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. 2014 Apr 24;6(5):1086–1095. doi: 10.1093/gbe/evu084

Mitochondrial Genome Rearrangements in the Scleractinia/Corallimorpharia Complex: Implications for Coral Phylogeny

Mei-Fang Lin 1,2,3,†, Marcelo Visentini Kitahara 4,†, Haiwei Luo 5, Dianne Tracey 6, Jonathan Geller 7, Hironobu Fukami 8, David John Miller 2,3,*, Chaolun Allen Chen 1,9,10,*
PMCID: PMC4040992  PMID: 24769753

Abstract

Corallimorpharia is a small Order of skeleton-less animals that is closely related to the reef-building corals (Scleractinia) and of fundamental interest in the context of understanding the potential impacts of climate change in the future on coral reefs. The relationship between the nominal Orders Corallimorpharia and Scleractinia is controversial—the former is either the closest outgroup to the Scleractinia or alternatively is derived from corals via skeleton loss. This latter scenario, the “naked coral” hypothesis, is strongly supported by analyses based on mitochondrial (mt) protein sequences, whereas the former is equally strongly supported by analyses of mt nucleotide sequences. The “naked coral” hypothesis seeks to link skeleton loss in the putative ancestor of corallimorpharians with a period of elevated oceanic CO2 during the Cretaceous, leading to the idea that these skeleton-less animals may be harbingers for the fate of coral reefs under global climate change. In an attempt to better understand their evolutionary relationships, we examined mt genome organization in a representative range (12 species, representing 3 of the 4 extant families) of corallimorpharians and compared these patterns with other Hexacorallia. The most surprising finding was that mt genome organization in Corallimorphus profundus, a deep-water species that is the most scleractinian-like of all corallimorpharians on the basis of morphology, was much more similar to the common scleractinian pattern than to those of other corallimorpharians. This finding is consistent with the idea that C. profundus represents a key position in the coral <-> corallimorpharian transition.

Keywords: naked coral hypothesis, gene order, mitochondrial genome, coral evolution

Introduction

Understanding the evolutionary history of the Scleractinia and relationships between corals and other members of the anthozoan subclass Hexacorallia should enable a better understanding of how it has been influenced by climate in the past and thus enable better predictions of the likely impacts of climate change (Romano and Palumbi 1996). Of the six Orders of hexacorals, only members of the Scleractinia develop continuous external calcified skeletons (Daly et al. 2003). The Scleractinia suddenly appear in the fossil record in the middle Triassic, about 240 Ma, but the range of morphological variation seen in the Middle Triassic fossils is comparable to that of extant scleractinians (Romano and Palumbi 1996). Molecular phylogenies based on both mitochondrial (mt) and nuclear (nucl) genes imply a deeper divergence (∼300 Ma—in the Late Carboniferous) of extant scleractinians into two major clades, the “Complexa” and the “Robusta” (Romano and Palumbi 1996; Romano and Cairns 2000; Chen et al. 2002; Le Goff-Vitry et al. 2004; Fukami et al. 2008; Barbeitos et al. 2010; Kitahara, Cairns, and Miller 2010; Kitahara, Cairns, Stolarski, et al. 2010; Kitahara, Cairns, et al. 2012; Kitahara et al. 2012; Kayal et al. 2013). By adding deep-water species to existing molecular data sets and applying an appropriately calibrated molecular clock, Stolarski et al. (2011) demonstrated that two exclusively deep-sea families, the Gardineriidae and Micrabaciidae, form a “basal” clade that diverged at around 425 Ma, prior to the Complexa/Robusta split, pushing the evolutionary origin of scleractinians deep into the Paleozoic. These results support the scenario that scleractinians are the descendants of soft-bodied (corallimorpharian-like) ancestors that survived the mass extinction at the Permian/Triassic boundary and subsequently gained the ability to deposit calcified skeletons (Stolarski et al. 2011).

The “naked coral” hypothesis, first put forward by Stanley and Fautin (2001) to explain the sudden appearance of diverse scleractinian fauna in the middle Triassic, is based on the idea that the skeleton has been an ephemeral trait during coral evolution. Under this hypothesis, the Scleractinia were skeleton-less in the early Triassic, a time when carbonate deposition was suppressed globally (Stanley 2003). Consistent with the idea of skeleton ephemerality, some coral species can undergo reversible skeleton loss under acid conditions (Fine and Tchernov 2007). Strong phylogenetic support for the “naked coral” hypothesis came from analyses based on the alignment of concatenated proteins encoded by 17 complete mt genomes from hexacorallians (Medina et al. 2006); in their analysis, scleractinians were paraphyletic, corallimorpharians being more closely related to the Complexa than are Robusta, the interpretation being that the Corallimorpharia arose by skeleton loss from a scleractinian ancestor at a time (during the mid-Cretaceous) of high oceanic CO2 levels (Medina et al. 2006).

