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Molecular Characterization of Visual Pigments in Branchiopoda and the Evolution of Opsins in Arthropoda Kazuyuki Kashiyama,* Takaharu Seki, Hideharu Numata,* and Shin G. Goto* *Graduate School of Science, Osaka City University, Osaka, Japan; and Division of Health Science, Osaka Kyoiku University, Kashiwara, Japan Studies on color vision in invertebrates have focused primarily on insect visual pigments, with little attention given to crustacean visual pigments. None of the blue–green-, blue-, or ultraviolet (UV)-sensitive–opsins have been identified in crustaceans. In addition, the discussion of visual pigments has been limited to long-wavelength-sensitive opsins in Pancrustacea. Here, we focused on Branchiopoda (Crustacea), which is a sister group of Hexapoda including insects. In the tadpole shrimp Triops granarius, the visual pigment chromophore was retinal. Multiple opsins were isolated from each of three branchiopod species, T. granarius, Triops longicaudatus, and the fairy shrimp Branchinella kugenumaensis (five, five, and four opsins from these species, respectively). Phylogenetic analyses and the presence of a lysine residue corresponding to position 90 in bovine rhodopsin suggested that three of the branchiopod opsins comprise UV-sensitive pigments. In addition, the phylogenetic relationships between insect and branchiopod UV-sensitive opsins revealed that the divergence of blue- and UV-sensitive pigments predates the Branchiopoda and Insecta divergence. The other branchiopod opsins show distant relationships to other known insect opsins and form novel clusters. The present results strongly suggest that the ancestral arthropod of the Chelicerata–Pancrustacea lineages possessed at least four types of opsins. The ancestors of Pancrustacea and the Insecta–Branchiopoda lineages possessed at least five and six types of opsins, respectively. Our results suggest that in the evolutionary process associated with each lineage, several opsins appeared and diversified with repeated gene duplication, of which some have been lost in some taxa. Introduction Among all animals, the ability to discriminate light of different wavelengths, that is, color vision, has evolved in vertebrates and arthropods. In Arthropoda, color vision has long been investigated in Hexapoda, especially in insects, whereas other groups such as Crustacea, Myriapoda, and Chelicerata have received little attention (Land and Nilsson 2002; Koyanagi et al. 2008). Multiple photoreceptor cells with distinct spectral properties have been physiologically identified in insects (Bennett et al. 1967; Menzel and Blakers 1976; White et al. 1983; Arikawa et al. 1987). Based on physiological properties, the photoreceptor cells are largely assigned to three spectral classes: long wavelength (LW) sensitive (maximally sensitive to light longer than 500 nm), blue sensitive (400–460 nm), and ultraviolet (UV) sensitive (325– 370 nm) (Chittka 1996). The spectral sensitivity of a photoreceptor cell is usually determined by its visual pigment but can also be affected by screening or sensitizing pigments (Arikawa, Mizuno, et al. 1999; Arikawa,Scholten, et al. 1999). The visual pigment, which is embedded in the membrane of a retinula cell, absorbs light in a certain spectral range and initiates a phototransduction cascade leading to vision (Nathans 1987). The pigment consists of a protein moiety, an opsin, and a light-sensitive prosthetic group, a chromophore. Upon absorbing a photon, the chromophore is isomerized from the 11-cis to the all-trans isomer, and the photoisomerization triggers a series of conformational changes in the opsin protein resulting in an active visual pigment (Nathans et al. 1986). Five retinal (RAL) congeners, that is, retinal, RAL1; 3,4-didehydroretinal, RAL2; (3R)-3-hydroxyretinal, (3R)-RAL3; (4R)-4-hydroxyretinal, Key words: opsin, Branchiopoda, Triops, Branchinella, visual pigments, retinal. E-mail: [email protected]. Mol. Biol. Evol. 26(2):299–311. 2009 doi:10.1093/molbev/msn251 Advance Access publication November 4, 2008 RAL4; and (3S)-3-hydroxyretinal, (3S)-RAL3, have been identified as chromophores among animals (Seki and Vogt 1998). The isolated 11-cis isomer is most sensitive to light of approximately 380 nm, but binding with an opsin can enhance its absorption maximum over a wider spectral range from about 360 to 640 nm (Kochendoerfer et al. 1999). A large number of insect opsins have been isolated in various orders, for example, Orthoptera (Towner et al. 1997), Lepidoptera (Chase et al. 1997; Kitamoto et al. 1998, 2000), Diptera (O’Tousa et al. 1985; Zuker et al. 1985; Cowman et al. 1986; Fryxell and Meyerowitz 1987; Montell et al. 1987; Zuker et al. 1987; Chou et al. 1996; Huber et al. 1997; Papatsenko et al. 1997), and Hymenoptera (Chang et al. 1996; Popp et al. 1996; Smith et al. 1997; Townson et al. 1998). In insects, multiple opsins with distinct spectral classes have been identified in each individual species, such as the swallowtail butterfly Papilio xuthus with five opsins and the fruit fly Drosophila melanogaster with seven. Molecular phylogenetic analyses revealed that insect opsins can be divided into four major subfamilies, three of which coincide with the physiologically identified LW-, blue-, and UV-sensitive visual pigment groups (Briscoe and Chittka 2001). The fourth subfamily, found only in Diptera, is composed of the blue–green-sensitive opsins including the D. melanogaster major pigment Rh1 (ninaE) and ocelli-specific Rh2 and is most closely related to LW-sensitive opsins. Information on visual pigments in Crustacea is rather limited, but several microspectrophotometric and electrophysiological studies have revealed multiple spectral classes of photoreceptor cells in the retinas of Malacostraca. For example, some stomatopod crustaceans possess at least 