Fig. 5 in Fig. 5 in Loss and Gain of Group I Introns in the Mitochondrial Gene of the Scleractinia (Cnidaria; Anthozoa).

Loss and Gain of Group I Introns in the Mitochondrial Cox1 Gene of the Scleractinia (Cnidaria; Anthozoa) Yaoyang Chuang1,2, Marcelo Kitahara3,4, Hironobu Fukami5, Dianne Tracey6, David J. Miller3,7,*, and Chaolun Allen Chen1,2,8,* 1Biodiversity Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan 2Institute of Oceanography, National Taiwan University, Taipei 106, Taiwan 3School of Pharmacy and Molecular Sciences, James Cook University, Townsville 4810, QLD, Australia 4Centro de Biologia Marinha, Universidade de São Paulo Rodovia Manoel Hyppólito do Rego, km 131,5 Praia do Cabelo Gordo 11600-000, Sao Sebastiao, SP, Brazil 5Department of Marine Biology and Environmental Science, University of Miyazaki, Miyazaki 889-2192, Japan 6National Institute of Water and Atmospheric Research, Wellington 6021, New Zealand 7ARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville 4810, QLD, Australia 8Taiwan International Graduate Program (TIGP)-Biodiversity, Academia Sinica, Nankang, Taipei 115, Taiwan (Received 19 January 2017; Accepted 12 April 2017; Published 4 May 2017; Communicated by Benny K.K. Chan) Yaoyang Chuang, Marcelo Kitahara, Hironobu Fukami, Dianne Tracey, David J. Miller, and Chaolun Allen Chen (2017) Group I introns encoding a homing endonuclease gene (HEG) that is potentially capable of sponsoring mobility are present in the cytochrome oxidase subunit 1 (cox1) gene of some Hexacorallia, including a number of scleractinians assigned to the “robust” coral clade. In an effort to infer the evolutionary history of this cox1 group I intron, DNA sequences were determined for 12 representative “basal” and “complex” corals and for 11 members of the Corallimorpharia, a sister order of the Scleractinia. Comparisons of insertion sites, secondary structures, and amino acid sequences of the HEG implied a common origin for cox1 introns of corallimorpharians, and basal and complex corals, but cox1 introns of robust corals were highly divergent, most likely reflecting independent acquisition. Phylogenetic analyses with a calibrated molecular clock suggested that cox1 introns of scleractinians and corallimorpharians have persisted at the same insertion site as that in the common ancestor 552 million years ago (mya). This ancestral intron was probably lost in complex corals around 213 to 190 mya at the junction between the Trassic and Jurassic. The coral cox1 gene remained intronless until new introns, probably from sponges or fungi, reinvaded different positions of the cox1 gene in robust corals around 135 mya in the Cretaceous, and then it subsequently began to lose them around 65.5 mya in some robust coral lineages coincident with the later Maastrichtian extinction at the Cretaceous-Tertiary boundary. Key words: Group I intron, Cytochrome oxidase I, Scleractinian, Corallimorpharian, Mass extinction. *Correspondence: Tel: 886-2-27899549. E-mail: [email protected] (CA Chen); [email protected] (DJ Miller). BACKGROUND While mitochondrial genomes of anthozoan cnidarians resemble those of other animals in a number of respects, they are unique among metazoans in typically containing one or more self-splicing group I introns (Beagley et al. 1996). NADH dehydrogenase subunit 5 (nad5) genes of all anthozoans so far examined contain a large group I intron that may be the result of a single transfer event (Beagley et al. 1998; Boore 1999; Fukami and Knowlton 2005; Lavrov and Lang 2005; Medina et al. 2006; Tseng et al. 2005; van Oppen et al. 2002). However, in some but not all anthozoans, a second group I intron is present in the cytochrome c oxidase subunit 1 (cox1) gene (Lin et al. 2011; Medina et al. 2006). The cox1 intron differs from that in the nad5 gene in that the Zoological Studies 56: 9 (2017) doi:10.6620/ZS.2017.56-09 1 former encodes an LAGLI-DADG