- Email: [email protected]
Deep-water sea anemone with a two-chromosome mitochondrial genome
Accepted Manuscript Deep-water sea anemone with a two-chromosome mitochondrial genome
Arseny Dubin, Sylvia Ighem Chi, Åse Emblem, Truls Moum, Steinar D. Johansen PII: DOI: Reference:
S0378-1119(19)30035-6 https://doi.org/10.1016/j.gene.2018.12.074 GENE 43525
To appear in:
Gene
Received date: Revised date: Accepted date:
4 September 2018 10 December 2018 20 December 2018
Please cite this article as: Arseny Dubin, Sylvia Ighem Chi, Åse Emblem, Truls Moum, Steinar D. Johansen , Deep-water sea anemone with a two-chromosome mitochondrial genome. Gene (2019), https://doi.org/10.1016/j.gene.2018.12.074
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Deep-water sea anemone with a two-chromosome mitochondrial genome
Arseny Dubin a, Sylvia Ighem Chi b, Åse Emblem b,c, Truls Moum a and Steinar
a
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D. Johansen a,b,*
Genomics group, Faculty of Biosciences and Aquaculture, Nord University, Bodø,
Norway; b Department of Medical Biology, Faculty of Health Sciences, UiT – Arctic
NU
University of Norway, Tromsø, Norway; c Research Laboratory and Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway
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Email addresses:
PT
ED
MA
030918
[email protected] (Arseny Dubin)
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[email protected] (Sylvia Ighem Chi) [email protected] (Åse Emblem) [email protected] (Truls Moum) [email protected] (Steinar D. Johansen)
* Corresponding author at: Genomics group, Faculty of Biosciences and Aquaculture, Nord University, N-8049 Bodø, Norway. E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT ABSTRACT
Mitochondrial genome organization of sea anemones appears conserved among species and families, and is represented by a single circular DNA molecule of 17 to 21 kb. The mitochondrial gene content corresponds to the same 13 protein components of the oxidative phosphorylation (OxPhos) system as in vertebrates.
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Hallmarks, however, include a highly reduced tRNA gene repertoire and the presence of autocatalytic group I introns. Here we demonstrate that the mitochondrial genome of the deep-water sea anemone Protanthea simplex deviates significantly from that of other known sea anemones. The P. simplex mitochondrial genome contains a heavily scrambled order of genes that are coded on both DNA strands and organized along two circular mito-chromosomes, MCh-I and MCh-II. We found MCh-I to be
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representative of the prototypic sea anemone mitochondrial genome, encoding 12 OxPhos proteins, two ribosomal RNAs, two transfer RNAs, and a group I intron. In contrast, MCh-II was found to be a laterally transferred plasmid-like DNA carrying
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the conserved cytochrome oxidase II gene and a second allele of the small subunit
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ribosomal RNA gene.
Keywords: Actiniaria; group I intron; mitochondrial genome rearrangement; mitochondrial plasmid; mtDNA; Protanthea simplex
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ACCEPTED MANUSCRIPT 1. Introduction
Cnidarians are basal non-bilateral metazoans that include species like sea anemones, stony corals, soft corals, hydroids, and jellyfishes. Cnidarian mitochondrial genomes (mtDNAs) are compact circular or linear DNAs between 16 kb and 21 kb in size, encoding the same set of 13 mitochondrial OxPhos proteins and two ribosomal
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RNAs (rRNAs) as most other metazoans (Osigus et al., 2013). The different hexacorallian orders (Actiniaria, Antipathalia, Corallimorpharia, Scleractinia and Zoantharia), however, possess several unique features in the content and organization of their circular mtDNAs (Medina et al., 2006; Brugler and France, 2007; Lin et al., 2014; Emblem et al., 2014; Chi and Johansen, 2017). These DNA molecules harbor only 1-2 transfer RNA (tRNA) genes, all genes are encoded by the same DNA strand,
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and the NADH dehydrogenase subunit 5 (ND5) gene always contains an autocatalytic group I intron (Beagley et al., 1996; Emblem et al., 2011). The about twenty completely sequenced sea anemone mtDNAs currently
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available represent species from 11 different families (Emblem et al., 2014; Foox et al., 2016; Zhang and Zhu, 2016; Chi et al., 2018). The mtDNAs harbor a closely
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related main-type mitochondrial gene organization, but an optional group I intron and additional protein coding genes may occur (see Goddard et al., 2006; Emblem et al., 2014; Chi et al., 2018). Here we report organization and evolutionary characterization
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of the complete mitochondrial genome of the deep-water sea anemone P. simplex,
hexacorals.
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which we found to deviate significantly from the general features known among other
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2. Materials and methods
2.1. Sampling and nucleic acid isolation
P. simplex was collected from a Lophelia pertusa cold-water coral reef outside Nord-Leksa, Norway (63º36’N; 9º24’E) at 150-200 m depth, using the research vessel «Gunnerus» and a Remotely Operated Vehicle «Minerva» (NTNU). Epidermal tissue samples from body wall and tentacles were immersed either in absolute ethanol or RNAlater® RNA Stabilization Solution (Life Technologies™) for DNA or RNA extraction respectively. All samples were stored at minus 20ºC until extraction. For 3
ACCEPTED MANUSCRIPT DNA extraction tissue samples were mechanically lysed in 300 μl of lysis buffer using Precellys 24 homogenizer (Bertin technologies™). The lysate was then additionally digested with proteinase K followed by phenol/chloroform extraction. The DNA was resuspended in water and stored at minus 20ºC until further use. Tissues prepared for RNA extraction were crushed in 500 μl of TRIzol using Precellys homogenizer. Acidic phenol/chloroform extraction was then performed, and RNA was re-dissolved in RNase-Free water and stored at minus 70ºC. To remove
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most of the ribosomal RNA, Low Input RiboMinus™ Eukaryote System v2 kit (Ambion™) was applied to P. simplex total RNA sample.