10 spectral types of photoreceptor cells (Cronin and Marshall 1989a, 1989b). In Crustacea, RAL1 is identified in malacostracan species (Goldsmith and Cronin 1993; Srivastava et al. 1996), but several crayfish use RAL2 as well as RAL1 (Suzuki et al. 1984; Hariyama and Tsukahara 1988; Zeiger and Goldsmith 1989). Opsins have also been characterized Ó 2008 The Authors This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/ uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 300 Kashiyama et al. in several malacostracan species (Hariyama et al. 1993; Sakamoto et al. 1996; Crandall and Cronin 1997; Crandall and Hillis 1997; Porter et al. 2007). Microspectrophotometric and electrophysiological studies suggest that the characterized malacostracan opsins, except opsins in the brachyuran crab, contribute to the reception of light in the LW range (Goldsmith 1978; Crandall and Cronin 1997; Porter et al. 2007). In phylogenetic analyses, these opsins are most closely related to insect LW-sensitive opsins (Porter et al. 2007). The brachyuran crab Hemigrapsus possesses at least two opsins, and they form a unique cluster in phylogenetic analyses (Sakamoto et al. 1996). A large number of opsins were identified in two ostracod species, but their nucleotide sequences are highly similar and all the sequences form a single cluster in the phylogenetic analysis (Oakley and Huber 2004). The spectral properties of the brachyuran crab and the ostracod pigments comprising these opsins have not been examined (Sakamoto et al. 1996; Oakley and Huber 2004). Information on Chelicerata opsins is quite limited. The horseshoe crab Limulus possesses at least two opsins, and they are expressed in different photoreceptor organs (lateral eyes and ocelli). They are members of the LW-sensitive opsins (Hubbard and Wald 1960; Nolte and Brown 1972; Smith et al. 1993). Recently, Koyanagi et al. (2008) cloned three opsins from the jumping spider, two of which belong to the LW-sensitive opsin group, and the third is considered to be UV sensitive. No information on Myriapod opsins is available. Phylogenetic relationships among Hexapoda, Crustacea, Myriapoda, and Chelicerata are ambiguous (Giribet and Ribera 2000). There are conflicting hypotheses about their relationship, but it is agreed that Chelicerata is the most or one of the most distant arthropod groups from Hexapoda (Giribet et al. 2001; Hwang et al. 2001; Peterson and Eernisse 2001; Mallatt et al. 2004). Several recent molecular studies have consistently revealed a close relationship between hexapods and crustaceans (Shultz and Regier 2000; Cook et al. 2001; Giribet et al. 2001; Regier and Shultz 2001; Mallatt et al. 2004). In addition, recent morphological studies (Strausfeld 2005; Harzsch 2006) and molecular phylogenetic analyses (Regier et al. 2005; Mallatt and Giribet 2006) suggest that Hexapoda originated within Crustacea. Consequently, the clade of Hexapoda and Crustacea is commonly referred to as ‘‘Pancrustacea.’’ Moreover, molecular phylogenetic analyses indicate that the sister group of Hexapoda is Branchiopoda (for review, see Glenner et al. 2006). This prompted us to investigate chromophore and opsins in Branchiopoda to expand the knowledge of evolution of visual pigments in Arthropoda. Class Branchiopoda is usually subdivided into three Orders, that is, Anostraca, Notostraca, and Cladocera. In the present study, we characterized opsins from two tadpole shrimps, Triops granarius and Triops longicaudatus, belonging to Notostraca, and from one fairy shrimp, Branchinella kugenumaensis, belonging to Anostraca. To characterize chromophore, we used only T. granarius. The compound eye is the main photoreceptive organ for the branchiopod adults like many other crustaceans (Meyer-Rochow 2001), and several species including Triops and Branchinella also have the ocellus or ocelli. The structure of the compound eye has been well investigated in Artemia (Wildt and Harzsch 2002), Daphnia (Young and Downing 1976), and Triops (Diersch et al. 1999; Harzsch and Walossek 2001). No opsins were identified in any of the branchiopod species. Color sensitivity studies of the compound eye have been performed only in Daphnia (Young 1974; Consi and Macagno 1985; Smith and Macagno 1990). Information on Branchiopod visual pigments will be a key link to understanding Crustacea and Insecta color vision and is indispensable for understanding the evolution of color vision in arthropods. Materials and Methods Animals From mid-June to mid-July in 2004–2006, the tadpole shrimp T. granarius and the fairy shrimp B. kugenumaensis were collected from paddy fields in Sakai City (34.56°N, 135.53°E) in Osaka Prefecture, Japan, and T. longicaudatus was collected from paddy fields in Ritto City (35.04°N, 136.00°E) in Shiga Prefecture, Japan. They were kept at 25 ± 1 °C under a 16 h light:8 h dark cycle and fed on TetraMin or TetraPlankton (Tetra Werke, Melle, Germany). High-Performance Liquid Chromatography (HPLC) Analysis of T. granarius Eye Retinoids Compound eyes and ocelli were collected from 40 adults of T. granarius under a microscope with light, immediately frozen in liquid nitrogen, and stored at 80 °C until the analysis. Retinoids were extracted by the oxime method (Suzuki and Makino-Tasaka 1983) according to the procedures of Irie et al. (1991). HPLC analysis was carried out using an Intelligent HPLC pump (PU-2080 Japan Spectroscopic, Tokyo, Japan) equipped with a silica gel column (6/ 150 mm; YMC-Pack A-012-3 S-3 SIL, Yamamura Chemical Laboratories, Kyoto, Japan). Two UV detectors (Jasco 875-UV and UV-970, Japan Spectroscopic) were used to monitor the absorbance at 330 and 350 nm, and the chromatogram was recorded using a network data processing system (Smart Chrom, Kya Tech Corporation, Tokyo, Japan). After collecting the eyes, all procedures were performed under dim red light (.610 nm). RNA Isolation and