family homing endonuclease (HE) that is potentially capable of mobilizing the intron (Beagley et al. 1996; Fukami et al. 2007), whereas the latter does not. Group I introns containing an HE gene (HEG) can create a double-strand break at specific nucleotide sequences and invade themselves or with introns, invade other genes (Belfort 1990; Dujon 1989; Perlman and Butow 1989). A homing cyclical model of parasitic genetic elements describes the life cycle of these elements and their strategies to avoid purifying selection (Goddard and Burt 1999; Gogarten and Hilario 2006). The distribution of introns in the mitochondrial cox1 genes of anthozoans is extremely patchy -- for example, introns are present in 13 of 41 genera (20 of 73 species) of “robust” corals, conventionally assigned to the suborder Faviina. With one exception, phylogenies of the robust coral cox1 gene and its intron are concordant, suggesting at most 2 insertions and many subsequent losses (Beagley et al. 1996; Fukami et al. 2007). The source(s)/donor(s) of introns in anthozoan cox1 genes are unclear. Beagley et al. (1996) suggested a common origin from a symbiotic dinoflagellate (genus Symbiodinium) or endolithic fungus living in close association with corals (Bentis et al. 2000; Raghukumar and Raghukumar 1991). In contrast, a separate origin (from a sponge or fungal donor) was proposed for cox1 group I introns in the suborder Faviina, the major group of “robust” corals (Fukami et al. 2007). While the hypothesis of 2 insertions and many subsequent losses accounts for the limited available data on anthozoan cox1 introns, the extent to which it is more generally applicable remains to be tested, as data are available for few members of the “basal” and “complex” lineages of scleractinians or their sister group, the corallimorpharians (Fukami et al. 2008; Kitahara et al. 2010; Lin et al. 2014; Medina et al. 2006). Corallimorpharians, a small order of Anthozoa composed of 40-45 species, are morphologically similar to scleractinians but lack a calcareous skeleton, and understanding their evolutionary relationship with corals has been a challenging endeavor for decades (Fukami et al. 2008; Kitahara et al. 2010; Kitahara et al. 2014; Lin et al. 2014; Medina et al. 2006). However, recent study based on 291 orthologous single copy protein-coding nuclear genes reveals a topology consistent with scleractinian monophyly and corallimorpharians as the sister clade of scleractinians (Lin et al. 2016) To better understand the evolutionary history of group I introns, cox1 intron data were obtained for 12 species of Scleractinia, from 7 genera representing 6 families in the “complex” and “basal” clades (Fukami et al. 2008; Kitahara et al. 2010), and from 11 species of Corallimorpharia, including 8 genera representing 3 families. Analyses of insertion sites, primary DNA sequences, and secondary structures, together with comparisons of inferred phylogenies for introns and their host cox1 genes, were consistent with a common origin for cox1 introns of corallimorpharians, actiniarians, anthipatharians, and some basal and complex scleractinians, but also the loss of this intron and subsequent reinvasion of the cox1 locus by distinct group I introns in robust corals. MATERIALS AND METHODS Cox1 exon and group I intron sequences Samples and sources of cox1 sequences used in this study are summarized in table 1. Samples were assigned to groups of corallimorpharians, and “basal”, “complex”, and “robust” scleractinian corals based on Fukami et al. (2008) and Kitahara et al. (Fukami et al. 2008; Kitahara et al. 2010). In addition, cox1 genes belonging to sponges (Porifera) and other anthozoans were included in the molecular evolution analyses. Total genomic DNA was extracted using the CHAOS buffer method (Fukami et al. 2004). A primer set (COX1COMF: 5’-GGT ACG TTA TAT TTA GTA TTT GGG ATT GG-3’ and COX1COMR: 5’-GGA GGA GAA ACA TGA ACC