2.2.
DNA and RNA sequencing
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Genomic DNA and total RNA were respectively subjected to whole genome and transcriptome Ion Torrent PGM sequencing. All library preparations, template reactions, and sequencing steps were performed according to the standard protocols.
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To construct 400 bp libraries, 100 ng of DNA and RNA were separately utilized. DNA library was prepared using Ion Xpress TM Plus gDNA Fragment Library
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Preparation kit manual. Chemical fragmentation was performed using Ion Shear™ Plus Reagents (Life Technologies™). Template dilution factor was determined based on qPCR concentrations using Ion Library Quantitation Kit procedure (Life
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technologies). Whole transcriptome library was constructed using Ion Total RNA-Seq Kit v2. Total RNA was polyA-selected using the mRNA DIRECT Purification Kit
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(ThermoFisher Scientific, Waltham, MA, USA) and subsequently fragmented enzymatically. The fragmented total RNA was subjected to reverse transcription with
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a reverse transcriptase mix (10X SuperScript® III Enzyme Mix). Final preparations and sequencing were performed using the Ion PGM™ Sequencing 400 Kit according the manufacturer's protocol and on 316 v.2 chips. Selected mtDNA regions were subjected to PCR amplification, plasmid cloning, and Sanger sequencing using specific primers (Table S1). Plasmid cloning was performed using the TOPO® TA Cloning® Kit with One Shot® TOP10 Competent Cells (Life Technologies™). Fifteen ng of PCR product were ligated overnight at 4oC with 50 ng of the vector, and subsequently transformed into DH5 E. coli competent cells. Plasmid DNA was subsequently purified using PureLink® Quick Plasmid Miniprep Kit (Invitrogen™),
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ACCEPTED MANUSCRIPT visualized on 1 % agarose gel, purified using QIAEX II Gel Extraction Kit (Qiagen™), and then Sanger sequenced (BigDye v3.1).
2.3. Data analyses
The P. simplex mtDNA sequence was assembled from whole genomic readpool by means of the Mitochondrial Baiting and Iterative Mapping (MITObim)
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pipeline (Hahn et al. 2013) and the CLC Genomics Workbench (Qiagen™). MEGA v7.0.20 software was used for sequence alignments and subsequent phylogenetic analysis (Kumar et al., 2016). The gene sequences were aligned with MUSCLE alignment tool with default MEGA parameters. All sequence alignments were model tested prior to the construction of the trees. Neighbour Joining (NJ), Maximum
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Parsimony (MP) and Maximum Likelihood (ML) methods were used for comparison. The topologies of the trees were evaluated by 1000 bootstrap replicates. RNAseq analysis was carried out on the filtered and trimmed whole transcriptome data, and
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mapping of individual genes was performed on the CLC Genomic workbench with
3. Results and discussion
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default setting (CLCBio).
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3.1. P. simplex mitochondrial genome consists of two distinct mito-chromosomes
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P. simplex is a small cold-water sea anemone recorded down to 500 m depth (www.marlin.ac.uk), and our sample was collected at 150-200 m on a Lophelia cold-
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water coral reef in Nord-Leksa (Norway). The complete mtDNA sequence (21,326 bp; GenBank accession numbers MH500774 and MH500775) was determined on both strands using Ion Torrent PGM and Sanger sequencing platforms. Interestingly, the mitochondrial genome was found as two separate circular DNAs, assigned mitochromosome I (MCh-I) of 17,134 bp and mito-chromosome II (MCh-II) of 4,192 bp (Fig. 1; Table 1). Ion Torrent PGM-generated transcriptome data supported mitochondrial gene expression from MCh-I (Table S2). Twelve of the annotated OxPhos protein genes, two rRNA genes, and two tRNA genes were located on MCh-I (Fig. 1a; Table 1). The essential and highly conserved cytochrome oxidase II (COII) gene was missing. While most genes were 5
ACCEPTED MANUSCRIPT coded by the same forward DNA strand, four mitochondrial genes (ND2, ND4L, SSU-a, and tRNA-M) were located on the opposite strand. Furthermore, the MCh-I gene order was scrambled compared to that of other known sea anemones. Finally, a giant group I intron (corresponding to 15,261 bp) was present within the ND5 gene, and harbored all additional MCh-I sequences (15 genes) embedded in its P8 ribozyme segment (Fig. 1b, left panel). Due to the circular organization of the mtDNA, the ND5
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exon 2 and exon 1 were only separated by a 33 bp spacer but in a permuted order (Fig. S1). This organization suggests intron removal by RNA back-splicing, essential as recently detected in the corresponding ND5-717 intron of Corallimorpharia hexacoral mitochondria (Chi, 2017). The P. simplex ND5 group I ribozyme was found closely related to the sea anemone consensus at the secondary structure level, but less similar in primary sequence (Fig. 1b, right panel).
