yes unknown (Cronin and Marshall oerstedii 1989; Brown 1996; Cronin et al. 1996; Porter et al. 2007, 2009; Cronin et al. 2010) Neogonodactylus AIF73507 UV1 yes unknown (Marshall and Oberwinkler oerstedii 1999; Bok 2013; Bok et al. 2014) Neogonodactylus AIF73508 UV2 yes unknown (Marshall and Oberwinkler oerstedii 1999; Bok 2013; Bok et al. 2014) Neogonodactylus AIF73509 UV3 yes unknown (Bok 2013; Bok et al. 2014) oerstedii Copepoda Harpacticoida Tigriopus ADZ45237 opsin not unknown (Colbourne et al. 2011) californicus applicable ALLOTRIOCARIDA Branchiopoda Anostraca Branchinella BAG80984 Rha yes unknown (Kashiyama et al. 2009) kugenumaensis Branchinella BAG80987 Rhb yes unknown (Kashiyama et al. 2009) kugenumaensis Branchinella BAG80985 Rhc yes unknown (Kashiyama et al. 2009) kugenumaensis

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Branchinella BAG80986 Rhd yes unknown (Kashiyama et al. 2009) kugenumaensis Notostraca Triops granarius BAG80976 RhA yes yes (Kashiyama et al. 2009) Triops granarius BAG80977 RhB yes yes (Kashiyama et al. 2009) Triops granarius BAG80978 RhC yes yes (Kashiyama et al. 2009) Triops granarius BAG80979 RhD yes yes (Kashiyama et al. 2009) Triops granarius BAG80980 RhE yes yes (Kashiyama et al. 2009) Triops BAG80981 RhA yes unknown (Kashiyama et al. 2009) longicaudatus Triops BAG80982 RhB yes unknown (Kashiyama et al. 2009) longicaudatus Triops BAG80983 RhC yes unknown (Kashiyama et al. 2009) longicaudatus Triops BAG80998 RhD yes unknown (Kashiyama et al. 2009) longicaudatus Triops BAG80999 RhE yes unknown (Kashiyama et al. 2009) longicaudatus Insecta Odonata, Anisoptera Sympetrum BAQ54698 RhLWA1 unknown unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54699 RhLWA2 yes unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54700 RhLWB1 unknown unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54701 RhLWC1 unknown unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54702 RhLWD1 unknown yes S. frequens (Futahashi et frequens al. 2015), S. rubicundulum (Mobbs et al. 1981) Sympetrum BAQ54703 RhLWE1 yes unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54704 RhLWF1 yes unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54705 RhLWF2 yes unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54706 RhLWF3 yes unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54707 RhLWF4 yes unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54708 RhSWa1 unknown unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54709 RhSWb1 yes unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54710 RhSWc1 yes unknown (Futahashi et al. 2015) frequens

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Sympetrum BAQ54711 RhSWc2 yes unknown (Futahashi et al. 2015) frequens Sympetrum BAQ54712 RhSWc2 yes unknown Futahashi et al. 2015 frequens Sympetrum BAQ54713 RhUV yes unknown S. frequens (Futahashi et frequens al. 2015), S. rubicundulum (Ruck 1965; Mobbs et al. 1981; Meinertzhagen et al. 1983), S. striolatum and S. vulgatum (Labhart and Nilsson 1995) Sympetrum BAQ54697 Rh7-like unknown unknown (Futahashi et al. 2015) frequens Odonata, Zygoptera Ischnura asiatica BAQ54909 RhLWA1 unknown unknown (Futahashi et al. 2015) Ischnura asiatica BAQ54910 RhLWA2 yes unknown (Futahashi et al. 2015) Ischnura asiatica BAQ54911 RhLWC1 unknown unknown (Futahashi et al. 2015) Ischnura asiatica BAQ54912 RhLWD1 unknown yes (Futahashi et al. 2015) Ischnura asiatica BAQ54913 RhLWE1 unknown yes (Futahashi et al. 2015) Ischnura asiatica BAQ54914 RhLWF1 yes unknown (Futahashi et al. 2015) Ischnura asiatica BAQ54915 RhLWF2 yes unknown (Futahashi et al. 2015) Ischnura asiatica BAQ54916 RhLWF3 yes unknown (Futahashi et al. 2015) Ischnura asiatica BAQ54917 RhLWF4 yes unknown (Futahashi et al. 2015) Ischnura asiatica BAQ54918 RhSWb1 yes unknown (Futahashi et al. 2015) Ischnura asiatica BAQ54910 RhSWb2 yes unknown (Futahashi et al. 2015) Ischnura asiatica BAQ54920 RhUV yes unknown I. asiatica (Futahashi et al. 2015), I. heterosticta (Huang et al. 2014), I. elegans (Henze et al. 2013) Ischnura asiatica BAQ54908 Rh7-like unknown unknown (Futahashi et al. 2015) Orthoptera, Caelifera Schistocerca CAA56377 Lo1 yes unknown (Schmeling et al. 2014) gregaria Schistocerca CAA56378 Lo2 yes no (Wilson 1978; Schmeling gregaria et al. 2014) Schistocerca BAP16681 LoUV yes unknown (Schmeling et al. 2014) gregaria Orthoptera, Ensifera Gryllus AEG78683 GreenA no yes (Henze et al. 2012) bimaculatus Gryllus AEG78684 GreenB yes no G. bimaculatus (Zufall et bimaculatus al. 1989; Henze et al. 2012; Frolov et al. 2014), G. campestris (Labhart et al. 1984)