CAT TCT AAG-3’) was designed to amplify complete cox1 exon fragments. A polymerase chain reaction (PCR) was performed with the following thermal cycle: 5 min at 95°C, followed by 5 cycles of 30 s at 94°C, 30 s at 50°C, and 90 s at 72°C, and then by 30 cycles as just described but with an annealing temperature of 55°C instead of 50°C, with a final extension at 72°C for 10 min. An internal primer set (CO1in-F: 5’-CCA TGC TTT TAA CGG ATA GAA ATT-3’ and CO1in-R: 5’-GCA CAT AAT GAA AAT GGG CTA CAA-3’) was designed to assist DNA sequencing if an intron existed. Sequence analysis, open reading frame (ORF), and secondary structure prediction of cox1 group I introns In order to examine the characteristics and origins of group I introns in scleractinians and page 2 of 18Zoological Studies 56: 9 (2017) corallimorpharians, cox1 exons and introns of other anthozoan orders (including the Zoantharia, Actiniaria, and Antipatharia) and Porifera, and of Cinachyrella levantinensis and Plakortis angulospiculatus, were retrieved from GenBank. In total, 94 cox1 sequences with 42 containing group I introns were used for the following analyses. Sequence alignment of cox1 exons and putative Table 1. Sequence information in this study. Information includes the name, location, whether or not it contains an intron, the NCBI accession number, and the reference Species Location cox1 intron NCBI Accession Porifera Cinachyrella levantinensis Israel + AM076987 Plakortis angulospiculatus Florida + EU237487 Octocorallia Acanella eburnea New England - NC_011016 Briareum asbestinum Florida - DQ640649 Dendronephthya gigantea Korea - NC_013573 Keratoisidinae sp. New England - NC_010764 Pseudopterogorgia bipinnata Florida - DQ640646 Zoanthidea Palythoa sp. Florida + DQ640650 Savalia savaglia France + NC_008827 Ceriantharia Ceriantheopsis americana Florida - DQ662399 Antipatharia Chrysopathes formosa California - NC_008411 Leiopathes glaberrima Greece + FJ597644 Actiniaria Metridium senile California + NC_000933 Nematostella sp. Florida - NC_008164 Corallimorpharia Amplexidiscus fenestrafer Taiwan + KP938435 Corallimorphus profundus Taiwan + KP938440 Corynactis califirnica Hawaii + KP938436 Discosoma nummiformis Taiwan + KP938434 Discosoma sp. Bali + NC_008072 Pseudocorynactis sp. Hawaii + KP938437 Rhodactis indosinesis Taiwan + KP938438 Rhodactis mussoides Taiwan + KP938439 Rhodactis sp. Bali + DQ640647 Ricordea florida Florida + NC_008159 Ricordea yuma Taiwan + KP938441 Scleractinia Basal Gardineria hawaiiensis New Caledonia + GQ868677 Complex Acropora tenuis Australia - NC_003522 Agaricia humilis Florida - NC_008160 Alveopora sp. Taiwan - KJ634271 Anacropora matthai Indonesia - NC_006898 Astreopora explanata Taiwan - NC_024090 Astreopora myriophthalma Taiwan - NC_024092 Dendrophyllia sp. Japan + KY887482 Euphyllia sp. Taiwan - KY887483 Fungiacyathus stephanus Taiwan + JF825138 Galaxea sp. Taiwan - KY887484 Goniopora columna Taiwan + JF825141 Goniopora sp. Taiwan + KY887485 Isopora palifera Indonesia - KJ634270 Isopora togianensis Indonesia - NC_024089 Leptoseris cucullata Panama - AB441221 page 3 of 18Zoological Studies 56: 9 (2017) Species Location cox1 intron NCBI Accession Montipora cactus Taiwan - NC_006902 Pachyseris sp. Taiwan - KY888878 Pavona clavus Panama - NC_008165 Porites compressa Taiwan + KY888879 Porites okinawanesis Japan + JF825142 Porites porites Florida + NC_008166 Siderastrea radians Florida + NC_008167 Pseudosiderastrea formosa Taiwan + NC_026530 Stephanocoenia michelinii Panama + AB441228 Tubastrea sp. Taiwan + AB441238 Robust Acanthastrea echinata Taiwan AB117250 Anthemiphyllia patera New Caledonia - HM018604 Astrangia sp. Florida - DQ643832 Blastomussa wellsi Palau + AB289563 Caulastraea furcata Japan + AB289579 Cynarina lacrymalis Pacific + AB289568 Cyphastrea serailia Japan AB117257 Deltocyathus suluensis Australia - HM018631 Diploastrea heliopora Japan + AB289567 Diploria