cDNA Synthesis Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and was dissolved in DNase-free water for reverse transcriptase-polymerase chain reaction (RTPCR) and rapid amplification of cDNA ends (RACE) and in formamide for Northern blots. DNA was digested with DNase I, amplification grade (Invitrogen) for RTPCR and RACE. For RT-PCR, single-stranded cDNA (ss cDNA) was synthesized using oligo(dT)12–18 primer and moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. For 3# and 5# RACE, a Super SMART PCR cDNA Synthesis Kit (Clontech, Palo Alto, CA) was used to synthesize double-stranded cDNA (ds cDNA). Branchiopoda Visual Pigment and Arthropod Opsin Evolution 301 Primers All the primers used in the present study were designed using Primer3 program (Rozen and Skaletsky 2000; http://primer3.sourceforge.net/), and their sequences were shown in supplementary table S1 (Supplementary Material online). Clone Development and 3# and 5# RACEs In the present study, single-step PCR, two-step PCR, 3# RACE, and 5# RACE were combined to develop opsin clones. Total RNAs of T. granarius were extracted from an anteriorpart ofthecarapace forRT-PCRandfrom thecompound eyes and ocelli for RACE. RNA of T. longicaudatus was extracted from the compound eyes and ocelli and for B. kugenumaensis, from the whole head including the compound eyes and ocelli. Clone development was achieved by PCR using 0.5 U of Platinum Taq DNA polymerase (Invitrogen) at a final concentration of 1 PCR Buffer minus Mg (Invitrogen), 0.2 mM of deoxynucleoside triphosphate (dNTP) mixture which contains all 4 deoxynucleotides (dATP, dCTP, dGTP, and dTTP), and 1.5 mM of MgCl2. The designed primer (0.5 lM; listed in supplementary table S1 in Supplementary Material online), 1 universal primer mix (UPM), and/or 0.5 lM of nested universal primer (NUP) were used for the PCR reaction. The latter two primers were provided in the SMART RACE cDNA Amplification Kit (Clontech). Details of clone development are described below. Three opsin fragments (RhA, RhB, and RhC) were obtained by PCR from T. granarius ss cDNA. RhA fragment was amplified with Op-F1 and Op-R1 primers. PCR conditions comprised 3 min at 95 °C, 42 cycles of 15 s at 95 °C, 15 s at 40 °C, and 30 s at 72 °C. RhB fragment was obtained with the same primer set and PCR conditions comprising 3 min at 95 °C, 40 cycles of 15 s at 95 °C, 15 s at 45 °C, and 45 s at 72 °C. RhC fragment was derived by a two-step PCR. The first PCR was performed similar to that for RhB fragment amplification. The second PCR was performed with a 0.05% final concentration of the first PCR product, and Op-F2 and Op-R1 primers, under PCR conditions of 3 min at 95 °C, 35 cycles of 15 s at 95 °C, 15 s at 45 °C, and 35 s at 72 °C. Two additional opsin fragments (RhD and RhE) were also obtained by the two-step PCR in T. granarius. For RhD, the first PCR was performed with ds cDNA, Op-F1 primer, and UPM, and PCR conditions comprised 3 min at 95 °C, 35 cycles of 15 s at 95 °C, 15 s at 44 °C, and 40 s at 72 °C. The second PCR was performed with 0.05% of the first PCR product, BkOpc-F primer, and NUP, and PCR conditions comprised 3 min at 95 °C, 40 cycles of 15 s at 95 °C, 15 s at 56 °C, and 40 s at 72 °C. For RhE, the first PCR was performed with ds cDNA, TlOpE-F, and UPM, and PCR conditions comprised 3 min at 95 °C, 35 cycles of 15 s at 95 °C, 15 s at 60 °C, and 30 s at 72 °C. The second PCR was performed with 0.05% of the first PCR product, TlOpE-F, and NUP, and PCR conditions comprised 3 min at 95 °C, 35 cycles of 15 s at 95 °C, 15 s at 65 °C, and 30 s at 72 °C. Using the SMART RACE cDNA Amplification Kit (Clontech), 3# and 5# RACE for RhA, RhB, and RhC were performed using ds cDNA, gene- specific primers (TgOpA-F and -R, TgOpB-F and -R, and TgOpC-F and -R, respectively), and UPM. PCR conditions comprised 5 cycles of 5 s at 94 °C and 3 min at 72 °C; 5 cycles of 5 s at 94 °C, 10 s at 70 °C, and 3 min at 72 °C; and 25 cycles of 5 s at 94 °C, 10 s at 68 °C, and 3 min at 72 °C. Using the same kit, 5# RACE for RhD and RhE were also performed using ds cDNA, gene-specific primers (TgOpDR and TgOpE-R, respectively), and UPM. PCR conditions comprised 5 cycles of 5 s at 94 °C and 3 min at 72 °C; 5 cycles of 5 s at 94 °C, 10 s at 70 °C, and 3 min at 72 °C; and 25 cycles of 5 s at 94 °C, 10 s at 68 °C, and 3 min at 72 °C. RhA, RhB, RhC, and RhD fragments in T. longicaudatus were obtained by a two-step PCR. The first PCR was performed with T. longicaudatus ds cDNA, Op-F1 primer, and UPM. PCR conditions comprised 3 min at 95 °C, 35 cycles of 15 s at 95 °C, 15 s at 56 °C, and 40 s at 72 °C. The second PCR was performed with 0.05% of the first PCR product, the designed primer (TgOpA-F, TgOpB-F, TgOpC-F, and BkOpc-F for RhA, RhB, RhC, and RhD, respectively), and NUP, and PCR conditions comprised 3 min at 95 °C, 40 cycles of 15 s at 95 °C, 15 s at 56 °C, and 30 s at 72 °C. RhE fragment was coamplified in 3# RACE for RhC. Two steps of 5# RACE reactions were performed for T. longicaudatus RhA. The first PCR was performed with ds cDNA, TlOpA-R, and UPM. PCR conditions comprised 5 cycles of 5 s at 94 °C and 2 min at 72 °C; 5 cycles of 5 s at 94 °C, 10 s at 68 °C, and 2 min at 72 °C; and 28 cycles of 5 s at 94 °C, 10 s at 64 °C, and 2 min at 72 °C. The second PCR was performed with 0.05% of the first PCR product, TlOpA-R, and NUP. PCR conditions comprised 3 min at 95 °C and 35 cycles of 15 s at 95 °C, 15 s at 58 °C, and 90 s at 72 °C. Similar to RhA, 5# RACE for RhC was performed except that the primer was TlOpC-R. The product of 5# RACE for RhB was generated with ds cDNA, TlOpB-R, and UPM, and PCR conditions comprised 5 cycles of 5 s at 94 °C and 2 min at 72 °C; 5 cycles of 5 s at 94 °C, 10 s at 70 °C, and 2 min at 72 °C; and 25 cycles of 5 s at 94 °C, 10 s at 65 °C, and 2 min at 72 °C. Rha fragment in B. kugenumaensis was obtained by PCR with ss