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Gryllus AEG78685 Blue yes no G. bimaculatus (Zufall et bimaculatus al. 1989; Henze et al. 2012; Frolov et al. 2014), G. campestris (Labhart et al. 1984; Blum and Labhart 2000) Gryllus AEG78686 UV yes no? G. bimaculatus (Zufall et bimaculatus al. 1989; Henze et al. 2012; Frolov et al. 2014), G. campestris (Labhart et al. 1984) Hemiptera Nephotettix BAO03864 LW unknown unknown (Matsumoto et al. 2014) cincticeps Nephotettix BAO03865 Blue unknown unknown (Matsumoto et al. 2014) cincticeps Nephotettix BAO03866 UV unknown unknown (Matsumoto et al. 2014) cincticeps Nilaparvata lugens BAO03855 LW yes yes (Matsumoto et al. 2014) Nilaparvata lugens BAO03856 UV1 yes no (Matsumoto et al. 2014, personal communication February 2015) Nilaparvata lugens BAO03857 UV2 yes no (Matsumoto et al. 2014, personal communication February 2015) Psocodea Pediculus humanus XP_002427337 op1 unknown not (Kirkness et al. 2010) applicable Pediculus humanus XP_002422743 op2 unknown not (Kirkness et al. 2010) applicable Pediculus humanus XP_002432663 op3 unknown not (Kirkness et al. 2010) applicable Hymenoptera Apis mellifera AAA69069 LW1 yes no (Goldsmith 1960; Autrum and v. Zwehl 1964; Menzel and Blakers 1976; Chang et al. 1996; Wakakuwa et al. 2005; Velarde et al. 2005) Apis mellifera DAA05740 LW2 no yes (Goldsmith and Ruck 1958; Velarde et al. 2005) Apis mellifera AAC13417 Blue yes no (Goldsmith and Ruck 1958; Autrum and v. Zwehl 1964; Menzel and Blakers 1976; Townson et al. 1998; Wakakuwa et al. 2005; Velarde et al. 2005)

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Apis mellifera AAC13418 UV yes yes (Goldsmith and Ruck 1958; Goldsmith 1960; Autrum and v. Zwehl 1964; Menzel and Blakers 1976; Townson et al. 1998; Wakakuwa et al. 2005; Velarde et al. 2005) Bombus impatiens AAS55402 LW1 unknown unknown (Spaethe and Briscoe 2004) Bombus impatiens AAS55406 LW2 unknown unknown (Spaethe and Briscoe 2004) Bombus impatiens XP_003494923 Blue yes unknown (Skorupski and Chittka 2010; Sadd et al. 2015) Bombus impatiens AAV67326 UV yes yes (Spaethe and Briscoe 2005; Skorupski and Chittka 2010) Ceratosolen solmsi AGH15790 LW1 unknown females: (Wang et al. 2013) unknown, males: not applicable Ceratosolen solmsi AGH15791 LW2 unknown females: (Wang et al. 2013) unknown, males: not applicable Ceratosolen solmsi AGH15792 Blue unknown females: (Wang et al. 2013) unknown, males: not applicable Ceratosolen solmsi AGH15793 UV unknown females: (Wang et al. 2013) unknown, males: not applicable Coleoptera Tribolium NP_001155991 LW yes not (Jackowska et al. 2007; castaneum applicable Park et al. 2008; Richards et al. 2008) Tribolium XP_970344 UV yes not (Jackowska et al. 2007; castaneum applicable Park et al. 2008; Richards et al. 2008)