clivosa Panama - AB117226 Echinophyllia aspera Japan + AB289572 Echinophyllia echinoporoides Palau + AB289573 Echinopora pacificus Japan - AB117261 Favia fragum Brazil - AB117223 Favia stelligera Japan - AB117264 Hydnophora grandis Palau - AB117286 Lobophyllia corymbosa Japan + AB117241 Madracis mirabilis Panama - EU400212 Madrepora oculata Taiwan - JX236041 Meandrina braziliensis Brazil - AB117297 Montastraea annularis Panama - AB117260 Montastraea cavernosa Panama - AB117288 Montastraea magnistellata Japan - AB117279 Mussismilia harttii Brazil - AB117232 Mycedium elephantotus Palau + AB289582 Mycetophyllia aliciae Panama - AB117235 Oulastrea crispata Taiwan - AB441197 Oulophyllia bennettae Palau + AB289581 Oxypora lacera Palau + AB289571 Paulastrea sp. Taiwan - KY887486 Pectinia paeonia Palau + AB289584 Physogyra lichtensteini Palau + AB289562 Platygyra lamellina Japan - AB117282 Pocillopora eydouxi Taiwan - KY887487 Polycyathus chaishanensis Taiwan - NC_015642 Scolymia cubensis Brazil - AB117237 Scolymia sp. Palau + AB289570 Scolymia vitiensis Palau + AB289569 Seriatopora caliendrum Taiwan - NC_010245 Seriatopora hystrix Taiwan - NC_010244 Solenastrea bournoni Panama - AB117291 Stylocoeniella sp. Japan - AB441225 Stylophora pistillata Taiwan - NC_011162 Symphyllia radians Japan + AB289578 Trachyphyllia geoffroyi Florida - AB117287 Table 1. (Continued) page 4 of 18Zoological Studies 56: 9 (2017) ORFs were performed using MEGA 5.05 and Gblocks (Talavera and Castresana 2007; Tamura et al. 2007). ORFs were translated in Vector NTI using the Acropora tenuis genetic code to detect ORFs of longer than 100 amino acids in the intron (van Oppen et al. 1999). Secondary structures of group I introns were estimated using the DNA Mfold server (http://mfold.bioinfo.rpi.edu/; (Zuker 2003) and our current understanding of the group I intron recognition process (Lisacek et al. 1994). Numbers of nonsynonymous substitutions (Ka) and synonymous substitutions (Ks) were calculated using DnaSP vers. 5 software to estimate the selection forces on exon and intron sequences (Librado and Rozas 2009). Phylogeny construction and comparisons All cox1 exon sequences used to reconstruct the phylogenetic relationship of scleractinian corals and the relative anthozoan taxa were listed in table 1. The best-fitting models in the maximumlikelihood (ML) analyses were evaluated using ModelTest vers. 3.7 (Posada and Crandall 1998). A general time-reversible substitution model with a proportion of invariance and gamma distribution model (GTR+I+G) of a DNA evolution model were determined using the Akaike Information Criterion (AIC) test for the ML analysis. The ML analysis with Shimodaira and Hasegawa (SH)- like branch support (Guindon et al. 2010) was conducted using the PhyML 3.0 online server. The Bayesian (BA) phylogeny was constructed using Mrbayes (Ronquist and Huelsenbeck 2003) with a substitution model evaluated with Mrmodeltest ver. 3.7 (Nylander 2004). The Bayesian tree was constructed with 6 simultaneous Markov chains for 107 generations with trees sampled every 1000 generations and 2500 initial trees discarded as burn-in. In order to test the consistency of cox1 exon and intron evolution, phylogenetic analyses of cospeciation comparisons between exons and introns were reconstructed with the ML and BA algorithms. Phylogenetic relationships of corallimorpharians, and the basal, complex, and robust clades of scleractinians were separately estimated because of the difficulty of aligning HEG sequences among basal, complex, and robust corals. The GTR+G model was determined using the AIC test for the cox1 exon and the HEG of corallimorpharians and the basal and complex corals, while the Hasegawa, Kishino, and Yano and gamma distribution (HKY+G) model and GTR+I were determined for the cox1 exon and HEG of robust corals. Patristic distance correlations between exon and intron phylogenies were estimated