Molecular Phylogenetics and Evolution 65 (2012) 323–328

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Molecular Phylogenetics and Evolution

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Short Communication Novel organization of the mitochondrial genome in the deep-sea coral, Madrepora oculata (Hexacorallia, Scleractinia, Oculinidae) and its taxonomic implications

Mei-Fang Lin a,c,g, Marcelo Visentini Kitahara b, Hiroyuki Tachikawa d, Hironobu Fukami e, ⇑ David John Miller c,g, Chaolun Allen Chen a,f,h, a Biodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan b Centro de Biologia Marinha, Universidade de São Paulo, São Sebastião 11600-970, Brazil c School of Pharmacy and Molecular Sciences, James Cook University, Townsville 4810, Australia d Natural History Museum and Institute, Chiba 955-2, Japan e Department of Marine Biology and Environmental Science, University of Miyazaki, Miyazaki 889-2192, Japan f Institute of Oceanography, National Taiwan University, Taipei 106, Taiwan g ARC Centre of Excellence for Coral Reef Studies, James Cook University, Townsville 4810, Australia h Taiwan International Graduate Program (TIGP)-Biodiversity, Academia Sinica, Nangang, Taipei 115, Taiwan article info abstract

Article history: Madrepora is one of the most ecologically important genera of reef-building scleractinians in the deep sea, Received 25 January 2012 occurring from tropical to high-latitude regions. Despite this, the taxonomic afﬁnities and relationships Revised 29 May 2012 within the genus Madrepora remain unclear. To clarify these issues, we sequenced the mitochondrial Accepted 4 June 2012 (mt) genome of the most widespread Madrepora species, M. oculata, and compared this with data for Available online 1 July 2012 other scleractinians. The architecture of the M. oculata mt genome was very similar to that of other scleractinians, except for a novel gene rearrangement affecting only cox2 and cox3. This pattern of gene orga- Keywords: nization was common to four geographically distinct M. oculata individuals as well as the congeneric Scleractinian species M. minutiseptum, but was not shared by other genera that are closely related on the basis of Madrepora Mitochondrial genome cox1 sequence analysis nor other oculinids, suggesting that it might be unique to Madrepora. Gene rearrangement Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction 2007), and comprises five recent species (Cairns, 2009). M. oculata is the type species and is probably the most-widespread scleractin- Almost half of extant scleractinians are azooxanthellate (i.e. live ian species, being essentially ubiquitous in deep water except in without dinoflagellate symbionts) and are often referred to as cold- the polar seas (Cairns and Zibrowius, 1997). M. oculata is highly or deep-water corals because they generally occur in waters polymorphic, six forms being recognized (alpha, beta, gamma, deeper than 50 m (Cairns et al., 1999; Cairns, 2007). Although less galapagensis, vitiae, and formosa; Cairns, 1991, 1995; Cairns et al., obvious than the shallow water coral reefs of tropical waters, the 1999) based on variations in color, branching patterns, the texture deep sea reefs constructed by some azooxanthellate scleractinians of the coenosteum and the size of septa; however, note that these serve as habitat, feeding, recruitment, and nursery grounds for characters are variable even within sympatric specimens (e.g., numerous marine organisms (Roberts et al., 2009) and have Galápagos specimens; Cairns, 1991). recently attracted the attention of both the scientific community Although traditionally classified in the Family Oculinidae and the general public, particularly the fishing industry. However, (Cairns et al., 1999; Cairns, 2009), recent molecular phylogenetic relatively few deep-water scleractinians – representatives of the analyses suggest that the family is polyphyletic (representatives genera Lophelia, Solenosmilia, Goniocorella, Madrepora, Oculina, were scattered across four different clades, these most likely repre- and Enallopsammia – are considered to fulfill the ecological and senting four different families; Kitahara et al., 2010), and that the geological criteria of true reef-building species (Stolarski and genus Madrepora should be elevated to a higher taxonomic level Vertino, 2007; Roberts et al., 2009). (Le Goff-Vitry et al., 2004; Kitahara et al., 2010). Molecular phylog- Of the deep-sea reef-builders, Madrepora has a fossil record dat- enetics based on partial cox1 data suggests that Madrepora oculata ing from the Lower Cretaceous (ca. 70 Mya; Stolarski and Vertino, may be more closely related to Caryophyllia (family Caryophyllii- dae) and pocilloporids than to other oculinids (Kitahara et al., 2010; Stolarski et al., 2011). ⇑ Corresponding author at: Biodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan. Fax: +886 2 28958059. Comparison of mitochondrial (mt) genomes has contributed to E-mail address: [email protected] (C.A. Chen). the clarification of phylogenetic relationships among animals (Boore,