cDNA, Op-F1, and Op-R1 primers. PCR conditions comprised 3 min at 95 °C, 40 cycles of 15 s at 95 °C, 15 s at 40 °C, and 20 s at 72 °C. Rhb and Rhc fragments were derived by a two-step PCR. For Rhb, the first PCR was performed with ds cDNA, TgOpA-F2, and UPM, and PCR conditions comprised 3 min at 95 °C, 35 cycles of 15 s at 95 °C, 15 s at 48 °C, and 30 s at 72 °C. The second PCR was performed with 0.05% of the first PCR product, TgOpA-F primer, and NUP, and PCR conditions comprised 3 min at 95 °C, 35 cycles of 15 s at 95 °C, 15 s at 55 °C, and 30 s at 72 °C. For Rhc, the first PCR was performed similar to that used for developing Rha fragment, and the second PCR was performed with 0.05% of the first PCR product, and Op-F2 and Op-R1 primers. The second PCR conditions were 3 min at 95 °C, 40 cycles of 15 s at 95 °C, 15 s at 40 °C, and 15 s at 72 °C. Rha and Rhc 3# RACE products were generated from ds cDNA using a gene-specific primer (BkOpa-F and BkOpc-F, respectively) and UPM. PCR conditions comprised 3 min at 95 °C, 40 cycles of 15 s at 95 °C, 15 s at 55 °C, and 30 s at 72 °C. The 3# RACE for Rhc co-amplified a novel opsin fragment (Rhd). Products of 5# RACE for Rha, Rhb, Rhc, and Rhd were generated from 302 Kashiyama et al. ds cDNA using a gene-specific primer (BkOpa-R, BkOpbR, BkOpc-R, and BkOpd-R, respectively) and UPM. PCR conditions comprised 5 cycles of 5 s at 94 °C and 2 min at 72 °C; 5 cycles of 5 s at 94 °C, 10 s at 70 °C, and 2 min at 72 °C; and 27 cycles of 5 s at 94 °C, 10 s at 65 °C, and 2 min at 72 °C. Sequencing PCR and RACE products were purified with a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI), and subcloned into pGEM-T Easy Vector (pGEMT Easy Vector Systems, Promega). Plasmids were purified with a Wizard Plus SV Minipreps DNA Purification System (Promega) and sequenced on an ABI PRISM 310 Genetic Analyzer or 3130 Genetic Analyzer with the Big Dye Terminator v1.1 or v3.1 Cycle Sequence Kit (Applied Biosystems, Foster City, CA). Additional primers (pwTgOpA-R, B-R, C-R, D-R, and E-R; listed in supplementary table S1, Supplementary Material online) were used for sequencing opsin clones from T. granarius. To remove the possibility of Taq polymerase errors, we sequenced several independent clones for each gene to confirm the sequence (2–3 clones derived from the PCR products, 3–8 clones from the 3# RACE products, and 3–10 clones from the 5# RACE products). We sequenced, however, only one or two clones for each of multiple Rhb sequences. Sequences from these products were combined to create full length of cDNA sequences. Nucleotide sequences of the branchiopod opsins were deposited in DNA Data Bank of Japan (DDBJ)/GenBank/European Molecular Biology Laboratory (EMBL) under the accession nos. AB293428– AB293438 and AB298789–298801. Northern Blot Total RNA extracted from the compound eyes of T. granarius, T. longicaudatus, and B. kugenumaensis adults was fractionated on a 1.0% agarose formaldehyde gel according to the protocol of Sambrook and Russel (2001) and blotted onto a nylon membrane (Hybond-Nþ, GE Healthcare, Buckinghamshire, United Kingdom) with a Turboblotter Rapid Downward Transfer System (Schleicher & Schuell, Keene, NH). DNA probes were generated by the PCR DIG Probe Synthesis Kit (Roche, Mannheim, Germany). The RNA on the membrane was immobilized at 120 mJ using a TL-2000 Ultraviolet Translinker (UVP, Upland, CA), and hybridized at 50 °C with DNA probes. Hybridization, washing, blocking, and immunological detection were performed using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche). Chemiluminescent signals were detected by a Lumino-image analyzer LAS-1000 (Fujifilm, Tokyo, Japan). RT-PCR Expression Analysis The ocellus and the compound eyes are situated at different positions in an anterior part of the carapase in Triops and they are separable under a microscope. Total RNA was extracted from several ocelli of T. granarius, and transcripts of RhA, RhB, RhC, RhD, and RhE were detected by RTPCR. Gene-specific primers (exTgOpA-F and -R, exTgOpB-F and -R, exTgOpC-F and -R, exTgOpD-F and R, and exTgOpE-F and -R, for RhA, RhB, RhC, RhD, and RhE, respectively; listed in supplementary table S1, Supplementary Material online) were designed from the coding regions of complete nucleotide sequences. PCR conditions comprised 3 min at 95 °C, 35 cycles of 15 s at 95 °C, 15 s at 55 °C, and 1 min at 72 °C. The PCR products covered almost entire cDNAs. Samples without reverse transcription, that is, samples without reverse transcriptase, were used as negative controls to eliminate the possibility of amplifying contaminated genomic DNA. Phylogenetic Inferences Although 14 opsins were obtained from three branchiopod species in the present study, two partial sequences of RhD and RhE in T. longicaudatus were excluded from the phylogenetic analyses. Multiple Rhb sequences in B. kugenumaensis were also excluded from the phylogenetic analyses, because each of the Rhb sequences was obtained from one or two clones. In addition to 11 branchiopod opsins, 60 opsin sequences from other crustaceans, insects, and chelicerates were used in the analyses. Two opsins from cephalopods, Octopus dofleini and Sepia officinalis, were used as outgroups. Although multiple opsins have been clarified in two ostracod crustaceans (Oakley and Huber 2004), they are excluded from the analyses because their sequences were too short to analyze. Finally, 73 deduced amino acid sequences were aligned in ClustalX 1.83 (Thompson et al. 1997). N- and C-termini of several crustacean opsins were not obtained, and the alignment of N-terminus showed less clarity. Therefore, we excluded both the termini, and a total of 281 sites including gap sites, corresponding to amino acids 57–327 in T. granarius RhA, were analyzed. The amino acid sequence covered from the first transmembrane domain to the part of the seventh (last) one. In