using Mesquite vers. 2.75 to compare the similarity of tree topologies of exons and HEGs (Maddison 2011). The Kishino-Hasegawa (KH) test, Shimodaira-Hasegawa (SH) test, and Approximately Unbiased (AU) test were conducted in the Consel program to estimate the confidence level of topological differences (Shimodaira and Hasegawa 2001). Molecular dating of the cox1 exon tree The divergence time of every clade was calculated using Beast vers. 1.6.1 which allows a relaxed molecular clock among different lineages (Drummond and Rambaut 2007). The Yule birthrate process was chosen as prior, and the distribution of the divergence on each node was set to a normal distribution with a 5% standard error. The GTR+I+G model was selected as the most appropriate evolutionary model to evaluate likelihood ratio tests for molecular clock estimates. In total, 5 × 108 generations were performed and saved every 5 × 104 generations to calculate their phylogenetic relationships. The first 2500 of 104 topologies were discarded as burnin, while the remainder was saved to calculate posterior probabilities. Four reference points were chosen to evaluate the divergence time of each clade. Time of reference points were as follow: Dendrophylliidae (127 million years ago, mya), Acropora (59 mya), Stylophora (68 mya; (BaronSzabo 2006), and Astrangia/Solenastrea (70 mya). In order to avoid over-evaluation of time, we constrained the origin of scleractinians to 460 mya (Stolarski et al. 2011). RESULTS Molecular characteristics of group I introns in cox1 loci Lengths of introns, putative open reading frames (ORFs), noncoding regions, locations and start/stop codons of ORFs in scleractinians, corallimorpharians, and other members of the Anthozoa and Porifera are summarized in table 2. Eleven species, representing 8 genera in 3 families of corallimorpharians all contained the cox1 intron. Among scleractinian corals, the “basal” coral, Garderneria hawawiiensis, and 11 “complex” coral page 5 of 18Zoological Studies 56: 9 (2017) Table 2. Molecular characteristics of cox1 introns and their open reading frames (ORFs), including the length of the intron, length of the ORF, length of the non-coding region, location of the ORF in the cox1 intron, and its start/stop codon according to predictions from Kitahara et al. (2014) and Lin et al. (2014) Taxon Length of Intron Length of ORF Length of Noncoding Location of ORF Start/Stop Codon Porifera Cinachyrella levantinensis 1143 1029 114 1-1029 TTA/TAA Plakortis angulospiculatus 1503 1038 195 359-1396 TTT/TAA Zoanthidea Palythoa sp. 1308 747 561 391-1137 ATG/TAA Savalia savaglia 1250 723 527 441-1163 ATG/TAG Actiniaria Metridium senile 853 672 181 155-826 ATG/TAA Corallimorpharia Amplexidiscus fenestrafer 1206 1005 201 71-1075 ATA/TAA Corallimorphus profundus 1182 786 396 47-832 ATA/TAG Corynactis califirnica 1265 1008 257 71-1078 ATA/TAG Discosoma nummiformis 1208 711 497 71-781 ATA/TAA Discosoma sp. 1206 1005 201 71-1075 ATA/TAA Pseudocorynactis sp. 1177 993 184 71-1063 ATA/TAA Rhodactis indosinesis 1204 726 478 71-796 ATA/TAA Rhodactis mussoides 1206 1005 201 71-1075 ATA/TAA Rhodactis sp. 1206 1029 177 71-1075 ATA/TAA Ricordea florida 1215 1017 198 90-1106 ATA/TAA Ricordea yuma 1198 975 223 110-1084 GTG/TAA Scleractinia (Basal complex group) Gardineria hawaiiensis 1140 981 159 48-1028 ATA/TAA Scleractinia (Complex group) Dendrophyllia sp. 972 831 141 49-879 ATA/TAA Fungiacyathus stephanus 970 828 142 48-875 ATA/TAA Goniopora sp. 970 831 139 48-878 ATA/TAA Porites porites 971 831 140 48-878 ATA/TAA Porites compressa 970 831 139 48-878 ATA/TAA Siderastrea radians 994 855 139 48-902 ATA/TAA Pseudosiderastrea formosa 970 855 115 48-902 ATA/TAA Stephanocoenia michelinii 940 792 148 47-838 ATA/TAG Tubastrea sp. 970 831 139 48-878 ATA/TAA Scleractinia (robust group) Blastomussa wellsi 1107 933 174 49-981 ATA/TAA Caulastraea furcata 1128 1005 123 27-1031 ATA/TAA