Opsin evolution in the Ambulacraria. D'Aniello, S; Delroisse, J; Valero-Gracia, A; Lowe, EK; Byrne, M; Cannon, JT; Halanych, KM; Elphick, MR; Mallefet, J; Kaul-Strehlow, S; Lowe, CJ; Flammang, P; Ullrich-Lüter, E; Wanninger, A; Arnone, MI © 2015 The Authors. CC-BY-NC-ND For additional information about this publication click this link. http://qmro.qmul.ac.uk/xmlui/handle/123456789/13563 Information about this research object was correct at the time of download; we occasionally make corrections to records, please therefore check the published record when citing. For more information contact [email protected] *Manuscript Click here to view linked References Opsin evolution in the Ambulacraria 1 2 3 D Aniello S1,§,*, Delroisse J2,3,§, Valero-Gracia A1, Lowe EK1,4, Byrne M5, Cannon 4 ’ 5 6 6,7 6 3 8 9 10 7 JT , Halanych KM , Elphick MR , Mallefet J , Kaul-Strehlow S , Lowe CJ , 8 9 Flammang P2, Ullrich-Lüter E11, Wanninger A12 and Arnone MI1 10 11 12 13 1 14 Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 15 80121 Napoli, Italy. 16 2 Biology of Marine Organisms and Biomimetics, Research Institute for Biosciences, University of Mons, Avenue du 17 18 Champs de Mars 6, 7000 Mons, Belgium. 19 3 School of Biological & Chemical Sciences, Queen Mary University of London, London, E1 4NS, UK. 20 4 BEACON Center for the Study of Evolution in Action, Michigan State University, East Lansing, Michigan, USA 21 22 5 Schools of Medical and Biological Sciences, The University of Sydney, Sydney, NSW, Australia. 23 6 Department of Biological Sciences and Molette Biology Laboratory for Environmental and Climate Change 24 25 Studies, Auburn University, Auburn, USA. 26 7 Department of Zoology, Naturhistoriska Riksmuseet, Stockholm, Sweden; Friday Harbor Laboratories, University 27 28 of Washington, Friday Harbor, WA 98250, USA. 29 8 Laboratory of Marine Biology, Earth and Life Institute, Université Catholique de Louvain, Louvain-La-Neuve, 30 31 Place Croix du Sud 3, bt L7.06.04, 1348 Louvain-la-Neuve, Belgium. 32 9 Department of Molecular Evolution and Development, University of Vienna, Althanstrasse 14, 1090 Vienna, 33 34 Austria. 35 10 Hopkins Marine Station of Stanford University, Pacific Grove, CA 93950, USA. 36 11 Museum fuer Naturkunde Berlin, Invalidenstr 43, 10115 Berlin, Germany. 37 12 38 Department of Integrative Zoology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria. 39 40 41 42 43 44 45 46 47 48 49 50 § 51 These authors contributed equally to this work. 52 53 54 *Corresponding author: [email protected] 55 56 57 58 59 60 61 62 63 64 1 65 Abstract 1 2 3 Opsins - G-protein coupled receptors involved in photoreception - have been 4 5 extensively studied in the animal kingdom. The present work provides new insights into 6 opsin-based photoreception and photoreceptor cell evolution with a first analysis of 7 8 opsin sequence data for a major deuterostome clade, the Ambulacraria. Systematic data 9 10 analysis, including for the first time hemichordate opsin sequences and an expanded 11 12 echinoderm dataset, led to a robust opsin phylogeny for this cornerstone superphylum. 13 14 Multiple genomic and transcriptomic resources were surveyed to cover each class of 15 16 Hemichordata and Echinodermata. In total, 119 ambulacrarian opsin sequences were 17 found, 22 new sequences in hemichordates and 97 in echinoderms (including 67 new 18 19 sequences). We framed the ambulacrarian opsin repertoire within eumetazoan diversity 20 21 by including selected reference opsins from non-ambulacrarians. Our findings 22 23 corroborate the presence of all major ancestral bilaterian opsin groups in Ambulacraria. 24 25 Furthermore, we identified two opsin groups specific to echinoderms. In conclusion, a 26 27 molecular phylogenetic framework for investigating light-perception and 28 photobiological behaviours in marine deuterostomes has been obtained. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 Keywords: Opsin; Photoreceptor Cell Evolution; Ambulacraria; Echinoderm; 54 55 Hemichordate; Phylogeny; Echinopsin. 56 57 Manuscript type: Original data paper. 58 59 60 61 62 63 64 2 65 Introduction 1 2 3 In animals, the prototypical molecules involved in photoreception and vision are opsin 4 5 proteins (Nilsson, 2004). Opsins are G-protein coupled receptors (GPCR) that consist of 6 an apoprotein plus a covalently bound to a chromophore (11-retinal) (Terakita, 2005). 7 8 The nitrogen atom of the amino group of residue K296, situated in helix VII, binds to 9 10 the retinal molecule through a Schiff-base linkage, forming a double bond with the 11 12 carbon atom at the end of this molecule (Hargrave et al., 1983). Residue K296 is, 13 14 therefore, crucial for light absorption, and its presence or absence can be used as a 15 16 molecular fingerprint to judge whether or not a GPCR is a bona fide opsin. 