Although the “naked coral” scenario is supported by analyses of protein sequence data, phylogenetics based on mt nucleotide sequences instead strongly support scleractinian monophyly (Stolarski et al. 2011; Kayal et al. 2013; Kitahara et al. 2014). The fundamental disagreement between phylogenies based on nucleotide (fig. 1A) or amino acid (fig. 1B) sequence data for mt proteins stems from the fact that none of the available models for sequence evolution adequately account for the observed data (Kitahara et al. 2014). One possible explanation for this is the occurrence of a “catastrophic” event—a major and unpredictable change, such as sudden impairment of mt DNA repair processes (which are believed to be an ancestral trait within Anthozoa (Pont-Kingdon et al. 1998; Shearer et al. 2002; Brockman and McFadden 2012).

Fig. 1.—

Fig. 1.—

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Alternative phylogenetic hypotheses for relationships between Scleractinia and Corallimorpharia based on mt genome nucleotide sequences (A) or the amino acid sequences of the proteins that they encode (B). The trees were modified from Kitahara et al. (2014). Note that, for both (A) and (B) scenarios, support for the node separating Corallimorpharia from Scleractinia (the root of the gray part of the tree) was over 97% under both maximum-likelihood analysis and Bayesian inference.

Given the intractability of coral/corallimorph relationships using conventional molecular phylogenetics, we explored the informativeness of mt genome architecture in this context. mt gene rearrangements occur relatively infrequently and have proven useful in resolving evolutionary relationships, both shallow and deep, across a broad range of organisms (e.g., Gai et al. 2008; Brockman and McFadden 2012; Kilpert et al. 2012). This study is based on the complete mt genomes of a total of 12 corallimorpharians (8 of which are novel), representing 3 of 4 currently described families (Daly et al. 2007; Fautin et al. 2007), and 32 scleractinians, and includes both the early diverging coral Gardineria hawaiiensis (Stolarski et al. 2011), and corallimorpharian, Corallimorphus profundus, which is considered to be the most coral-like of corallimorpharians based on morphological grounds (Moseley 1877; den Hartog 1980; Riemann-Zürneck and Iken 2003). The results indicate that, by contrast with the Scleractinia, extensive rearrangements of the mt genome have occurred within Corallimorpharia. The most surprising finding, however, was that the mt genome of C. profundus is scleractinian-like, and is organized very differently to those of all other corallimorpharians for which data are available. Both nucleotide and amino acid sequenced-based phylogenetics unequivocally place C. profundus as an early diverging corallimorpharian, indicating that this organism most closely reflects the coral <-> corallimorpharian transition.

Materials and Methods

DNA Extraction, Polymerase Chain Reaction, Long Polymerase Chain Reaction, Cloning, and Sequencing

Genomic DNA was extracted from corallimorpharian samples that had been preserved in 95% (V/W) ethanol following Chen et al. (2002)—sampling information is summarized in table 1. Long-range polymerase chain reaction (L-PCR; Cheng et al. 1994) was used to amplify large (6–9 kb) and overlapping fragments covering the entire mt genomes of corallimorpharians and corals. For each species, either two- or three-specific primer pairs were designed on the basis of previously available partial sequence data for of rns, rnl, and COI (Folmer et al. 1994; Romano and Palumbi 1997; Chen and Yu 2000; Lin et al. 2011) (supplementary table S1, Supplementary Material online). Reactions were set up in a total volume of 50 μl: 10× LA PCR buffer, 2.5 mM MgCl2, 2.5 mM of each dNTP, 2.5 units of TaKaRa La Taq, 0.5 μm of each primer, and approximately 0.5 μg of genomic DNA. The L-PCR conditions were slightly modified from those recommended by the polymerase manufacturer as follows: 94 °C for 1 min, then 30 cycles of 10 s at 98 °C, 45 s at 62–63 °C, 14.25 min at 68 °C, and 10 min at 72 °C. PCR products were recovered from the agarose gel using the TOPO XL gel purification method, cloned into a pCR-XL-PCR vector system using topoisomerase I (Invitrogen), and transformed into Escherichia coli (Top10) by electroporation. The nucleotide sequences were determined for complementary strains of two to six clones from each sample using primer walking on the same PCR product by an ABI 377 Genetic Analyzer (Applied Biosystems). The M13 forward and reverse primers were used to obtain the initial sequences from the ends of each insertion. The consensus sequences from three sequenced clones were present for each species.