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Lepidoptera Manduca sexta O02464 Manop1 yes unknown (Boëthius et al. 1968; RhP520 Höglund and Struwe 1970; Carlson and Philipson 1972; Pappas and Eaton 1977; White et al. 1983; Cutler et al. 1995; Chase et al. 1997; White et al. 2003) Manduca sexta O96107 Manop3 yes no (Boëthius et al. 1968; RhP450 Höglund and Struwe 1970; Carlson and Philipson 1972; Pappas and Eaton 1977; White et al. 1983; Cutler et al. 1995; Chase et al. 1997; White et al. 2003) Manduca sexta O02465 Manop2 yes unknown (Boëthius et al. 1968; RhP357 Höglund and Struwe 1970; Carlson and Philipson 1972; Pappas and Eaton 1977; White et al. 1983; Cutler et al. 1995; Chase et al. 1997; White et al. 2003) Diptera Drosophila AAA28733 Rh1 yes no (Harris et al. 1976; melanogaster Scavarda et al. 1983; Nichols and Pak 1985; O'Tousa et al. 1985; Zuker et al. 1985; Mismer and Rubin 1987; Pollock and Benzer 1988; Zuker et al. 1988; Feiler et al. 1992; Chou et al. 1996) Drosophila AAA28734 Rh2 no yes (Hu et al. 1978; Feiler et al. melanogaster 1988; Pollock and Benzer 1988; Zuker et al. 1988; Feiler et al. 1992; Chou et al. 1996) Drosophila CAB06821 Rh6 yes no (Huber et al. 1997; Salcedo melanogaster et al. 1999; Yasuyama and Meinertzhagen 1999; Helfrich-Förster et al. 2002; Malpel et al. 2002; Sprecher et al. 2007; Mazzoni et al. 2008; Sprecher and Desplan 2008)

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Drosophila AAC47426 Rh5 yes no (Chou et al. 1996; melanogaster Papatsenko et al. 1997; Salcedo et al. 1999; Malpel et al. 2002; Sprecher et al. 2007; Mazzoni et al. 2008; Sprecher and Desplan 2008) Drosophila AAA28854 Rh3 yes no (Fryxell and Meyerowitz melanogaster 1987; Zuker et al. 1987; Pollock and Benzer 1988; Feiler et al. 1992; Chou et al. 1996; Mazzoni et al. 2008) Drosophila AAA28856 Rh4 yes no (Montell et al. 1987; melanogaster Pollock and Benzer 1988; Feiler et al. 1992; Chou et al. 1996; Mazzoni et al. 2008) Drosophila NP_524035 Rh7 unclear unclear (Bleyl 2008; Grebler 2010; melanogaster Kistenpfennig 2012) Musca domestica XP_005182995 Rh1a unknown no (Kirschfeld et al. 1988; Scott et al. 2014) Musca domestica XP_011291215 Rh1b unknown no (Kirschfeld et al. 1988; Scott et al. 2014) Musca domestica XP_005182983 Rh1c unknown no (Kirschfeld et al. 1988; Scott et al. 2014) Musca domestica XP_005191160 Rh2 unknown yes (Kirschfeld et al. 1988; Scott et al. 2014) Musca domestica XP_005186097 Rh6 yes no (Hardie et al. 1979; Hardie 1986; Kirschfeld et al. 1988; Scott et al. 2014), Calliphora (Smola and Meffert 1979; Schmitt et al. 2005) Musca domestica XP_005175090 Rh5 yes no (Hardie 1986; Kirschfeld et al. 1988; Scott et al. 2014), Calliphora (Smola and Meffert 1979; Schmitt et al. 2005) Musca domestica XP_005190072 Rh3 yes no (Hardie et al. 1979; Hardie 1984, 1986; Kirschfeld et al. 1988; Scott et al. 2014), Calliphora (Smola and Meffert 1979; Schmitt et al. 2005) Musca domestica XP_005181535 Rh7 unknown unknown (Scott et al. 2014)

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Table S3 Genomes and transcriptomes searched for visual r-opsins and peropsins.