1999; van Oppen et al., 2002; Chen et al., 2008a,b), gene arrangement reported, each with important phylogenetic implications. These vari- has been generally the most informative mt character (reviewed in ations include the presence of an idiosyncratic atp8 in Seriatopora and Boore et al., 2005). Several well-established evolutionary lineages a duplicated trnW in some pocilloporids (Chen et al., 2008b), the are deﬁned by common organization of mt genes (reviewed in Boore insertion of a distinct group I intron in the cox1 gene of some ‘‘robust’’ and Brown, 1998) and unique arrangements distinguish subgroups corals (Fukami et al., 2007) and the extended 30-end found of the cox1 within nematodes, mollusks (reviewed in Boore and Brown (1998)) gene of Euphyllia (Lin et al., 2011). and cnidarians (Hexacorallia; Beagley et al., 1998; Medina et al., To clarify the phylogenetic status of Madrepora, the complete 2006; Brugler and France, 2007). In terms of mt gene order, many mt genome of M. oculata was sequenced, revealing a novel gene scleractinians conform to a consensus ﬁrst reported in rearrangement that constitutes only the second known deviation Acropora tenuis (van Oppen et al., 2002;seeFig. 2A upper). Within from the scleractinian consensus. This pattern of mt genome the Scleractinia, only one example of apparent gene reorganization organization appears to be common across Madrepora, but is is known – the case of Lophelia pertusa (Emblem et al., 2011; see not shared by genera that are closely related on the basis of Fig. 2A lower) – but a number of minor variations have also been cox1 data (i.e. pocilloporids and some caryophyllids) or other

Fig. 1. Madrepora oculata branch fragment and calicular views of the specimen used for determination of the complete sequence of the mitochondrial genome, and the sampling locations examined in this study. Collection sites are indicated by solid black squares. M.-F. Lin et al. / Molecular Phylogenetics and Evolution 65 (2012) 323–328 325 members of Oculinidae, the Family in which Madrepora is classi- 94 °C, 45 s at 52 °C, and 1.5 min at 68 °C, followed by a ﬁnal exten- cally placed. Madrepora apparently represents a distinct lineage, sion step at 68 °C for 3 min. PCR products from the gene rearrange- but whether it merits family status is as yet unclear. ment survey and cox1 region from each sample were directly sequenced using the same method described above.