Table 1.

Characteristics of the mt Genomes of Corallimorpharians, Scleractinians, and Other Anthozoans

Order Scleractinian Clades Species Total Length (bp) Nucleotide (%) Gene Size (bp) Species Collection Site and GenBank No.
A + T C + G atp6 atp8 cob COI COI intron COII COIII nd1 nd2 nd3 nd4 nd4l nd5 nd6 rnl rns trnM trnW IGS (length)
Corallimorpharia Actinodiscus nummiformis 20,922 60.9 39 699 210 1,161 1,581 1,209 756 789 984 1,098 357 1,476 300 1,839 612 2,350 1,304 71 70 3,032 Wanlitung, Taiwan
Amplexidiscus fenestrafer 20,188 61 39 699 210 1,161 1,561 1,207 756 789 984 1,098 357 1,476 300 1,839 612 2,349 1,304 71 70 2,729 Taioshi, Taiwan
Corallimorphus profundusa 20,488 60.3 39.6 699 237 1,149 1,734 1,183 744 789 984 1,098 357 1,521 300 1,839 630 2,434 1,253 71 70 3,396 Southern Ocean
Corynactis californicaa 20,632 60.2 39.8 699 219 1,179 1,602 1,266 765 789 984 1,098 357 1,479 300 1,839 612 2,552 1,256 71 69 2,906 California, USA
Discosoma sp. 1 20,908 61 38.9 699 210 1,161 1,581 1,208 756 789 984 1,098 357 1,476 300 1,839 615 2,340 1,224 71 70 4,284 NC_008071
Discosoma sp. 2 20,912 61 38.9 699 210 1,161 1,581 1,207 756 789 984 1,098 357 1,476 300 1,839 615 2,342 1,068 71 70 4,289 NC_008072
Pseudocorynactis sp. 21,239 60.9 39 699 213 1,230 1,575 1,178 756 789 984 1,098 357 1,476 300 1,839 612 2,537 1,223 71 70 3,177 Birch Aquarium at SIO
Rhodactis indosinesis 20,092 60.9 39.1 699 210 1,161 1,581 1,205 756 789 984 1,098 357 1,476 300 1,839 612 2,350 1,303 71 71 2,624 Wanlitung, Taiwan
R. mussoides 20,826 61 39 699 210 1,161 1,581 1,207 756 789 984 1,098 357 1,476 300 1,839 615 2,355 1,304 71 70 3,007 Taioshi, Taiwan
Rhodactis sp. 20,093 61 39 699 210 1,161 1,581 1,207 756 789 984 1,098 357 1,413 300 1,839 612 2,348 1,240 71 70 3,358 NC_008158
Ricordea florida 21,376 62.1 37.9 699 210 1,140 1,623 1,180 756 789 987 1,098 357 1,476 300 1,839 606 2,447 1,218 71 70 4,510 NC_008159
Ri. yuma 22,015 62.4 37.6 699 213 1,140 1,599 1,199 756 789 984 1,098 357 1,476 300 1,839 606 2,444 1,262 71 70 5,148 Wanlitung, Taiwan
Scleractinia Basal Gardineria hawaiiensisa 19,429 60.3 39.7 699 264 1,197 1,584 1,136 738 789 981 1,098 357 1,452 300 1,836 615 2,400 1,159 71 70 2,278 New Caledonia
Complex Acropora tenuis 18,338 62.1 37.9 699 219 1,155 1,602 744 780 984 1,098 357 1,476 300 1,836 594 2,261 1,176 71 70 3,615 NC_003522
Agaricia humilis 18,735 59.6 40.4 699 196 1,152 1,581 744 789 984 1,098 357 1,479 300 1,836 594 1,577 1,136 70 69 4,773 NC_008160
Alveopora sp. 18,146 62.2 37.8 699 237 1,158 1,602 744 789 984 1,098 357 1,476 300 1,836 594 2,261 1,125 71 70 3,444 KJ634271
Anacropora matthai 17,888 61.6 38.4 699 219 1,158 1,602 744 789 984 1,098 357 1,476 300 1,836 594 2,261 1,174 71 70 3,155 NC_006898
Astreopora explanata 18,106 62.2 37.8 699 219 1,146 1,587 744 789 984 1,098 357 1,476 300 1,836 594 2,243 1,176 71 70 3,416 KJ634269
Astreopora myriophthalma 18,106 62.1 37.8 699 219 1,146 1,587 744 789 984 1,098 357 1,476 300 1,836 594 2,244 1,176 71 70 3,415 KJ634272
Euphyllia ancora 18,875 62.3 37.8 699 219 1,155 2,301 744 789 984 1,098 357 1,476 300 1,863 594 2,308 1,177 71 70 3,369 NC_015641
Fungiacyathus stephanusa 19,381 62.2 37.8 699 231 1,161 1,629 962 744 789 984 1,098 357 1,476 300 1,839 594 2,366 1,114 71 70 3,596 NC_015640
Goniopora columna 18,766 62.9 37.1 699 216 1,164 1,652 947 744 789 984 1,098 357 1,476 300 1,836 594 2,227 1,029 69 70 3,214 NC_015643
Isopora palifera 18,725 61.7 38.2 699 219 1,158 1,602 744 789 984 1,098 357 1,476 300 1,836 594 2,259 1,175 71 70 3,993 KJ634270
Isopora togianensis 18,637 61.8 38.2 699 219 1,158 1,602 744 789 984 1,098 357 1,476 300 1,836 594 2,259 1,177 71 70 3,903 KJ634268