17 Recent investigations on opsin phylogeny resolved six distinct groups present in 18 19 metazoans: ciliary opsins, rhabdomeric opsins, Go-opsins, neuropsins, peropsins, and 20 21 RGR (RPE-retinal G protein-coupled receptor) opsins (Porter et al., 2012; Feuda et al. 22 23 2012; Terakita et al., 2012). A vast number of opsins are also expressed in non-ocular 24 25 tissues (Porter et al., 2012; Plachetzki et al., 2005; Koyanagi et al., 2005; Terakita et 26 27 al., 2012). 28 With regard to opsin evolution in the deuterostomes, genomic and transcriptomic data 29 30 of a number of chordates have been used to identify and characterize their opsins (e.g. 31 32 Holland et al., 2008; Kusakabe et al., 2001). However, little attention has been paid to 33 34 Ambulacraria, the sister group to all extant chordates, (i.e. cephalochordates, 35 36 urochordates, and vertebrates, Edgecombe et al., 2011), a key clade to reconstruct the 37 38 opsin set of the common ancestor of extant deuterostomes. 39 The present study integrates opsin sequences from two ambulacrarian sub-lineages: 40 41 enteropneust Hemichordata, (Harrimaniidae, Spengelidae, Ptychoderidae and 42 43 Torquaratoridae), and the pentameral Echinodermata comprising five classes 44 45 (Crinoidea, Ophiuroidea, Asteroidea, Holoturoidea and Echinoidea). 46 47 The phylogenetic relationship of echinoderms and hemichordates as sister groups within 48 Ambulacraria, as shown in Figure 1, was already suggested by Metschnikoff (1881), 49 50 and supported by Nielsen (2012). The monophyly of Ambulacraria is also well 51 52 supported by molecular phylogenetic analyses (Cannon et al., 2014; Telford et al., 53 54 2014). Moreover, Cannon and colleagues showed that the six hemichordate subgroups 55 56 cluster into two monophyletic taxa, Enteropneusta and Pterobranchia (Rhabdopleuridae 57 58 and Cephalodiscidae). Finally, Figure 1 conforms to the Asterozoa hypothesis 59 separating the Echinozoa (Echinoidea + Holothuroidea) and the Asterozoa (Asteroidea 60 61 62 63 64 3 65 + Ophiuroidea), which is now well supported by recent molecular phylogenies (Cannon 1 2 et al., 2014; Telford et al., 2014; O’Hara et al., 2014). 3 4 Other than a few structural investigations of eye-like structures in some asteroid species 5 6 (e.g. the starfish optic cushion) and in enteropneust larvae (Brandenburger et al., 1973; 7 8 Nezlin and Yushin, 2004; Braun et al., 2015), the molecular mechanisms of echinoderm 9 10 and hemichordate photoreception remained enigmatic until recently. 11 12 Immunohistochemical studies indicated the presence of a putative rhodopsin in the 13 14 asteroid Asterias forbesi and in the ophiuroid Ophioderma brevispinum (Johnsen, 15 16 1997). Subsequently, Raible et al. (2006) analyzed the ‘rhodopsin-type’ G-protein- 17 18 coupled receptors family in an echinoid genome (Strongylocentrotus purpuratus). They 19 20 predicted six bona fide opsin sequences, four of which were reported independently by 21 22 Burke et al. (2006). Later, Ooka et al. (2010) cloned an “encephalopsin” orthologue in 23 24 25 the sea urchin Hemicentrotus pulcherrimus. Recently, more opsin sequences have been 26 found in sea urchins (S. purpuratus; Paracentrotus lividus), starfish (Asterias rubens), 27 28 and brittle stars (Ophiocomina nigra, Amphiura filiformis) (Delroisse et al., 2013, 2014, 29 30 2015 a,b ; Ullrich-Lüter et al., 2011, 2013). These studies highlighted the expression of 31 32 ciliary and rhabdomeric opsins in various echinoderm tissues. Also, a large opsin gene 33 34 repertoire was identified in the brittle star A. filiformis, pinpointing notable differences 35 with findings from the previously published sea urchin genome (Delroisse ., 2014). 36 et al 37 However, a comprehensive description of opsin diversity in echinoderms is still lacking 38 39 and almost nothing is known about hemichordate opsins. 40 41 Therefore, to characterize and describe the diversity of the opsin family in the 42 43 Ambulacraria, we conducted a detailed analysis of 6 genomic and 24 transcriptomic 44 45 sequence databases. This work represents the first attempt to describe and characterize 46 47 the evolution of the opsin “toolkit” in the ambulacrarian lineage. We performed a 48 49 phylogenetic study using the largest dataset of ambulacrarian opsin sequences to date, 50 51 including representatives of a previously neglected group, Hemichordata. 52 53 54 55 56 Materials and methods 57 58 59 60 Data mining 61 62 63 64 4 65 Strongylocentrotus purpuratus opsins belonging to all the paralogous classes (Supp. 1 2 File 1) were used as starting query sequences for tBLASTx against transcriptomic and 3 genomic databases including public databases (NCBI, JGI, Ensemble, Echinobase 4 5 (www.echinobase.org/), BioInformatique CNRS-UPMC (http://octopus.obs-vlfr.fr/) and 6 7 Genoscope (http://www.genoscope.cns.fr/spip/Generation-de-ressources.html). The 8 9 parameters used across all our tBLASTx searches were the following: Matrix: 10 11 Blosum62; gap penalties: existence: 11; extension: 1; neighboring words threshold: 13; 12 13 window for multiple hits: 40.