2. Materials and methods 2.3. Mitochondrial genome annotation 2.1. Coral sample collections Nucleotide sequences were verified and assembled using The M. oculata colony fragment used for complete mt genome SeqManII (DNAstar vers. 5.0) and then analyzed in Vector NTI vers. sequencing (Fig. 1 upper, the colony fragment and the corallite) 9.0 (InforMax, Invitrogen Life Science). Open reading frames (ORFs) was collected from a deep-sea expedition in the South China Sea of considerable length (>50 amino acids) were initially translated (21°3502400N, 117°1702400E, at 353 m in depth) by an otter trawl of using a cnidarian mt genetic code (NCBI translation Table 4) and the research vessel Fisheries Researcher I, of the Taiwan Fishery Re- compared to sequences in the GenBank database using BLAST X search Institute (TFRI), Council of Agriculture. Pacific M. oculata (Gish and States, 1993). Alignment of identical putative ORFs and specimens were collected in New Caledonian waters (23°010S, rRNA genes was performed using MEGA vers. 4.0 (Tamura et al., 168°170E, at 550 m in depth) during the Norfolk 2 expedition 2007) with a weighted matrix of Clustal W (Thompson et al., (sta. DW 2142), and off Katsuura, Chiba, Japan. The Indian Ocean 1994). Transfer (t)RNAs structures were predicted by tRNAscan- specimen was collected near Western Australia (between SE search server vers. 1.21 (Lowe and Eddy, 1997). The 31°5705400S, 115°0601800E at 928 m and 31°5605900S, 115°0700500E, arrangement of the protein-coding gene, tRNAs, rRNA, and at 1170 m in depth) (Fig. 1). A colony fragment of M. minutiseptum intergenic spacers was illustrated using Vector NTI vers. 9.0 examined herein was also collected in Japanese waters, near le- (InforMax, Invitrogen Life Science). channel. All samples were preserved in CHAOS solution as described in Fukami et al. (2004). Skeleton vouchers were deposited 2.4. Phylogenetic analyses in the National Museum of Natural Science (NMNS), Taichung, Tai- wan; Chiba Museum (CMNH), Chiba, Japan; and Muséum National Based on the final cox1 alignment (595 bp), the most appropri- d’Histoire Naturelle (MNHN), Paris, France. ate model of nucleotide substitution was estimated by the hierar- chical likelihood ratio test implemented in MrModeltest (Nylander, 2004) as GTR + G + I. Phylogenetic reconstructions were performed 2.2. DNA extraction, polymerase chain reaction (PCR) amplification, using Garli vers. 1.0 (Zwickl, 2006) for maximum likelihood (ML) and sequencing and Mr. Bayes vers. 3.1.2 (Huelsenbeck and Ronquist, 2001) for Bayesian inference (BAY). The ML analysis was performed with Genomic DNA was extracted following Chen et al. (2002). Three 100 bootstrap replicates, and BAY consisted of two runs each of fragments with a size range of 3–9 kb, covering the entire mt gen- 106 generations with topologies saved each 100 generations. The ome of M. oculata were amplified using a long-PCR technique first 2000 saved topologies were discarded as burnin, and the (Cheng et al., 1994), with primers designed (Supplementary mate- remaining were used to calculate posterior probabilities. rial) from specific sequences of M. oculata obtained using the large- (rnl) (Romano and Palumbi, 1997), small-subunit ribosomal (r)RNA (rns)(Chen and Yu, 2000), and cytochrome c oxidase subunit I 3. Results and discussion (cox1)(Folmer et al., 1994; Lin et al., 2011) primers, respectively. The first two pairs of long-PCR primers were designed to target se- As indicated above, despite its current classification, molecular quences from the rns to the cox1 genes (F4P and R4P primers), and phylogenetics implies that M. oculata may be more-closely related from the cox1 to the rnl genes (F5P and R5P). These primers ampli- to pocilloporids and some caryophylliids than to other oculinids fied around 3 and 5 kb, respectively. The third primer set (F1M and (Le Goff-Vitry et al., 2004; Kitahara et al., 2010; Stolarski et al., R1M) covered the remaining mt genome of M. oculata. Long-PCRs 2011). In an attempt to clarify its evolutionary position, the com- were set up in a volume of 50 ll, containing 10 LA PCR buffer, plete mt genome sequence of M. oculata was determined. The

2.5 mM MgCl2, 2.5 mM of each dNTP, 2.5 units of TaKaRa La Taq™, 15,841 bp mt genome of M. oculata has the same gene content as 0.5 lM of each primer, and approximately 0.5 lg of genomic DNA. other scleractinians; a total of 17 genes of which the nad5 is inter- Long-PCR conditions were as follows: an initial denaturation step rupted by a group I intron of 10,137 bp that contains 11 genes of 94 °C for 1 min, then 30 cycles of 10 s at 98 °C, 45 s at 62– (Fig. 2A, type 2). As in most hexacorallians (see Chen et al., 2008b), 63 °C, and 14.25 min at 68 °C; followed by 10 min at 72 °C. PCR the 13 protein-coding genes, two rRNAs, and two tRNAs are all tran- products were cloned using a TOPO XL PCR cloning kit (Invitrogen, scribed from the same strand. The sense strand of the mt genome of USA) following the manufacturer’s protocol. Nucleotide sequences M. oculata is composed of 22.71% A, 10.85% C, 19.68% G, and 46.75% were determined for complementary strains from two to six clones T. The overall (A + T) content (69.46%) content is typical of ‘‘robust’’ of each PCR product by an ABI 377 Genetic Analyzer (Applied Bio- scleractinians, and very similar to values reported for pocilloporids systems, USA). (68.3–70.1%; Chen et al., 2008b). However, the (A + T) content varied The F4P and R4P primers were also used for a gene rearrange- considerably across the mt genome (Table 1), ranging from 56.3% in ment survey among caryophylliid representatives. However, spe- the tRNA (methionine) (trnM) to 78.8% in ATP synthase subunit 6 cific primers (FG3 and RG3) targeting cox2- and cox3-coding (atp6). genes (1.5 kb) were designed to assess the gene rearrangement The protein-coding genes in the mt genome of M. oculata com- in M. minutiseptum (Supplementary material). prised 3888 codons, and the amino acid usage was not significantly In those Madrepora specimens for which a complete mt genome different to other scleractinians for which data are available was not sequenced, the partial cox1 gene was amplified using uni- (p > 0.05, Mann–Whitney test). The most frequently used codons versal primers (Folmer et al., 1994). were UUU (446), UUA (333), and GUU (282), most likely as a PCRs were performed in a final volume of 50 ll following consequence of the high (A + T) content, and CGC (1), UGC (2), instructions of the Advantage 2 PCR kit (Clontech, USA). Cycling and ACC (2) were the least used codons. Nine of the 13 protein cod- conditions consisted of 1 min at 94 °C, then 35 cycles of 30 s at ing genes used methionine (ATG) as the translation initiation 326 M.-F. Lin et al. / Molecular Phylogenetics and Evolution 65 (2012) 323–328