Montipora cactus 17,887 61.6 38.4 699 219 1,158 1,602 744 789 984 1,098 357 1,476 300 1,836 594 2,266 1,172 71 70 3,151 NC_006902
Pavona clavus 18,315 59.5 40.5 699 219 1,152 1,581 744 789 984 1,098 357 1,476 300 1,836 606 2,299 1,169 70 69 3,566 NC_008165
Porites okinawanesis 18,647 63.8 36.2 699 216 1,161 1,531 966 744 789 984 1,098 357 1,476 300 1,836 594 2,301 1,029 71 70 3,124 NC_15644
Porites porites 18,648 63.7 36.2 699 216 1,161 1,578 966 744 789 984 1,098 357 1,476 300 1,836 594 2,271 1,060 71 70 3,077 NC_008166
Siderastrea radians 19,387 63.1 36.9 699 234 1,155 1,584 989 744 789 984 1,098 357 1,476 300 1,836 594 2,242 1,296 71 70 3,568 NC_008167
Robust Astrangia sp. 14,853 68.1 31.9 678 198 1,140 1,551 685 780 948 1,092 342 1,440 300 1,812 561 1,178 532 72 70 1,474 NC_008161
Colpophyllia natans 16,906 66.4 33.5 678 198 1,140 1,566 685 780 948 1,104 342 1,440 300 1,815 561 1,885 1,012 72 70 2,310 NC_008162
Lophelia pertusaa 16,150 65.1 34.9 699 159 1,161 1,566 618 780 948 1,092 345 1,446 300 1,836 507 1,829 907 71 70 1,816 NC_015143
Madracis mirabilis 16,951 68.4 31.7 678 224 1,140 1,587 759 780 978 1,092 345 1,446 300 1,815 564 1,937 910 71 70 2,255 NC_011160
Madrepora oculataa 15,839 69.6 30.3 681 198 1,140 1,560 792 780 948 1,092 345 1,446 300 1,815 567 1,998 1,163 71 70 873 JX_236041
Montastraea annularis 16,138 66.4 33.5 678 198 1,140 1,578 708 780 948 1,287 342 1,440 300 1,815 561 1,973 903 73 69 1,345 NC_007224
Montastraea faveolata 16,138 66.4 33.6 678 198 1,140 1,578 708 780 948 1,287 342 1,440 300 1,815 561 1,973 903 72 69 1,346 NC_007226
Montastraea franksi 16,137 66.4 33.6 678 198 1,140 1,578 708 780 948 1,287 342 1,440 300 1,815 561 1,973 903 72 69 1,345 NC_007225
Mussa angulosa 17,245 66.3 33.7 678 198 1,140 1,575 685 780 948 1,104 342 1,440 300 1,815 561 550 695 72 70 4,292 NC_008163
Pocillopora eydouxi 17,422 69.8 30.1 678 213 1,140 1,550 801 780 978 1,308 345 1,491 300 1,839 564 1,917 909 71 70 2,468 NC_009798
Polycyathus chaishanensis 15,357 70.9 29.1 678 198 1,140 1,574 708 780 948 1,092 342 1,440 300 1,812 561 1,893 905 72 70 844 NC_015642
Seriatopora caliendrum 17,010 69.7 30.3 678 237 1,140 1,548 759 780 978 1,092 345 1,446 300 1,839 564 1,902 916 71 70 2,345 NC_010245
S. hystrix 17,059 69.9 30.2 678 237 1,140 1,548 759 780 978 1,092 345 1,446 300 1,839 564 1,904 916 71 70 2,392 NC_010244
Stylophora pistilata 17,177 70.2 29.9 678 249 1,140 1,548 837 780 978 1,092 345 1,446 300 1,839 564 1,936 914 71 70 2,390 NC_011162
Other Anthozoa Chrysopathes formosa 18,398 60.5 39.6 714 213 1,143 1,593 750 750 984 1,146 357 1,476 300 1,851 633 2,588 1,168 71 70 2,591 NC_008411
Savalia savaglia 20,764 51.7 48.3 699 219 1,161 1,521 1,239 753 789 990 1,158 357 1,515 300 1,848 666 2,644 1,197 71 3,637 NC_008827
Nematostella sp.a 16,389 60.9 39.1 699 231 1,179 1,587 744 789 984 1,110 357 1,476 300 1,816 600 602 693 71 70 3,081 NC_008164
Metridium senilea 17,443 61.8 38.1 690 219 1,182 1,593 853 747 789 1,005 1,158 357 1,476 300 1,803 609 2,189 1,082 71 70 2,103 NC_000933
Briareum asbestinum 18,632 62.9 37.1 708 218 1,143 1,582 762 786 972 1,164 354 1,449 294 1,818 558 2,224 581 71 882 DQ_640649
Pseudopterogorgia bipinnata 18,733 62.7 37.3 708 219 1,144 1,597 762 786 972 1,093 354 1,449 294 1,818 558 2,211 924 71 815 DQ_640646
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Note.—Sources of publically available data and collection sites in the case new sequences are also listed. IGS here refers to the total length (bp) of IGSs in each of the mt genomes. Data for the octocoral mtMuts gene are not included. NC_008158 rns gene is 1,239 bp based on the analyses in this study.