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MCh-II harbored the COII gene. We also recognized a second apparently functional SSU rRNA gene (SSU-b) and two truncated pseudogenes (SSU-b and
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ND4), all encoded by the same DNA strand on MCh-II (Fig. 1a; Table 1). The SSUCOII-ND4 synteny corresponded to the main-type mtDNA organization of sea anemones (Emblem et al., 2014). A larger non-coding region was located between
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ND4 and SSU-b, which contained complex sets of repetitive sequence features (DR-a and DR-b). DR-a consisted of 5 copies of an 80-bp direct repeat motif,
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including an internal 40-bp inverted repeat (IR) feature (Fig. 1a). We speculate that DR-a is part of an origin of replication element. Interestingly, this non-coding region
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also contained a 75 bp sequence motif with 100% identity to a sequence within the MCh-I (Fig. 1a). Presence of an inter-chromosomal sequence motif (InterCS) may
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indicate cross-talking between MCh-I and MCh-II.
3.2. Mito-chromosomes I and II have different evolutionary origins
Sequence alignment and secondary structure assessments of SSU-a and SSU-b rRNAs encoded by MCh-I and MCh-II, respectively, suggested both to be functional since nucleotide positions and structures known to be essential in translation were highly conserved (Fig. S2). We noted a unique 37 nt direct repeat duplication within SSU-a, which could lead to an alternative RNA secondary folding of Domain 3’M (Fig. S3). Interestingly, among the 54 sea anemone species assessed (Fig. 2) the
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ACCEPTED MANUSCRIPT duplication was only present in P. simplex and Gonactinia prolifera (both belong taxonomically to family Gonactiniidae), and in the related Viatrix globulifera (family Boloceroididae). It is tempting to suggest that the SSU-a rRNA sequence duplication could interfere with mitochondrial ribosome function. Nucleotide BLAST searches using a 770 bp mitochondrial SSU rRNA gene region available for a larger number of sea anemones were performed (Table S3;
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Table S4). Here, SSU-a corresponded to those of the sea anemone family Gonactiniidae (where P. simplex belongs taxonomically), whereas SSU-b is closer to the distantly related sea anemone family Actiniidae. We then investigated the molecular relationship between the mitochondrial SSU rRNA, ND1, COII, and ND4 genes by phylogenetic reconstruction. A representative Neighbour Joining tree of the mitochondrial SSU rRNA gene (Fig. 2) was based on a 770 bp sequence obtained
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from about 50 additional species of sea anemones. SSU-a (MCh-I) and SSU-b (MChII) represented distinct origins within the sea anemones. While SSU-a clustered to Gonactiniidae species, SSU-b was closely related to that of Actiniidae species.
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Additional analyses based on the complete SSU rRNA and COII genes (Fig. S4), and ND1 and ND4 genes (Fig. S5) gave supporting conclusions. These findings strongly
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indicate an Actiniidae-like origin of MCh-II.
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3.3. Horizontal transfer of mito-chromosome II?
We envision the following scenario to explain the unique two-chromosome
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mitochondrial genome organization in P. simplex. (1) The ancestral sea anemone was probably represented by the common mtDNA organization, but due to unknown
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environmental factors a dramatic rearrangement event occurred. Interestingly, minor within-order mtDNA rearrangements have previously been associated with deepwater habitats in stony corals (Emblem et al., 2011; Lin et al., 2012) and mushroom corals (Lin et al., 2014). (2) This re-arrangement resulted in a scrambled gene organization (MCh-I) that utilized both mtDNA strands as coding templates. The latter feature is so far unprecedented among hexacorals, but common in octocorals (Osigus et al., 2013). (3) The essential COII gene was lost during the mitochondrial genome rearrangement, but rescued by lateral transfer of MCh-II. (4) The donor was a distantly related Actiniidae-like sea anemone. The COII gene transfer occurred either through a plasmid-like vector (MCh-II), or by transfer of the complete Actiniidae-like 7
ACCEPTED MANUSCRIPT mitochondrial genome that subsequently was reduced to MCh-II by functional redundancy. (5) MCh-I and MCh-II co-evolved to ensure efficient OxPhos activities in P. simplex. We speculate that the MCh-II encoded SSU-b became important for COII translation in P. simplex, perhaps due to functional restrictions linked to structural plasticity in the SSU-a Domain 3’M (Fig. S5). Interestingly, a switch in RNA structural composition has been reported in mammalian mitochondrial
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ribosomes (Rorbach et al., 2016). (6) Finally, the InterCS element became involved in inter-chromosomal coordination.
Acknowledgements
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We thank Jussi Evertsen at Norwegian University of Sciences and Technology (NTNU) for collecting P. simplex, Tor Erik Jørgensen and Anita Ursvik for support in
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Ion Torrent sequencing, and members of our research teams at Nord University and University of Tromsø for interesting discussions. This work is supported by grants from the Tromsø Research Foundation, the Research Council of Norway, the
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University of Tromsø, and Nord University.
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References
Beagley, C.T., Okada, N.A., Wolstenholme, D.R., 1996. Two mitochondrial group I
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introns in a metazoan, the sea anemone Metridium senile: one intron contains genes for subunits 1 and 3 of NADH dehydrogenase. Proc. Natl. Acad. Sci. USA, 93, 5619-5623. Brugler, M.R., France, S.C., 2007. The complete mitochondrial genome of the black coral Chrysopathes formosa (Cnidaria: Anthozoa: Antipatharia) supports classification of antipatharians within the subclass Hexacorallia. Mol. Phylegenet. Evol. 42, 776-788. Chi, S.I., 2017. Mitochondrial group I introns in hexacorals. PhD thesis, UiT – The Arctic University of Norway. ISBN 978-82-7589-556-9.