the alignment, 91.5% and 92.2% of sites are variable when outgroups (cephalopods) were included and excluded, respectively. For DDBJ/GenBank/EMBL accession numbers, see supplementary table S2 (Supplementary Material online). Three types of phylogenetic analyses were performed for the amino acid sequences, that is, a Neighbor-Joining method (Saitou and Nei 1987) as implemented by MEGA 3.1 (Kumar et al. 2004), a maximum likelihood method (Felsenstein 1981) as implemented by PhyML-aLRT v1.1 (Guindon and Gascuel 2003; Anisimova and Gascuel 2006), and a Bayesian inference as implemented by MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). In the Neighbor-Joining analysis, all gap sites in the aligned sequences were deleted using a complete deletion mode. The Poisson corrected distance was employed, and the rates among the sites were set to be uniform. The best-fit model for the maximum likelihood was determined among candidate models of protein evolution using ProtTest v1.4 (Abascal et al. 2005), and the model of WAG þ I þ G þ F (Whelan and Goldman 2001) was recovered according to the Akaike Information Branchiopoda Visual Pigment and Arthropod Opsin Evolution 303 Table 1 The ratio of the peak height at 330 nm to that at 350 nm (H330/H350) of peaks a, b, e, and f as shown in figure 1 and the peak ratio (H330/H350) of standard samples Triops granarius Authentic Standard Peaks H330/H350 FIG. 1.—High-performance liquid chromatogram of retinoids in the compound eyes and ocelli of adult Triops granarius detected at 350 nm. Isomers of retinaloximes and ROLs are assigned to peaks of syn (a) and anti (a#) 11-cis, syn (b) and anti (b#) all-trans, syn 9-cis (c), and syn 13-cis (d) retinaloximes, and 11- and/or 13-cis (e) and all-trans (f) ROLs. See table 1 for identification of peaks a, b, e, and f. Scale bars indicate absorbance at 350 nm. Criterion. Proportion of invariable sites was estimated from the data set. The model of rate heterogeneity was set at four categories. The gamma distribution parameter alpha was estimated from the data set. Bayesian methods coupled with Markov chain Monte Carlo (BMCMC) inference was used with the Poisson model. The rates among the sites were set as gamma. Two independent BMCMC analyses were run with each consisting of four chains. The program was run with a 5.0 105 generation Markov chain from a random tree. Trees were saved every 100 generations for a total size of 5,000 in the initial sample. Graphical inspection of tree log-likelihood in this sample revealed that stationarity was reached within 5.0 105 generations, and the average standard deviation of split frequencies became lower than 0.01. Thus, we discarded the first quarter generations as burn-in and used the remaining generations. The statistical confidence of a particular cluster of sequences in the Neighbor-Joining and maximum likelihood trees was evaluated by the bootstrap test (Felsenstein 1985) with 500 and 100 replicates, respectively. Bayesian posterior probabilities were also employed (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003). The branching pattern of the opsins was superimposed on the evolutionary pattern of arthropods, that is, Chelicerata branched off first followed by Malacostraca and Branchiopoda-Insecta lineages according to the recent molecular phylogenetic studies (Giribet et al. 2001; Hwang et al. 2001; Peterson and Eernisse 2001; Mallat et al. 2004; Mallatt and Giribet 2006). Results Retinoid Composition in Eyes of Adult T. granarius An HPLC chromatogram of retinoids extracted using the oxime method from 40 heads of adult T. granarius is shown in figure 1. Based on the elution profile and retention times (cf., Irie and Seki 2002; Irie et al. 2004), peak a was assigned to syn-11-cis, peak b to syn-all-trans, peak c to syn-9-cis, peak d to syn-13-cis, peak a# to anti-11-cis, a 0.73 b e f 0.66 1.99 1.44 Peaks Syn 11-cis RAL Oxime Syn all-trans RAL Oxime 11-cis ROL All-trans ROL H330/H350 H330/H350 of RAL1 of RAL2 Oxime or ROL1 Oxime or ROL2 0.74 0.58 0.63 2.13 1.97 0.52 0.84 0.74 NOTE.—For detailed explanation, see text and figure 1. and peak b# to anti-all-trans isomers of either retinal (RAL1) oxime or 3,4-didehydroretinal (RAL2) oxime. Further, peak e was assigned to 11-cis and/or 13-cis and peak f to all-trans isomers of retinol (ROL1) or 3,4-didehydroretinol (ROL2). To identify the peaks with either RAL1 or RAL2 oxime, or with either ROL1 or ROL2, the ratio of the peak height monitored at 330 nm to that at 350 nm (H330/H350) of peaks a and b or e and f were calculated. The values were compared with the peak ratios (H330/ H350) of authentic geometric isomers of RAL1 and RAL2 oximes or ROL1 and ROL2, respectively. The summary results shown in table 1 suggest that every peak was clarified as either RAL1 oxime isomers or ROL1 isomers. Branchiopod Opsins Fourteen sequences were determined from three branchiopod species. In T. granarius, five complete nucleotide sequences were clarified, and the deduced amino acid sequences showed high similarity to other arthropod opsin sequences in Blast searches of GenBank (Altschul et al. 1997). A total length of 1,365 bp encoding 385 amino acids (aa), 1,514 bp encoding 394 aa, 1,321 bp encoding 374 aa, 1,345 bp encoding 384 aa, and 1,509 bp encoding 378 aa, were designated RhA, B, C, D, and E, respectively. In T. longicaudatus, three complete and two partial nucleotide sequences were determined. A total length of 1,401, 1,507, 1,326, 334, and 508 bp were designated RhA, B, C, D, and E because each deduced amino acid sequence showed high homology (more than 90% identity) to T. granarius RhA, B, C, D, and E, respectively (data not shown). RhA, B, and C in T. longicaudatus encode 387 aa, 394 aa, and 374 aa, respectively. Four complete nucleotide sequences were determined in B. kugenumaensis. Three of them were 1,271 bp encoding 368 aa, 1,277 bp encoding 378 aa, and 1,276 bp encoding 379 aa and were designated Rha, Rhc, and Rhd, respectively, because their similarity to Triops RhA-E was low (less than 80% identity) (data not shown). Variations in the nucleotide sequence were observed in the multiple clones from 5# and 3# RACE for the fourth sequence (Rhb). The variation occurred especially in 3# and 5# untranslated regions (UTRs), and most substitutions in the coding region were synonymous, 304 Kashiyama et al. FIG. 2.