Fig. 2. Linearized scleractinian mitochondrial gene order and phylogenetic tree. (A) Typical scleractinian mt genome organization (type I), compared to that in Madrepora (type II) and Lophelia pertusa (type III). The nad5 intron is indicated by a thick black bar, and the genes involved in rearrangement events are shaded gray. The arrows indicate the transcriptional orientation of the mt genomes. (B) Phylogenetic tree estimated by the maximum-likelihood (ML) analysis and Bayesian inference of the partial cox1 gene. Numbers at the nodes correspond to the values for ML and Bayesian posterior probabilities, respectively. M.-F. Lin et al. / Molecular Phylogenetics and Evolution 65 (2012) 323–328 327

Table 1 Mitochondrial genome organization of Madrpora oculata (15,841 bp).

Gene region Position Length (bp) AT (%) Start/ stop codon IGS length (bp)a IGS (A + T)-content (%) trnM 1–71 71 56.3 0 rnl 72–2069 1998 70.9 0 nd5(50) 2070–2780 711 70.5 AUG 111 73.9, group I intron nd1 2892–3839 948 67.5 AUG/UAA 24 95.8, igs I cob 3864–5004 1140 70.4 AUG/UAA 220 74.1, igs2 nd2 5224–6315 1092 70.3 AUU/UAA 0 nd6 6316–6882 567 72.8 AUG/UAA 37 78.4, igs3 atp6 6920–7600 681 72.7 AUG/UAG 1 nd4 7600–9045 1446 69.2 GUG/UAG 0 rns 9046–10,208 1163 68.7 0 cox2 10,209–11,000 792 67.3 AUG/UAA 165 81.8, igs4 cox3 11,166–11,945 780 66.5 AUG/UAA 176 64.2, igs5 nd4l 12,122–12,421 300 72.3 GUG/UAG 1 nd3 12,421–12,765 345 70.4 GUG/UAA 155 63.9, igs6 nd5(30) 12,920–14,023 1104 71.6 GUA 1 trnW 14,023–14,092 70 60 2 atp8 14,095–14,292 198 78.8 AUG/UAA 1 cox1 14,292–12 1560 65.6 AUG/UAG 10

a Nucleotide sequence of the overlapping genes is indicated by the negative IGS value.