aAzooxanthellate species.

Genome Annotation and Sequence Analysis

Sequences were verified and assembled using SeqManII (DNAstar v5.0) or Sequencher v4.8 (Gene Codes Corporation) and then analyzed in Vector NTI v9.0 (InforMax). Open-reading frames (ORFs) of length more than 50 (amino acids) were translated using National Center for Biotechnology Information translation table 4 and compared with the databases using BlastX (Gish and States 1993). No novel ORFs were identified on this basis. MEGA v5.0 (Tamura et al. 2011) with a weighted matrix of Clustal W (Thompson et al. 1994) was used to align the identical putative ORFs and rRNA genes with previously published data. The 5′- and 3′-ends of the rRNA genes were predicted using the program SINA on the Silva ribosomal RNA database site (www.arb-silva.de/, last accessed February 1, 2014) using the default settings (Pruesse et al. 2012). tRNAs were predicted using tRNAscan-SE search server v1.21 (Lowe and Eddy 1997). rRNA loci were identified on the basis of sequence similarity. Finally, Vector NTI v9.0 was used to generate maps of the mt genomes based on the assembled sequence data.

Gene Order Phylogeny

The double cut and join (DCJ) distance metric (Yancopoulos et al. 2005), implemented in GRAPPA (Moret et al. 2002; Zhang et al. 2009), was used to calculate the pairwise DCJ and breakpoint distances (BPDs) from the gene order data and to generate pairwise distance matrixes. Gene order phylogenies (DCJ and BPD) were estimated with FastME (Desper and Gascuel 2002).

Because gene order is a single character with multiple states (Shi et al. 2010), bootstrapping is not applicable, hence the reliability of each branch was estimated by applying a jackknife resampling technique that in each iteration randomly removed 25% of the initial orthologous gene sets. Note that, because the data set consisted of only 13 protein-coding genes, higher removal rates (e.g., 50%) are unable to resolve the tree branching order. Jackknifing was used to generate 1,000 matrices, which were imported into FastME and used to obtain 1,000 DCJ- and BPD-based trees. Finally, the CONSENSE program in the PHYLIP software package (Felsenstein 1989) was used to calculate majority-rule consensus trees with percent values at each node. Each value represents the percentage of trees supporting a clade defined by a node.