Taxon Sample Sequence References Accession no. Visual organs

MYRIAPODA (Outgroup) Chilopoda generic, head and SRX462011 (Fernández et al. Scutigeromorpha anterior part of transcriptome (assembly provided only lateral eyes 2014) Scutigera coleoptrata the trunk by the authors) OLIGOSTRACA Branchiura whole specimens lateral and median Arguloida transcriptome (Sahoo et al. 2013) SRA053334 of both sexes eyes Argulus siamensis Ostracoda, Podocopa http://dx.doi.org/10. Podocopida median eye transcriptome (Oakley et al. 2013) only median eye 5061/dryad.tb40v Heterocypris sp. Ostracoda, Myodocopa whole embryos http://dx.doi.org/10. lateral and median Myodocopida transcriptome (Speiser et al. 2014) (males) 5061/dryad.277g0 eyes Euphilomedes carcharodonta MULTICRUSTACEA Copepoda six developmen- Calanoida tal samples of Calanus finmarchicus whole individuals: embryo (egg), transcriptome (Lenz et al. 2014) GAXK00000000.1 only median eye early and late nauplii, early and late copepodites and adult females

table continued on next page

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Taxon Sample Sequence References Accession no. Visual organs

ALLOTRIOCARIDA Branchiopoda fused lateral naturally inbred (Colbourne et al. Cladocera genome ACJG00000000.1 eyes and median isoclonal isolate 2011) Daphnia pulex eye Remipedia Nectiopoda 10 complete (von Reumont et al. transcriptome GAJM00000000.1 no eyes Xibalbanus (Speleonectes) specimens 2014) tulumensis Collembola lateral and Symphypleona generic, adult transcriptome (Misof et al. 2014) SRX314895 median eyes Sminthurus viridis Insecta lateral and Zygentoma generic transcriptome (Misof et al. 2014) SRX314908 median eyes Tricholepidion gertschi Insecta lateral and Archaeognatha generic, adult transcriptome (Misof et al. 2014) SRX314868 median eyes Machilis hrabei Insecta lateral and Ephemeroptera generic, adult transcriptome (Misof et al. 2014) SRX314815 median eyes Ephemera danica Insecta a single male, inbred http://www.butt (Dasmahapatra et al. five generations of genome erflygenome.org/ only lateral eyes 2012) Heliconius melpomene sib mating assembly v1.1

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Table S4 Visual r-opsins identified in the genomes and transcriptomes listed in Supplementary Table S3, and references on the expression of opsins.

expression in Species Unique No. Opsin lateral median References name eyes eyes MYRIAPODA (Outgroup) Chilopoda Scutigeromorpha Scutigera 16909 MW yes not (Meyer-Rochow et al. coleoptrata applicable 2006), see comment on interpretation of data in Nilsson and Kelber (2007) OLIGOSTRACA Branchiura Arguloida Argulus siamensis 401512162 rOp1 unknown unknown Argulus siamensis 401532453 rOp2 unknown unknown Argulus siamensis 401513687 rOp3 unknown unknown Argulus siamensis 401520393 rOp4 unknown unknown Argulus siamensis 401516392 rOp5 unknown unknown Argulus siamensis 401535242 rOp6 unknown unknown Argulus siamensis 401521393 rOp7 unknown unknown Ostracoda, Podocopa Podocopida Heterocypris sp. contig06320 rOp1§ not yes applicable Ostracoda, Myodocopa Myodocopida Euphilomedes UN1394 rOp1 unknown unknown carcharodonta Euphilomedes UN2129 rOp2 unknown unknown carcharodonta MULTICRUSTACEA Copepoda Calanoida Calanus GBFB01096267 rOp1 not unkown finmarchicus applicable Calanus GBFB01009482 rOp2 not unknown finmarchicus applicable ALLOTRIOCARIDA Branchiopoda Cladocera Daphnia pulex LOPA1 LOpA1 unknown unknown Daphnia pulex LOPA2 LOpA2 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPA3 LOpA3 unknown unknown (Colbourne et al. 2011)