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ACCEPTED MANUSCRIPT Chi, S.I., Johansen, S.D., 2017. Zoantharian mitochondrial genomes contain unique complex group I introns and highly conserved intergenic regions. Gene, 628, 24-31. Chi, S.I., Urbarova, I., Johansen, S.D., 2018. Expression of homing endonuclease gene and insertion-like element in sea anemone mitochondrial genomes: lesson learned from Anemonia viridis. Gene, 652, 78-86.
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Emblem, Å., Karlsen, B.O., Evertsen, J., Johansen, S.D., 2011. Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a long-term evolving group I intron. Mol. Phylogenet. Evol. 61, 495-503.
Emblem, Å., Okkenhaug, S., Weiss, E. S., Denver, D.R., Karlsen, B.O., Moum, T., Johansen, S.D., 2014. Sea anemones possess dynamic mitogenome structures.
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Mol. Phylogenet. Evol. 75, 184-193.
Foox, J., Brugler, M., Siddall, M.E., Rodríguez, E., 2016. Multiplexed pyrosequencing of nine sea anemone (Cnidaria: Anthozoa: Hexacorallia:
Anal. 27, 2826-2832.
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Actiniaria) mitochondrial genomes. Mitochondrial DNA A DNA Mapp. Seq.
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Goddard, M.R., Leigh, J., Roger, A.J., Pemberton, A.J., 2006. Invasion and persistence of a selfish gene in the Cnidaria. PLoS One, 1, e3. Hahn, C., Bachmann, L., Chevreux, B., 2013. Reconstructing mitochondrial genomes
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directly from genetic next-generation sequencing reads – a baiting and iterative mapping approach. Nucleic Acids Res. 41, e129.
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Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870-1874.
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Lin, M.F., Kitahara, M.V., Luo, H., Tracey, D., Geller, J., Fukami, H., Miller D.J., Chen, C.A., 2014. Mitochondrial genome rearrangements in the scleractinia/ corallimorpharia complex: implications for coral phylogeny. Genome Biol. Evol. 6, 1086-1095. Lin, M.F., Kitahara, M.V., Tachikawa, H., Fukami, H., Miller D.J., Chen, C.A., 2012. Novel organization of the mitochondrial genome in the deep-sea coral Madropora oculata (Hexacorallia, Scleractinia, Oculinidae) and its taxonomic implications. Mol. Phylogenet. Evol. 65, 323-328. Medina, M., Collins, A.G., Takaoka, T.L., Kuehl, J.V., Boore, J.L., 2006. Naked corals: skeleton loss in Scleractinia. Proc Natl Acad Sci USA 103, 9096-9100. 9
ACCEPTED MANUSCRIPT Osigus, H.J., Eitel, M., Bernt, M., Donath, A., Schierwater, B., 2013. Mitogenomics at the base of Metazoa. Mol. Phylogenet. Evol. 69, 339-351. Rorbach, J., Gao, F., Powell, C.A., D’Souza, A., Lightowlers, R.N., Minczuk, M., Chrzanowska-Lightowlers, Z.M., 2016. Human mitochondrial ribosomes can switch their structural RNA composition. Proc. Natl. Acad. Sci. USA, 113, 12198-12201.
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Zhang, L., Zhu, Q., 2016. Complete mitochondrial genome of the sea anemone, Anthopleura midori (Actiniaria: Actiniidae). Mitochondrial DNA A DNA Mapp. Seq. Anal. 27, 1-2.
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Figure legends
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Figure 1. Mitochondrial genome features in Protanthea simplex. (a) Gene contents and organizations of the circular mito-chromosomes I and II (MCh-I and MCh-II), presented as linear maps. Intergenic regions (IGRs) are indicated above the maps.
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Location of obligatory intron (ND5-717) is shown. MCh-I: all genes are coded by the forward DNA strand (left to right), except ND2, SSU-a, ND4L, and M. InterCS, 75 bp
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inter-chromosomal sequence flanked by a 10-bp direct repeat. MCh-II: all genes (SSU-b and COII) and pseudogenes (SSU-b and ND4) are coded by the forward
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DNA strand (left to right). The main non-coding region (IGR-18) is expanded below the map. DR-a and DR-b, direct repeat motifs; IR, inverted repeat motif. Gene
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abbreviations: SSU and LSU, mitochondrial small- and large-subunit ribosomal RNA genes; ND1-6, NADH dehydrogenase subunit 1 to 6 genes; COI-III, cytochrome c oxidase subunit I to III genes; A6 and A8, ATPase subunit 6 and 8 genes; Cyt B, cytochrome B gene; M; tRNA gene for fMet indicated by the standard one-letter symbol for amino acids. (b) Secondary structure diagram of the catalytic core region of ND5-717 from P. simplex (left panel) and a consensus structure based on 16 sea anemone species (right panel). The P8 extension in P. simplex ND5-717 contains 15 essential mitochondrial gene sequences. The ND5-717 core deviates at 21 positions (red highlights) within the core structure positions that are 100% conserved among all other studied sea anemone ND5-717 introns. 10
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Figure 2. SSU rRNA-based phylogeny of sea anemones. A Neighbour Joining (NJ) phylogenetic tree is shown. The tree was based on an alignment of 770 nucleotide positions from 54 sea anemones (Actinaria), 7 colonial anemones (Zoantharia), and one black coral (Antopatharia). Bootstrap values (above 50%) of 1000 replications are shown at the internal nodes. Red filled circles indicate highly significant branch
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points (bootstrap values above 95%). Relative distance is indicated below the tree (bar corresponding to a relative distance of 0,04). Note that the branch lengths of Alicia, Viatrix, and Gonactiniidae species (yellow box) are collapsed. Arrow (orange) indicates the intruduction of repeat sequence within the Domain 3’M. Species classified within the family Actiniidae are indicated (blue box). According to current taxonomy Phymanthus, Heteractis and Stichodactyla are not members of the
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Actiniidae family. P. simplex SSU-a (MCh-I) and SSU-b (MCh-II) are highlighted.