—Opsin transcripts in the compound eyes and ocelli. (A) Northern blot in the compound eyes of Triops granarius, Triops longicaudatus, and Branchinella kugenumaensis. RNA size markers are shown on the left. (B) RT-PCR analysis in the adult ocelli of T. granarius. Asterisk indicates negative controls (no reverse transcription) for identifying genomic DNA contamination. resulting in little variation in the amino acid sequence. The nucleotide sequence inferred from the multiple sequences encodes 379 aa (data not shown). RAL-binding (a lysine residue, K) and disulfidebinding (cysteine residues, Cs) sites that are important for functional opsins were well conserved in the branchiopod opsins (data not shown; Karnik and Khorana 1990; Oakley and Huber 2004; Koyanagi et al. 2008). Pfam (www.sanger.ac.uk/Software/Pfam/; Bateman et al. 2004) identified seven transmembrane helices (accession number: PF00001) in all of the characterized opsins (data not shown). Opsin Messenger Ribonucleic Acid (mRNA) Expression in Compound Eyes and Ocelli To verify mRNA expression in the visual system, total RNA from the compound eyes of each species was extracted, and Northern blots were performed. Transcripts of all genes (RhA, B, C, D, and E) were clearly detected in each species (fig. 2A). The length of each transcript corresponds approximately to the complete nucleotide sequences. To further investigate opsin expression in ocelli, RTPCR expression analysis was performed only in T. granarius. Transcripts for all genes were detected as shown (fig. 2B). of insect, crustacean (malacostracan), and chelicerate LW- and insect blue–green-sensitive opsins. None of the 11 opsins from the three branchiopod species were categorized into this group. The second group (G2) consisted of insect blue- and UV-sensitive opsins, chelicerate UVsensitive opsins, and five branchiopod opsins. A cluster of B. kugenumaensis Rhc and Rhd was basal to this group with a high bootstrap support (96%). A cluster of Triops RhCs and B. kugenumaensis Rha was positioned at the base of the insect UV clade. The third group that was clustered to the G2 group with a low reliability (57% bootstrap support) consisted of two brachyuran crab and six branchiopod opsins. This group was further subdivided into two clades (G3 and G4), but the reliability was not high (51% bootstrap support). One (G3) consisted of H. sanguineus BcRh1 and BcRh2 and Triops RhAs, and the other (G4) consisted of Triops RhBs and T. granarius RhD and RhE. When partial sequences of T. longicaudatus RhD and RhE were included in the analyses, they formed a cluster with T. granarius RhD and RhE, respectively (data not shown). In addition, B. kugenumaensis Rhbs clustered with T. granarius RhD when they were included in the analyses (data not shown). When partial sequences of ostracod opsins were similarly analyzed, these opsins were closely related to B. kugenumaensis Rhc and Rhd (data not shown). A tree reconstructed by a maximum likelihood method was almost identical to the tree constructed by the NeighborJoining method with slight differences in branching patterns at the tips of the tree (data not shown). The analysis also supported the clades of G1, G2, G3, and G4 and their branching patterns, with similar bootstrap supports (data not shown). The topology of the Bayesian tree was almost identical with that of the Neighbor-Joining tree, but G1 clade branched off first followed by G3, G4, and G2 clades with a high degree of reliability (data not shown). Amino Acid Comparison in Opsins Responsible for Blue and UV Sensitivities In all phylogenetic analyses, a cluster of Triops RhCs and B. kugenumaensis Rha was basal to the insect UV clade, and a cluster of B. kugenumaensis Rhc and Rhd was basal to all insect blue- and UV-sensitive and chelicerate UV-sensitive opsins. Here, we focused on a lysine residue that is responsible for invertebrate UV vision (Salcedo et al. 2003). All known insect UV-sensitive opsins have a lysine residue at the site corresponding to glycine 90 in bovine rhodopsin (fig. 4). On the other hand, the amino acid in insect blue-sensitive opsins was asparagine or glutamic acid. In the opsins characterized in the three branchiopod species, RhCs and Rha possessed a lysine residue at this site (fig. 4). The amino acid in B. kugenumaensis Rhc and Rhd was glutamine. Phylogenetic Relationships of Invertebrate Opsins Figure 3 represents the molecular phylogenetic relationships of invertebrate opsins, reconstructed by the Neighbor-Joining method. These opsins were largely divided into three groups. The first group (G1) consisted Evolution of Opsins in Arthropoda In the evolution of Arthropoda, Chelicerata, and Pancrustacea lineages diversified and Pancrustacea clade later Branchiopoda Visual Pigment and Arthropod Opsin Evolution 305 FIG. 3.—Neighbor-Joining tree for invertebrate opsins. Bootstrap values of more than 60% are shown at the node. Branch lengths are proportional to the scale provided in substitutions per site. The spectral classes of opsins are given on the right (LW, long wavelength; BG, blue–green; and blue, UV, ultraviolet). For further explanation, see text. Accession numbers, the known maximum absorption (kmax) values, and references for kmax values are shown in supplementary table S2 (Supplementary Material online). 306 Kashiyama et al. FIG. 4.