codon, while nad2 used isoleucine (ATT), and nad3, nad4, and nad4l the same gene rearrangement, suggesting that mt gene order used valine (GTG). As in other cnidarians, all of the protein-coding may be common across the genus. genes had complete (TAA or TAG) stop codons, of which, in M. Phylogenetic analysis based on the protein-coding gene cox1 oculata, the former was more frequently used; this appears to be (Fig. 2B) confirmed both that Madrepora is a distinct lineage and the most common condition among scleractinians (see Flot and Til- the relationship to pocilloporids and some caryophylliids implied lier, 2007). by previous analyses (Fukami et al., 2008; Kitahara et al., 2010). In general, the protein-coding genes of M. oculata were more Although the status of the three other Madrepora species (M. similar in size to those ‘‘robust’’ scleractinians than to those of arbuscula, M. carolina, and M. porcellana) has yet to be examined, members of the ‘‘basal’’ or ‘‘complex’’ scleractinian clades (Chen a logical next step is to investigate mt gene order in Caryophyllia et al., 2008b; Kitahara et al., unpublished data). However, the grayi, C. atlantica, and Dasmosmilia lymani, caryophylliids that small-subunit rRNA gene (rns) was an exception to this general grouped with Madrepora in molecular phylogenetics. Note that rule; at 1163 bp the M. oculata rns was closer in length to its coun- complete mt genome sequences are available for a number of terparts in ‘‘complex’’ corals. Consistent with the hypothesis that pocilloporids (Chen et al., 2008a,b) and in each case these have the mt genomes of ‘‘robust’’ scleractinians are the smallest among the canonical scleractinian gene order. Results of PCR experiments hexacorallians (Lin et al., unpublished data), only six intergenic indicate that each of these caryophylliids has the canonical cox2– spacers (igs) are present in the mt genome of M. oculata, of which cox3 gene arrangement rather than that seen in Madrepora igs2 (between cob and nad2) is the largest, extending for 220 bp. No (Fig. 2A, type I), supporting the hypothesis that this atypical gene spacers are present between: trnM–rnl, rnl–nad5 (50), nad2–nad6, order is restricted to the genus Madrepora. nad4–rns, and rns–cox2. There are four cases of single base overlaps Preliminary sequence comparison of partial cox2 (614 bp) and between genes (atp6–nad4, nad4l–nad3, nad5 (30)–trnW, and atp8– cox3 (655 bp) datasets demonstrated high nucleotide similarity cox1), and cox1–trnM overlap by 10 bp (Table 1). (97.7% for cox2 and 97.8% for cox3) between M. oculata and M. min- The mt genome of M. oculata is so far unique amongst sclerac- utiseptum. However, the intergenic spacer between these genes tinians in that a gene rearrangement affects the order (but not (igs4) was 42 bp longer in the latter than in M. oculata. Sequence the orientation) of cox2 and cox3. In the case of M. oculata gene or- alignment (Supplementary material) indicates that the M. oculata der is (50)rns–cox2–cox3(30), whereas the typical scleractinian gene igs4 contains a small tandem repeat (2.9 copies of a 19 nt repeat) order is (50)rns–cox3–cox2(30)(Fig. 2A, type II). The only other that was not present in M. minutiseptum. Tandem repeats have known exception to the scleractinian consensus mt gene order is been indicated in mt gene rearrangement in reptiles (Macey the case of Lophelia pertusa, another deep-water colonial species, et al., 1998), sea cucumbers (Arndt and Smith, 1998) and lungless in which the gene order is very different (Emblem et al., 2011). L. salamanders (Mueller and Boore, 2005). Unlike the Lophelia mt pertusa has the canonical cox3–cox2 organization, but a block of genome in which gene rearrangement directly reflects the duplica- three genes ( cob, nad2, and nad6) has been shifted in position tion-random loss model (Emblem et al., 2011), no duplicated seg- (Fig. 2A, type III), most likely via tandem duplication and random ments (e.g., coding sequence duplications) were found in the M. loss (Emblem et al., 2011). As such, the differences in gene order oculata mt genome. However, further analysis of Madrepora and seen in M. oculata and L. pertusa are almost certainly the results other species is required to determine if such elements played a of separate events. role in gene rearrangement in scleractinian mtDNA. In order to check if the cox2–cox3 gene rearrangement M. oculata was restricted to Taiwanese populations, gene order was inves- tigated in additional specimens collected from Japan, New 4. Conclusions Caledonia, and western Australia (Fig. 1) by the design and use of specific PCR primers (Supplementary material). These experiments Four geographically distinct Madrepora oculata isolates shared a confirmed that the gene order is uniform in all of the M. oculata novel organization of mt genes in which the order (but not the ori- individuals surveyed from both the Pacific and Indian Oceans (Sup- entation) of the cox2 and cox3 genes differ from the scleractinian plementary material). Analysis of the corresponding fragment indi- consensus. This gene rearrangement was also observed in the cates that a second Madrepora species, M. minutiseptum, also has congeneric species M. minutiseptum but not by taxa that are 328 M.-F. Lin et al. / Molecular Phylogenetics and Evolution 65 (2012) 323–328 closely-related on the basis of cox1 sequence analysis (pocillopor- Chen, C., Dai, C.-F., Plathong, S., Chiou, C.-Y., Chen, C.A., 2008b. The complete ids and caryophylliids), nor by members of the Oculinidae (the mitochondrial genomes of needle corals, Seriatopora spp. (Scleractinia: Pocilloporidae): an idiosyncratic atp8, duplicated trnW gene, and Family within which Madrepora is traditionally placed). In molecu- hypervariable regions used to determine species phylogenies and recently lar analyses, the Oculinidae are polyphyletic, and Madrepora diverged populations. Mol. Phylogenet. Evol. 46, 19–33. constitutes a distinct lineage. Whilst these data suggest that Chen, C.A., Wallace, C.C., Wolstenholme, J., 2002. 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