Cynarina lacrymalis 1078 933 145 49-981 ATA/TAA Diploastrea heliopora 1076 933 143 49-981 ATA/TAA Echinophyllia aspera 1077 954 123 27-980 ATA/TAA Echinophyllia echinoporoides 1077 954 123 27-980 ATA/TAA Lobophyllia corymbosa 1077 954 123 27-980 ATA/TAA Mycedium elephantotus 1128 1005 123 27-1031 ATA/TAA Oulophyllia bennettae 1128 1005 123 27-1031 ATA/TAA Oxypora lacera 1078 933 145 49-981 ATA/TAA Pectinia paeonia 1128 1005 123 27-1031 ATA/TAA Physogyra lichtensteini 1077 933 144 49-981 ATA/TAA Scolymia sp. 1120 996 124 27-1022 ATA/TAA Scolymia vitiensis 1078 933 145 49-981 ATA/TAA Symphyllia radians 1077 954 123 27-980 ATA/TAA page 6 of 18Zoological Studies 56: 9 (2017) species, representing 7 genera of 6 families, had introns inserted at the same nucleotide position (nt) 893 in cox1 (herein called I893) as those of corallimorpharians, actinarians, and antipatharians (Table 3). In “robust” corals, 15 species, representing 13 genera of 4 families, had introns inserted at nt position 729 in cox1 (herein called I729), which is identical to the insertion position of sponges (Table 3). In zoanthids, cox1 introns were inserted at nt position 876 (herein called I876). Lengths of introns and ORFs ranged from 853/672 bp in the actiniarian, Metridinum senile, to 1503/1039 bp in the sponge, Plakortis angulospiculatus (Table 2). Intron and ORF lengths of “complex” corals were significantly shorter than those of “robust” corals (Mann-Whitney U-test p < 0.01) but no significantly different between complex corals and corallimorpharians (MannWhitney U-test, p = 0.09). Start and stop codons of the cox1 intron ORF were ATA and TAA, respectively, for most corallimorpharians and scleractinians, except those of Ricordea yuma (start codon = GTG) and Stephenoconia sp. (stop codon = TAG). An ORF composed of a specific domain of the LAGLI-DADG HE was identified, although nucleotide sequence alignments among different anthozoans were low. As observed by Fukami et al. (2007), amino acid sequences of the HE in robust corals were highly similar to those of sponges (mean p-distance = 22.2%, Table 4) (Fukami et al. 2007). However, they were highly divergent compared to those of basal, complex, and corallimorpharians (mean p-distance = 83.8%), whereas the latter three possessed relatively similar HEs (mean p-distance = 43.2%). Secondary structures of the cox1 intron for sponges and 7 anthozoans were predicted to confirm its identification in group I based on the consensus primary structure (Fig. 1). All cox1 introns contained 4 consensus primary structures (P, Q, R, and S) in core structures of group I intron and 9 or 10 paired regions of helices. Paired regions of the secondary structure also reflected the relationship based on primary sequences of introns. For example, I729 of both robust corals and the sponge had lost the P2 helix (Figs. 1A, B). Structures and positions of P1 to P9 were highly similar among I893s of basal and complex Table 3. Insertion sites of the cox1 intron in different groups of anthozoans and a sponge. Double arrows indicate insertion sites of the intron Taxa Sequence type Location Zoanthidea Palythoa sp. I876 Savalia savaglia I876 Actiniaria Metridium senile I893 Antipatharia Leiopathes glaberrima I893 Corallimorpharia Ricordea florida I893 Scleractinia Gardineria hawaiiensis I893 Siderastrea radians I893 Diploastrea heliopora I729 Sponge Cinachyrella levantinensis I792 Table 3. Insertion sites of the cox1 intron in different groups of anthozoans and a sponge. Double arrows indicate insertion sites of the intron. Taxa Sequence type Location 729 876 893 Zoanthidea Palythoa sp. GCCAT CCGGAGGTTT [149 nt] TGTGTGGGCT àß CACCACATGTTTACAGT - AGGGA I876 Savalia savaglia GCCAT CCGGAGGTTT [149 nt] TGTGTGGGCT àß CACCACATGTTTACAGT - AGGGA I876 Actiniaria Metridium senile GGCAT CCGGAAGTTT [149 nt] TGTGTGGGCA CATCACATGTTTACGGT àß TGGAA I893 Antipatharia Leiopathes glaberrima GCCAC