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ACCEPTED MANUSCRIPT Abbreviation list:
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MCh-I: MCh-II: mtDNA: ND5: OxPhos: PGM: rRNA: SSU:
cytochrome oxidase II inter-chromosomal sequence motif mito-chromosome I mito-chromesome II mitochondrial genome NADH dehydrogenase subunit 5 oxidative phosphorylation personal genome machine ribosomal RNA small subunit
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CO II: InterCS:
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ACCEPTED MANUSCRIPT Highlights
• • •
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•
The mtDNA consists of two circular mito-chromosomes; MCh-I and MCh-II MCh-I resembles a typical sea anemone mtDNA, but lacks the CO II gene MCh-I gene order is heavily rearranged and both DNA strands contain genes MCh-I contains a giant group I intron element of 15 kb within the ND5 gene MCh-II provides the CO II gene, and appears laterally transferred
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•
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Figure 1
Figure 2
Arseny Dubin, Sylvia Ighem Chi, Åse Emblem, Truls Moum, Steinar D. Johansen PII: DOI: Reference:
S0378-1119(19)30035-6 https://doi.org/10.1016/j.gene.2018.12.074 GENE 43525
To appear in:
Gene
Received date: Revised date: Accepted date:
4 September 2018 10 December 2018 20 December 2018
Please cite this article as: Arseny Dubin, Sylvia Ighem Chi, Åse Emblem, Truls Moum, Steinar D. Johansen , Deep-water sea anemone with a two-chromosome mitochondrial genome. Gene (2019), https://doi.org/10.1016/j.gene.2018.12.074
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Deep-water sea anemone with a two-chromosome mitochondrial genome
Arseny Dubin a, Sylvia Ighem Chi b, Åse Emblem b,c, Truls Moum a and Steinar
a
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D. Johansen a,b,*
Genomics group, Faculty of Biosciences and Aquaculture, Nord University, Bodø,
Norway; b Department of Medical Biology, Faculty of Health Sciences, UiT – Arctic
NU
University of Norway, Tromsø, Norway; c Research Laboratory and Department of Laboratory Medicine, Nordland Hospital, Bodø, Norway
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Email addresses:
PT
ED
MA
030918
[email protected] (Arseny Dubin)
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[email protected] (Sylvia Ighem Chi) [email protected] (Åse Emblem) [email protected] (Truls Moum) [email protected] (Steinar D. Johansen)
* Corresponding author at: Genomics group, Faculty of Biosciences and Aquaculture, Nord University, N-8049 Bodø, Norway. E-mail address: [email protected]
1
ACCEPTED MANUSCRIPT ABSTRACT
Mitochondrial genome organization of sea anemones appears conserved among species and families, and is represented by a single circular DNA molecule of 17 to 21 kb. The mitochondrial gene content corresponds to the same 13 protein components of the oxidative phosphorylation (OxPhos) system as in vertebrates.
SC RI PT
Hallmarks, however, include a highly reduced tRNA gene repertoire and the presence of autocatalytic group I introns. Here we demonstrate that the mitochondrial genome of the deep-water sea anemone Protanthea simplex deviates significantly from that of other known sea anemones. The P. simplex mitochondrial genome contains a heavily scrambled order of genes that are coded on both DNA strands and organized along two circular mito-chromosomes, MCh-I and MCh-II. We found MCh-I to be
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representative of the prototypic sea anemone mitochondrial genome, encoding 12 OxPhos proteins, two ribosomal RNAs, two transfer RNAs, and a group I intron. In contrast, MCh-II was found to be a laterally transferred plasmid-like DNA carrying
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the conserved cytochrome oxidase II gene and a second allele of the small subunit
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CE
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ribosomal RNA gene.
Keywords: Actiniaria; group I intron; mitochondrial genome rearrangement; mitochondrial plasmid; mtDNA; Protanthea simplex
2
ACCEPTED MANUSCRIPT 1. Introduction
Cnidarians are basal non-bilateral metazoans that include species like sea anemones, stony corals, soft corals, hydroids, and jellyfishes. Cnidarian mitochondrial genomes (mtDNAs) are compact circular or linear DNAs between 16 kb and 21 kb in size, encoding the same set of 13 mitochondrial OxPhos proteins and two ribosomal
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RNAs (rRNAs) as most other metazoans (Osigus et al., 2013). The different hexacorallian orders (Actiniaria, Antipathalia, Corallimorpharia, Scleractinia and Zoantharia), however, possess several unique features in the content and organization of their circular mtDNAs (Medina et al., 2006; Brugler and France, 2007; Lin et al., 2014; Emblem et al., 2014; Chi and Johansen, 2017). These DNA molecules harbor only 1-2 transfer RNA (tRNA) genes, all genes are encoded by the same DNA strand,
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and the NADH dehydrogenase subunit 5 (ND5) gene always contains an autocatalytic group I intron (Beagley et al., 1996; Emblem et al., 2011). The about twenty completely sequenced sea anemone mtDNAs currently
MA
available represent species from 11 different families (Emblem et al., 2014; Foox et al., 2016; Zhang and Zhu, 2016; Chi et al., 2018). The mtDNAs harbor a closely
ED
related main-type mitochondrial gene organization, but an optional group I intron and additional protein coding genes may occur (see Goddard et al., 2006; Emblem et al., 2014; Chi et al., 2018). Here we report organization and evolutionary characterization
PT
of the complete mitochondrial genome of the deep-water sea anemone P. simplex,
hexacorals.