—Phylogenetic relationships of insect and branchiopod opsins with the amino acid at the position corresponding to glycine 90 in bovine rhodopsin. The topology is supported by all of the phylogenetic analyses (the Neighbor-Joining, maximum likelihood, and Bayesian analyses). Amino acid at position 90 and the spectral classes of the opsins are depicted on the right. divided into the subclades of malacostracans and hexapods– branchiopods (Giribet et al. 2001; Hwang et al. 2001; Peterson and Eernisse 2001; Mallat et al. 2004; Mallatt and Giribet 2006). When the evolutionary patterns of opsins reconstructed by the Neighbor-Joining/maximum likelihood analyses were superimposed on the evolution of Arthropoda, the analysis suggested that at least four types of opsins already existed in the ancestor of the Chelicerata– Pancrustacea lineages (fig. 5). The analysis also suggested that the ancestor of the Pancrustacea and the ancestor of the Insecta–Branchiopoda lineages possessed at least five and six types of opsins, respectively (fig. 5). When we adopted the tree reconstructed by the Bayesian inference, the ancestor of the Chelicerata–Pancrustacea lineages possessed at least five types of opsins (data not shown). Discussion Among the five RAL congeners, RAL1 is a common congener in many vertebrate and invertebrate visual systems (Seki and Vogt 1998). In Crustacea, RAL1 is identified in the mantis shrimp (Goldsmith and Cronin 1993), the lobster, the crayfish (Srivastava et al. 1996), and the water flea (described in Smith and Macagno 1990). Hariyama and Tsukahara (1988) revealed that several crayfish species use RAL2 in winter besides RAL1. The present study revealed that T. granarius adults use RAL1 but not RAL2. This suggests that RAL1 is the most common RAL congener from Malacostraca to Branchiopoda in Crustacea. Insects use RAL1 and/or RAL3; RAL3 has been identified only in Insecta (Seki et al. 1987; Gleadall et al. 1989; Smith and Goldsmith 1990). Based on the distribution of RAL1 and RAL3 superimposed on the phylogenetic tree of insects, Seki and Vogt (1998) suggested that the original chromophore of insect visual pigments is RAL1. The present study further demonstrated that the original chromophore of visual pigments in the ancestors of Insecta– Branchiopoda is RAL1. However, further information on other species, especially on species belonging to Anostraca, is necessary to clarify this issue. Five opsins (RhA–E) from T. granarius, five (RhA–E) from T. longicaudatus, and four (Rha–Rhd) from B. kugenumaensis were identified and characterized in the present study. Variations in the nucleotide sequence of Rhb were observed in multiple clones. Oakley and Huber (2004) identified multiple opsin genes having a high homology among them in each of two ostracod crustaceans. The authors suggest that these species possess many opsin genes diversified by recent repeated gene duplications. This would hold true for Rhb because many of the nucleotide substitutions were synonymous, and the variations were identified in the 3# and 5# UTRs. Although errors introduced by Taq polymerase could produce variations in the nucleotide sequences, the variations would occur at random. Thus, the error cannot account for such nonrandom substitutions. Current genome project in the water flea Daphnia pulex (Cladocera, Branchiopoda) also shows that the species possesses multiple opsins having a high homology among them (wFleaBase, http://wfleabase.org/), which are closely related to RhAs in Triops or to Rhbs in Branchinealla. Branchiopoda Visual Pigment and Arthropod Opsin Evolution 307 FIG. 5.—Schematic drawing of the evolution of arthropod opsins inferred from the Neighbor-Joining and maximum likelihood methods. In this diagram, gene duplications (shown in open circles) were expected to occur at the latest stage in the evolutionary process. Parallelograms indicate organisms, and each branch in each parallelogram indicates the opsin group. Deduced amino acid sequences of all opsin genes characterized in three branchiopod species show high similarity with other arthropod opsins. A RAL-binding site and disulfide-binding sites, which are indispensable for functional opsins, are well conserved in all branchiopod opsins, and seven transmembrane domains were identified. In addition, transcripts of all opsin genes were detected in the compound eyes. These results indicate that the genes are not pseudogenes but functional genes. Many arthropods have multiple photoreceptor organs. In Drosophila (Pollock and Benzer 1988; Carulli et al. 1994), Limulus (Smith et al. 1993), and ostracod crustaceans (Oakley and Huber 2004), different opsin genes are expressed in different photoreceptor organs. Triops granarius possesses a simple photoreceptor called ocellus in addition to a pair of compound eyes. All five opsin genes were transcribed in the ocellus as well as in the compound eyes. These results suggest that the ocellus in Triops may function as a photoreceptor contributing to color vision, although the function of crustacean ocelli has not been well documented (Marshall et al. 1991a, 1991b; Diersch et al. 1999; Meyer-Rochow 2001; Wildt and Harzsch 2002), and there is no report on the role of the ocelli in Triops. In insects, opsins are divided into roughly three spectral classes (LW, blue, and UV) based on the results of electrophysiological experiments (Chittka, 1996). Many opsins have been characterized in insects, and the overall topology of the opsin phylogenetic tree corresponds with the spectral classes (Briscoe 2000; Briscoe and Chittka 2001). The compound eyes of several crustaceans also include distinct photoreceptor cells with distinct spectral properties (Cummins and Goldsmith 1981; Cronin and Marshall 1989a; Smith and Macagno 1990). No multiple opsins with distinct classes have been characterized in a single species of Crustacea (Sakamoto et al. 1996; Oakley and Huber 2004; Porter et al. 