CCAGAGGTTT [149 nt] TGTGTGGGCT CATCACATGTTCACGGT àß TGGAA I893 Corallimorpharia Ricordea florida GACAT CCAGAGGTAT [149 nt] TGTGTGGGCA CACCATATGTTTACGGT àß TGGAA I893 Scleractinia Gardineria hawaiiensis GGCAT CCCGAAGTTT [149 nt] TGGGTGGGCC CATCATATGTTTACGGT àß TGGAA I893 Siderastrea radians GGCAT CCAGAAGTTT [149 nt] TGTGTGGGCC CACCATATGTTTACGGT àß TGGGA I893 Diploastrea heliopora GGCAT àß CCTGAAGTTT ~ I729 Sponge Cinachyrella levantinensis GGCAT àß CCAGAAGTTT [149 nt] AGTTTGAGCC CATCACATGTTTACAGT TGGAA I792 page 7 of 18Zoological Studies 56: 9 (2017) corals and corallimorpharians except for the P5 region (Figs. 1C, D). I893s of antipatharians and actiniarians possessed similar secondary structures to corallimorpharians (Fig. 1E), whereas I876 of zoanthiarians had a branched form of the P9.1 paired region representing a unique type of cox1 intron compared to other anthozoans (Fig. 1F). Evolutionary rates of the exon and HEG To evaluate selection forces on the exon and HEG of the intron, we estimated the rate of nonsynonymous substitutions (Ka) versus synonymous substitutions (Ks) in exons and HEGs of corallimorpharians, complex corals, and robust corals. Similar small ratios were observed among cox1 exons of corallimorpharians and scleractinians, while larger differences were found in comparisons of HEGs among corallimorpharians and scleratinians (Fig. 2, Ka/Ks = 0.03-0.045 on average for cox1 exons; Ka/Ks = 0.254-0.489 on average for HEGs). Both the cox1 exon and HEG deviated from neutral variations (Ka/Ks = 1). A lower nonsynonymous ratio of substitutions for the cox1 exon suggested that more-purified selection of the cox1 exon was stronger than that of the HEG. Co-evolution of the cox1 exon and intron in scleractinians and corallimorpharians Results of the phylogenetic analyses are summarized in figure 3. Octocorals were used as outgroups for the phylogenetic analyses because of the sister group relationship between hexacorallians and octocorallians. Both the ML and BA analyses strongly supported the monophyly of the Scleractinia composed of “basal”, “complex”, and “robust” clades as proposed by Kitahara et al. (2010) and Stolarski et al. (2011). Corallimorpharian genera were grouped into a monophyletic clade, except for Corallimorphus profundus which formed a basal clade next to the Scleractinia, although statistical supports of the MA and BA to this node were not relatively high (86/61). Mapping the occurrence of cox1 introns onto the phylogenetic tree showed that I893 appeared in all genera of the order Corallimorpharia, the basal scleractinian, Gardineria hawaiinensis, and Fungiacyanthus, Porites, Goniopora, Turbinaria, Dendrophyllia, Siderastrea, Pseudosiderastrea and Stephanocoenia of the complex clade of the Order Scleractinia (herein called complex I) (Fig. 3). I893 was absent from the other lineage, complex II, which contained genera of the families Euphyllidae, Acroporidae, and Agaricidae. I729, in contrast to the distributional pattern of I893 in corallimorpharians, and basal and complex coral clades, had a sporadic but restricted distribution in the robust clade of Pacific scleractinian corals (Fig. 3). Co-evolution tests of cox1 exons and introns were separately conducted for corallimorpharians, basal and complex corals (Fig. 4A), and robust corals (Fig. 4B) due to the high divergence of primary DNA sequences between I893 and I729. cox1 exon and I893 phylogenies were largely congruent in corallimorpharians, and basal and complex corals (Patristic distance correlation = 0.96). The difference was in positions of Corallimorphus profundus and Porites compressa between these 2 trees (Fig. 4A). In contrast, in the case of robust corals, exon and intron phylogenies substantially differed (Patristic distance correlation = 0.67), suggesting significantly different evolutionary histories for I729 and the cox1 exons in the robust clade (Fig. 4B, Table 5). To test