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which we found to deviate significantly from the general features known among other
AC
2. Materials and methods
2.1. Sampling and nucleic acid isolation
P. simplex was collected from a Lophelia pertusa cold-water coral reef outside Nord-Leksa, Norway (63º36’N; 9º24’E) at 150-200 m depth, using the research vessel «Gunnerus» and a Remotely Operated Vehicle «Minerva» (NTNU). Epidermal tissue samples from body wall and tentacles were immersed either in absolute ethanol or RNAlater® RNA Stabilization Solution (Life Technologies™) for DNA or RNA extraction respectively. All samples were stored at minus 20ºC until extraction. For 3
ACCEPTED MANUSCRIPT DNA extraction tissue samples were mechanically lysed in 300 μl of lysis buffer using Precellys 24 homogenizer (Bertin technologies™). The lysate was then additionally digested with proteinase K followed by phenol/chloroform extraction. The DNA was resuspended in water and stored at minus 20ºC until further use. Tissues prepared for RNA extraction were crushed in 500 μl of TRIzol using Precellys homogenizer. Acidic phenol/chloroform extraction was then performed, and RNA was re-dissolved in RNase-Free water and stored at minus 70ºC. To remove
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most of the ribosomal RNA, Low Input RiboMinus™ Eukaryote System v2 kit (Ambion™) was applied to P. simplex total RNA sample.
2.2.
DNA and RNA sequencing
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Genomic DNA and total RNA were respectively subjected to whole genome and transcriptome Ion Torrent PGM sequencing. All library preparations, template reactions, and sequencing steps were performed according to the standard protocols.
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To construct 400 bp libraries, 100 ng of DNA and RNA were separately utilized. DNA library was prepared using Ion Xpress TM Plus gDNA Fragment Library
ED
Preparation kit manual. Chemical fragmentation was performed using Ion Shear™ Plus Reagents (Life Technologies™). Template dilution factor was determined based on qPCR concentrations using Ion Library Quantitation Kit procedure (Life
PT
technologies). Whole transcriptome library was constructed using Ion Total RNA-Seq Kit v2. Total RNA was polyA-selected using the mRNA DIRECT Purification Kit
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(ThermoFisher Scientific, Waltham, MA, USA) and subsequently fragmented enzymatically. The fragmented total RNA was subjected to reverse transcription with
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a reverse transcriptase mix (10X SuperScript® III Enzyme Mix). Final preparations and sequencing were performed using the Ion PGM™ Sequencing 400 Kit according the manufacturer's protocol and on 316 v.2 chips. Selected mtDNA regions were subjected to PCR amplification, plasmid cloning, and Sanger sequencing using specific primers (Table S1). Plasmid cloning was performed using the TOPO® TA Cloning® Kit with One Shot® TOP10 Competent Cells (Life Technologies™). Fifteen ng of PCR product were ligated overnight at 4oC with 50 ng of the vector, and subsequently transformed into DH5 E. coli competent cells. Plasmid DNA was subsequently purified using PureLink® Quick Plasmid Miniprep Kit (Invitrogen™),
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2.3. Data analyses
The P. simplex mtDNA sequence was assembled from whole genomic readpool by means of the Mitochondrial Baiting and Iterative Mapping (MITObim)
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pipeline (Hahn et al. 2013) and the CLC Genomics Workbench (Qiagen™). MEGA v7.0.20 software was used for sequence alignments and subsequent phylogenetic analysis (Kumar et al., 2016). The gene sequences were aligned with MUSCLE alignment tool with default MEGA parameters. All sequence alignments were model tested prior to the construction of the trees. Neighbour Joining (NJ), Maximum
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Parsimony (MP) and Maximum Likelihood (ML) methods were used for comparison. The topologies of the trees were evaluated by 1000 bootstrap replicates. RNAseq analysis was carried out on the filtered and trimmed whole transcriptome data, and
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mapping of individual genes was performed on the CLC Genomic workbench with
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default setting (CLCBio).
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3.1. P. simplex mitochondrial genome consists of two distinct mito-chromosomes
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P. simplex is a small cold-water sea anemone recorded down to 500 m depth (www.marlin.ac.uk), and our sample was collected at 150-200 m on a Lophelia cold-
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water coral reef in Nord-Leksa (Norway). The complete mtDNA sequence (21,326 bp; GenBank accession numbers MH500774 and MH500775) was determined on both strands using Ion Torrent PGM and Sanger sequencing platforms. Interestingly, the mitochondrial genome was found as two separate circular DNAs, assigned mitochromosome I (MCh-I) of 17,134 bp and mito-chromosome II (MCh-II) of 4,192 bp (Fig. 1; Table 1). Ion Torrent PGM-generated transcriptome data supported mitochondrial gene expression from MCh-I (Table S2). Twelve of the annotated OxPhos protein genes, two rRNA genes, and two tRNA genes were located on MCh-I (Fig. 1a; Table 1). The essential and highly conserved cytochrome oxidase II (COII) gene was missing. While most genes were 5
ACCEPTED MANUSCRIPT coded by the same forward DNA strand, four mitochondrial genes (ND2, ND4L, SSU-a, and tRNA-M) were located on the opposite strand. Furthermore, the MCh-I gene order was scrambled compared to that of other known sea anemones. Finally, a giant group I intron (corresponding to 15,261 bp) was present within the ND5 gene, and harbored all additional MCh-I sequences (15 genes) embedded in its P8 ribozyme segment (Fig. 1b, left panel). Due to the circular organization of the mtDNA, the ND5
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exon 2 and exon 1 were only separated by a 33 bp spacer but in a permuted order (Fig. S1). This organization suggests intron removal by RNA back-splicing, essential as recently detected in the corresponding ND5-717 intron of Corallimorpharia hexacoral mitochondria (Chi, 2017). The P. simplex ND5 group I ribozyme was found closely related to the sea anemone consensus at the secondary structure level, but less similar in primary sequence (Fig. 1b, right panel).