2007). In addition, neither putative blue- nor UV-sensitive opsins have been identified in Crustacea. The present study revealed that multiple opsins characterized from each of three branchiopod species can be phylogenetically classified into four distinct groups: The first is a group including the brachyuran crab opsins (G3 clade). The second group is composed of three types of Triops opsins (G4). The third group (RhC and Rha) is closely related to insect UVsensitive opsins. The fourth group composed of two B. kugenumaensis opsins was basal to all short-wavelength (blue- and UV-) sensitive opsins. Current genome project revealed that D. pulex also possesses opsins belonging to G2, G3, or G4 clade. As of now, however, we could not find the opsin belonging to G1 clade (wFleaBase). The wavelength of maximum absorption (kmax) of visual pigments has been determined in some arthropods. In D. melanogaster (Feiler et al. 1988, 1992; Zuker et al. 1988; Salcedo et al. 1999), Apis mellifera (Townson et al. 1998), and Schistocerca gregaria (Engels et al. 2000), the kmax values were obtained by electroretinogram measurements of a transgenic D. melanogaster in which the target opsin gene was expressed. The kmax values of opsins in P. xuthus (Kitamoto et al. 1998; Arikawa, Scholten, et al. 1999; Kitamoto et al. 2000; Kinoshita et al. 2006), Manduca sexta (Chase et al. 1997; White et al. 2003), and Limulus polyphemus (Hubbard and Wald 1960; Nolte and Brown 1972; Smith et al. 1993) were estimated from the correlation of opsin expression patterns with intracellular electrophysiological properties in situ. Phylogenetic analysis also clearly estimates their spectral classes (Briscoe and Chittka 2001). The present phylogenetic analysis reveals that Triops RhA is most closely related to the brachyuran crab opsins. From the opsin mRNA expression pattern and the electroretinogram measurements in the compound eyes, Sakamoto et al. (1996) suggested that the maximal absorption of 308 Kashiyama et al. visual pigments composed by these brachyuran crab opsins is in the blue–green region of the spectrum. The genes, however, are expressed simultaneously in all seven retinular cells (R1–R7), and there are no intracellular electrophysiological results in the ommatidia. Therefore, it is difficult to predict the spectral class of Triops RhA. Similarly, it was not possible to predict the spectral classes of Triops RhB, RhD, and RhE from their phylogenetic inferences. Branchinella kugenumaensis Rhc and Rhd were basal to all short-wavelength (blue- and UV-) sensitive opsins. Triops RhCs and B. kugenumaensis Rha appear to have a closer relationship to the insect UV-sensitive opsins than the blue-sensitive opsins. Salcedo et al. (2003) conducted a comparative phylogenetic analysis of closely related blueand UV-sensitive opsins in invertebrates and investigated an amino acid responsible for UV sensitivity using transgenic techniques. Finally, a lysine residue corresponding to the amino acid position 90 in bovine rhodopsin was determined to be essential for the UV sensitivity of invertebrate opsins. Indeed, all known insect opsins phylogenetically classified into the UV clade possess a lysine residue at amino acid position 90. Triops RhCs and B. kugenumaensis Rha also possess a lysine residue at this position, clearly suggesting that these opsins are UV-sensitive. On the other hand, a glutamine residue at amino acid 90 in each of B. kugenumaensis Rhc and Rhd suggests that these opsins are not UVsensitive. Blue- or UV-sensitive opsins have not been identified in Crustacea (Crandall and Cronin 1997; Porter et al. 2007). Therefore, the origin of these pigments can be discussed only within Insecta. Briscoe (2000) suggested that the divergence of these pigments predates the Holometabolous– Hemimetabolous split in insects. Our results show that the three branchiopod species possessing putative UV-sensitive pigments are in a monophyletic UV clade. The UV clade formed a cluster with insect blue-sensitive opsins. When the branching pattern was superimposed on the phylogenetic relationship of Insecta and Branchiopoda, it is suggested that the ancestor of Insecta–Branchiopoda lineages already possessed two types of opsins which were diversified later as insect blue-sensitive opsins and insect and branchiopod UV-sensitive opsins, that is, the divergence of blue- and UV-sensitive pigments predates the Branchiopoda and Insecta divergence in the Late Silurian or Early Devonian period (Glenner et al. 2006). The present study also revealed that some clusters do not include any insect opsins, suggesting that crustaceans (at least malacostracans and branchiopods) possess unique visual pigments. Such unique opsins were also found in LW-sensitive opsins in some orders in Insecta (Spaethe and Briscoe 2004). Koyanagi et al. (2008) proposed that at least three types of opsins existed in the ancestors of the Chelicerata–Pancrustacea lineages. It is not conclusive yet because more additional data are needed for valid conclusions, but our results revealed that the ancestral arthropod of the Chelicerata–Pancrustacea lineages possessed at least four types of opsins and that the ancestors of Pancrustacea and Insecta–Branchiopoda lineages possessed at least five and six types of opsins, respectively. In the evolutionary processes associated with each lineage, several opsins appeared and diversified with repeated gene duplication, some of which have been lost in some taxa, leading to the evolution of color vision. Supplementary Material Tables S1 and S2 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/). Acknowledgments We thank Dr M. Koyanagi of Osaka City University for his invaluable suggestions and critical reading of the manuscript. We are very grateful to anonymous reviewers for their invaluable suggestions. 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