§one of four closely related candidates

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Daphnia pulex LOPA4 LOpA4 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPA6 LOpA6 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPA8 LOpA8 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPA9 LOpA9 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPA10 LOpA10 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB1 LOpB1 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB2 LOpB2 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB3 LOpB3 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB4 LOpB4 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB5 LOpB5 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB6 LOpB6 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB7 LOpB7 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB8 LOpB8 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB9 LOpB9 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB13 LOpB13 unknown unknown (Colbourne et al. 2011) Daphnia pulex LOPB14 LOpB14 unknown unknown Daphnia pulex LOPB15 LOpB15 unknown unknown (Colbourne et al. 2011) Daphnia pulex UVOP UV1 unknown unknown (Colbourne et al. 2011), D. magna (Smith and Macagno 1990) Daphnia pulex BLOP UV2 unknown unknown (Colbourne et al. 2011), D. magna (Smith and Macagno 1990) Daphnia pulex UNOP1 UnOp1 unknown unknown (Colbourne et al. 2011) Daphnia pulex UNOP2 UnOp2 unknown unknown (Colbourne et al. 2011) Collembola Symphypleona Sminthurus viridis GATZ01005081 LW1 unknown unknown Sminthurus viridis GATZ01093387 LW2 unknown unknown Sminthurus viridis GATZ01074098 LW3 unknown unknown Sminthurus viridis GATZ01000283 MW1 unknown unknown Sminthurus viridis GATZ01005173 MW2 unknown unknown Sminthurus viridis GATZ01094783 MW3 unknown unknown Sminthurus viridis GATZ01090057 MW4 unknown unknown Sminthurus viridis GATZ01087993 MW5 unknown unknown Sminthurus viridis GATZ01010906 MW6 unknown unknown Sminthurus viridis GATZ01103981 MW7 unknown unknown Sminthurus viridis GATZ01103643 MW8 unknown unknown Insecta Archaeognatha Machilis hrabei GAUM01017353 LW1 unknown unknown Machilis hrabei GAUM01184980 LW2 unknown unknown Machilis hrabei GAUM01017675 MW unknown unknown Machilis hrabei GAUM01184193 UV unknown unknown Zygentoma Tricholepidion GASO01008719 LW1 unknown unknown gertschi

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Tricholepidion GASO01008718 LW2 unknown unknown gertschi Tricholepidion GASO01008717 LW3 unknown unknown gertschi Ephemeroptera Ephemera danica GAUK01106886 LW1 unknown unknown Ephemera danica GAUK01109703 LW2 unknown unknown Ephemera danica GAUK01002297 LW3 unknown unknown Ephemera danica GAUK01002300 LW4* unknown unknown Ephemera danica GAUK01002296 LW4* unknown unknown Ephemera danica GAUK01017324 SW1 unknown unknown Ephemera danica GAUK01003006 SW2 unknown unknown Ephemera danica GAUK01003005 SW3 unknown unknown Ephemera danica GAUK01003004 SW4 unknown unknown Ephemera danica GAUK01007309 UV* yes unknown (Meyer-Rochow 1982) Ephemera danica GAUK01007310 UV* yes unknown (Meyer-Rochow 1982) Lepidoptera Heliconius HMEL003344-PA LW yes not H. erato (Zaccardi et al. melpomene applicable 2006), H. erato, H. numata, and H. sara (Struwe 1972a, b) Heliconius HMEL010706-PA Blue yes not H. erato (Zaccardi et al. melpomene applicable 2006), H. erato, H. numata, and H. sara (Struwe 1972a, b) Heliconius HMEL010962-PA UV1 yes not H. melpomene, H. erato melpomene applicable (Briscoe et al. 2010; Bybee et al. 2012), H. erato (Zaccardi et al. 2006), H. erato, H. numata, and H. sara (Struwe 1972a, b) Heliconius HMEL022525-PA UV2 yes not H. melpomene, H. erato melpomene applicable (Briscoe et al. 2010; Bybee et al. 2012), H. erato (Zaccardi et al. 2006), H. erato, H. numata, and H. sara (Struwe 1972a, b) Heliconius HMEL022527-PA Rh7-like unknown not melpomene applicable

*same amino-acid sequence, differences in nucleotide sequence

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