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MCh-II harbored the COII gene. We also recognized a second apparently functional SSU rRNA gene (SSU-b) and two truncated pseudogenes (SSU-b and
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ND4), all encoded by the same DNA strand on MCh-II (Fig. 1a; Table 1). The SSUCOII-ND4 synteny corresponded to the main-type mtDNA organization of sea anemones (Emblem et al., 2014). A larger non-coding region was located between
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ND4 and SSU-b, which contained complex sets of repetitive sequence features (DR-a and DR-b). DR-a consisted of 5 copies of an 80-bp direct repeat motif,
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including an internal 40-bp inverted repeat (IR) feature (Fig. 1a). We speculate that DR-a is part of an origin of replication element. Interestingly, this non-coding region
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also contained a 75 bp sequence motif with 100% identity to a sequence within the MCh-I (Fig. 1a). Presence of an inter-chromosomal sequence motif (InterCS) may
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3.2. Mito-chromosomes I and II have different evolutionary origins
Sequence alignment and secondary structure assessments of SSU-a and SSU-b rRNAs encoded by MCh-I and MCh-II, respectively, suggested both to be functional since nucleotide positions and structures known to be essential in translation were highly conserved (Fig. S2). We noted a unique 37 nt direct repeat duplication within SSU-a, which could lead to an alternative RNA secondary folding of Domain 3’M (Fig. S3). Interestingly, among the 54 sea anemone species assessed (Fig. 2) the
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Table S4). Here, SSU-a corresponded to those of the sea anemone family Gonactiniidae (where P. simplex belongs taxonomically), whereas SSU-b is closer to the distantly related sea anemone family Actiniidae. We then investigated the molecular relationship between the mitochondrial SSU rRNA, ND1, COII, and ND4 genes by phylogenetic reconstruction. A representative Neighbour Joining tree of the mitochondrial SSU rRNA gene (Fig. 2) was based on a 770 bp sequence obtained
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from about 50 additional species of sea anemones. SSU-a (MCh-I) and SSU-b (MChII) represented distinct origins within the sea anemones. While SSU-a clustered to Gonactiniidae species, SSU-b was closely related to that of Actiniidae species.
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Additional analyses based on the complete SSU rRNA and COII genes (Fig. S4), and ND1 and ND4 genes (Fig. S5) gave supporting conclusions. These findings strongly
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indicate an Actiniidae-like origin of MCh-II.
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3.3. Horizontal transfer of mito-chromosome II?
We envision the following scenario to explain the unique two-chromosome
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mitochondrial genome organization in P. simplex. (1) The ancestral sea anemone was probably represented by the common mtDNA organization, but due to unknown
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environmental factors a dramatic rearrangement event occurred. Interestingly, minor within-order mtDNA rearrangements have previously been associated with deepwater habitats in stony corals (Emblem et al., 2011; Lin et al., 2012) and mushroom corals (Lin et al., 2014). (2) This re-arrangement resulted in a scrambled gene organization (MCh-I) that utilized both mtDNA strands as coding templates. The latter feature is so far unprecedented among hexacorals, but common in octocorals (Osigus et al., 2013). (3) The essential COII gene was lost during the mitochondrial genome rearrangement, but rescued by lateral transfer of MCh-II. (4) The donor was a distantly related Actiniidae-like sea anemone. The COII gene transfer occurred either through a plasmid-like vector (MCh-II), or by transfer of the complete Actiniidae-like 7
ACCEPTED MANUSCRIPT mitochondrial genome that subsequently was reduced to MCh-II by functional redundancy. (5) MCh-I and MCh-II co-evolved to ensure efficient OxPhos activities in P. simplex. We speculate that the MCh-II encoded SSU-b became important for COII translation in P. simplex, perhaps due to functional restrictions linked to structural plasticity in the SSU-a Domain 3’M (Fig. S5). Interestingly, a switch in RNA structural composition has been reported in mammalian mitochondrial
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ribosomes (Rorbach et al., 2016). (6) Finally, the InterCS element became involved in inter-chromosomal coordination.
Acknowledgements
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We thank Jussi Evertsen at Norwegian University of Sciences and Technology (NTNU) for collecting P. simplex, Tor Erik Jørgensen and Anita Ursvik for support in
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Ion Torrent sequencing, and members of our research teams at Nord University and University of Tromsø for interesting discussions. This work is supported by grants from the Tromsø Research Foundation, the Research Council of Norway, the
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University of Tromsø, and Nord University.
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References
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introns in a metazoan, the sea anemone Metridium senile: one intron contains genes for subunits 1 and 3 of NADH dehydrogenase. Proc. Natl. Acad. Sci. USA, 93, 5619-5623. Brugler, M.R., France, S.C., 2007. The complete mitochondrial genome of the black coral Chrysopathes formosa (Cnidaria: Anthozoa: Antipatharia) supports classification of antipatharians within the subclass Hexacorallia. Mol. Phylegenet. Evol. 42, 776-788. Chi, S.I., 2017. Mitochondrial group I introns in hexacorals. PhD thesis, UiT – The Arctic University of Norway. ISBN 978-82-7589-556-9.