the co-evolution of the exon and intron, the AU, KH, and SH tests were conducted to examine the congruence of the exon and intron phylogenetic trees. Statistical results of the AU, KH and SH Table 4. Genetic distances of the homing endonuclease gene (HEG) among different organisms. P-distance comparisons of HEG amino acid sequences among different groups of organisms. Distances are listed as percentages (%). nc, not compared Group Basal and complex corals Robust corals Corallimorpharians Actiniarians Sponges Basal and complex corals 20.6 Robust corals 82.6 6.9 Corallimorpharians 43.2 81.5 2.1 Actiniarians 66.0 82.4 65.1 nc Sponges 83.8 22.2 81.2 83.3 19.2 page 8 of 18Zoological Studies 56: 9 (2017) Fig. 1. Secondary structures of representative cox1 introns in anthozoans. A: Corallimorpharian (Rhodactis howesii); B: basal and complex corals (Gardeneris hawaiinesis); C: robust corals (Diploastrea heliopora); D: actiniarian (Metridinium senile); E: poriferian (Plakortis angulospiculatus); F: zoantharian (Savalia savaglia). Features of the secondary structure indicate the characteristics of group I introns: 10 helical elements P1~P10; consensus primary structures P, Q, R, and S in hollow letters; internal guide sequence, IGS. Initial and terminal sites of the predicted open reading frame are labeled “ORF start” and “ORF stop”, respectively. (A)Corallimorpharian (B)Complex corals (C)Robust corals (D)Actiniaria (E)Porifera (F)Zoanthids page 9 of 18Zoological Studies 56: 9 (2017) taxa disappeared towards the end of the Triassic, although the cause of the Rhaetian mass extinction remains controversial (Galli et al. 2005; Marzoli et al. 2004; Olsen et al. 2002). Kiessling et al. suggested that volcanism causing climate changes led to disturbances in the carbon cycle which could have been the reason for this extinction (Kiessling et al. 2007; Kiessling and Baron-Szabo 2004), including inducing high extinction rates among taxa of inshore habitats and reefs (Kiessling and Baron-Szabo 2004). Although re-invasion of I729 into the cox1 gene of robust corals occurred around 135 mya, the later Maastrichtian extinction (K-T extinction) which occurred at 65.5 mya might have triggered loss of I729 from some lineages of robust corals. Interestingly, major loss and invasion events of introns also occurred contemporarily with 2 major mass extinction events in the evolutionary history of scleractinians (Kiessling and BaronSzabo 2004). These correlations indicate that mass extinctions might have provided cox1 intronless species with a selective advantage of respiration efficiency allowing them to increase their distribution in harsh environments compared to their counterparts with the cox1 intron. A previous study indicated that mobile elements would have led hosts to experience greater selective forces unless they were harmless to their host genes (Domart-Coulon et al. 2001). Although the homing process might have provided a successful way to increase the preservation of invaded elements (Edgell et al. 2011; Gagan et al. 2000), it is believed that introns might decrease the efficiency of gene expression because of the prolonged lengths of their transcripts (Chen et al. 2005; Jeffares et al. 2008). The concentration of atmospheric oxygen rapidly declined from approximately 30% to 13% during the beginning of the Triassic to the Rhaetian extinction (Berner 2001, 2009; Berner et al. 2003; Glasspool and Scott 2010). It is believed that non-essential introns would have imposed costs to a gene by prolonging transcription (Lynch 2002). Cox1 is the catalytic center for reducing oxygen to water in the oxidative phosphorylation of aerobic respiration (Pierron et al. 2012). We speculated that faster transcription efficiency might have provided cox1intronless corals with a selective advantage under a scenario of declining oxygen concentrations. 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