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ACCEPTED MANUSCRIPT Chi, S.I., Johansen, S.D., 2017. Zoantharian mitochondrial genomes contain unique complex group I introns and highly conserved intergenic regions. Gene, 628, 24-31. Chi, S.I., Urbarova, I., Johansen, S.D., 2018. Expression of homing endonuclease gene and insertion-like element in sea anemone mitochondrial genomes: lesson learned from Anemonia viridis. Gene, 652, 78-86.
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Emblem, Å., Okkenhaug, S., Weiss, E. S., Denver, D.R., Karlsen, B.O., Moum, T., Johansen, S.D., 2014. Sea anemones possess dynamic mitogenome structures.
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Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870-1874.
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Lin, M.F., Kitahara, M.V., Luo, H., Tracey, D., Geller, J., Fukami, H., Miller D.J., Chen, C.A., 2014. Mitochondrial genome rearrangements in the scleractinia/ corallimorpharia complex: implications for coral phylogeny. Genome Biol. Evol. 6, 1086-1095. Lin, M.F., Kitahara, M.V., Tachikawa, H., Fukami, H., Miller D.J., Chen, C.A., 2012. Novel organization of the mitochondrial genome in the deep-sea coral Madropora oculata (Hexacorallia, Scleractinia, Oculinidae) and its taxonomic implications. Mol. Phylogenet. Evol. 65, 323-328. Medina, M., Collins, A.G., Takaoka, T.L., Kuehl, J.V., Boore, J.L., 2006. Naked corals: skeleton loss in Scleractinia. Proc Natl Acad Sci USA 103, 9096-9100. 9
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Zhang, L., Zhu, Q., 2016. Complete mitochondrial genome of the sea anemone, Anthopleura midori (Actiniaria: Actiniidae). Mitochondrial DNA A DNA Mapp. Seq. Anal. 27, 1-2.
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Figure legends
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Figure 1. Mitochondrial genome features in Protanthea simplex. (a) Gene contents and organizations of the circular mito-chromosomes I and II (MCh-I and MCh-II), presented as linear maps. Intergenic regions (IGRs) are indicated above the maps.
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Location of obligatory intron (ND5-717) is shown. MCh-I: all genes are coded by the forward DNA strand (left to right), except ND2, SSU-a, ND4L, and M. InterCS, 75 bp
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inter-chromosomal sequence flanked by a 10-bp direct repeat. MCh-II: all genes (SSU-b and COII) and pseudogenes (SSU-b and ND4) are coded by the forward
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DNA strand (left to right). The main non-coding region (IGR-18) is expanded below the map. DR-a and DR-b, direct repeat motifs; IR, inverted repeat motif. Gene
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abbreviations: SSU and LSU, mitochondrial small- and large-subunit ribosomal RNA genes; ND1-6, NADH dehydrogenase subunit 1 to 6 genes; COI-III, cytochrome c oxidase subunit I to III genes; A6 and A8, ATPase subunit 6 and 8 genes; Cyt B, cytochrome B gene; M; tRNA gene for fMet indicated by the standard one-letter symbol for amino acids. (b) Secondary structure diagram of the catalytic core region of ND5-717 from P. simplex (left panel) and a consensus structure based on 16 sea anemone species (right panel). The P8 extension in P. simplex ND5-717 contains 15 essential mitochondrial gene sequences. The ND5-717 core deviates at 21 positions (red highlights) within the core structure positions that are 100% conserved among all other studied sea anemone ND5-717 introns. 10
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Figure 2. SSU rRNA-based phylogeny of sea anemones. A Neighbour Joining (NJ) phylogenetic tree is shown. The tree was based on an alignment of 770 nucleotide positions from 54 sea anemones (Actinaria), 7 colonial anemones (Zoantharia), and one black coral (Antopatharia). Bootstrap values (above 50%) of 1000 replications are shown at the internal nodes. Red filled circles indicate highly significant branch
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points (bootstrap values above 95%). Relative distance is indicated below the tree (bar corresponding to a relative distance of 0,04). Note that the branch lengths of Alicia, Viatrix, and Gonactiniidae species (yellow box) are collapsed. Arrow (orange) indicates the intruduction of repeat sequence within the Domain 3’M. Species classified within the family Actiniidae are indicated (blue box). According to current taxonomy Phymanthus, Heteractis and Stichodactyla are not members of the
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Actiniidae family. P. simplex SSU-a (MCh-I) and SSU-b (MCh-II) are highlighted.
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ACCEPTED MANUSCRIPT Abbreviation list:
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MCh-I: MCh-II: mtDNA: ND5: OxPhos: PGM: rRNA: SSU:
cytochrome oxidase II inter-chromosomal sequence motif mito-chromosome I mito-chromesome II mitochondrial genome NADH dehydrogenase subunit 5 oxidative phosphorylation personal genome machine ribosomal RNA small subunit
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CO II: InterCS:
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ACCEPTED MANUSCRIPT Highlights
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The mtDNA consists of two circular mito-chromosomes; MCh-I and MCh-II MCh-I resembles a typical sea anemone mtDNA, but lacks the CO II gene MCh-I gene order is heavily rearranged and both DNA strands contain genes MCh-I contains a giant group I intron element of 15 kb within the ND5 gene MCh-II provides the CO II gene, and appears laterally transferred
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Figure 1
Figure 2