Results

Characteristics of mt Genomes of Corallimorpharians and Gardineria hawaiiensis

The molecular characteristics of the mt genomes of a representative range (8) of corallimorpharians and the “basal” scleractinian G. hawaiiensis are summarized in table 1, along with the publically available data for hexacorallians (42 species). All the corallimorpharian and scleractinian mt genomes, both those determined in this study and previous work, encode 13 protein-coding genes, 2 tRNA genes (trnM and trnW; but note that Seriatopora spp. have a duplicated trnW), the small (rns) and large (rnl) subunit ribosomal DNA genes, and a COI group I intron. Corallimorpharian mt genomes range in size from 20,093 bp in Rhodactis sp. to 22,015 bp in Ricordea yuma and are significantly larger than those of both Complexa and Robusta corals due not only to the presence of COI group I intron (table 1) but also to differences in size of the intergenic spacers (IGSs) between the three lineages (supplementary fig. S1, Supplementary Material online). In fact, the mt genome architectures of the Corallimorpharia are less dense than those of Scleractinia; mt genome size correlates with the total size of the IGS (r2 = 0.5371, P < 0.001; supplementary fig. S2, Supplementary Material online). Corallimorpharian mt genomes are characterized by the genes being discrete (i.e., nonoverlapping), whereas this is quite rare in the Scleractinia, where this in shown by only 2 (the complex corals, Siderastrea sp. and Fungiacyathus stephanus) of the 29 species for which data are available.

The mt genomes of scleractinians are smaller than those of corallimorpharians, but the size (19,429 bp) reported here for that of G. hawaiiensis is the largest known for a scleractinian. Two cases of gene overlap were observed in the G. hawaiiensis mt genome; ND4 and rns loci overlap by 1 bp, and ATP8 and COI overlap by 18 bp.

Gene Order and Rearrangements

The organization of the mt genomes of hexacorallian anthozoans is summarized as linear maps in figure 2 and potential rearrangement mechanisms discussed below. As in the Scleractinia, there is a canonical corallimorpharian gene arrangement (CII), but these two patterns are clearly distinct. Ten of 12 corallimorpharian mt genomes exhibited an identical gene arrangement (referred to as Type CII in fig. 3), the exceptions being those of Corynactis californica (Type CI) and C. profundus (Type CIII). In the Scleractinia, 27 of the 29 complete mt genomes have identical gene order, but again two cases of rearrangement are known (fig. 2). However, although noncanonical gene arrangements have been observed in both Corallimorpharia and Scleractinia, those in the latter involve relatively small changes (i.e., can be explained by single rearrangement events), the rearrangements within Corallimorpharia are much more extensive (fig. 2). At least four rearrangement events are required for the transition between Type CII and Type CI, up to six rearrangement events were identified between Type CII and Type CIII. In the case of scleractinians, far fewer rearrangement events can explain the two deviations from the canonical pattern (Type SII), which G. hawaiiensis shares with most of the Scleractinia. Madrepora oculata (Type SIII) differs from the SII pattern only in having the order of the COII–COIII genes changed, whereas in Lophelia pertusa (Type SI), a block of genes (COB-ND2-ND6) has been rearranged (Type SI). The most surprising finding was that, in terms of gene organization, the mt genome of the deep sea corallimorph C. profundus (Type CIII) was more similar to the canonical scleractinian organization (Type SII) than it was to other corallimorpharians. Only two rearrangements of blocks of genes are required to explain the SII–CIII transition (fig. 2). Thus, although Corallimorphus is unquestionably a corallimorpharian in terms of the sequences of mt genes, the organization of those genes is scleractinian-like, implying that it might represent a key transitional state.

Fig. 2.—

Fig. 2.—

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Linear maps showing mt genome architecture in Corallimorpharia, Scleractinia, and other members of the anthozoan subclass Hexacorallia. Names of each Order are indicated in bold. The arrow indicates the direction of transcription. The positions of the 5′- and 3′-ends of the ND5 intron are indicated by black squares. Corresponding blocks of genes are marked with color; for clarity, lines showing how genes or gene blocks differ in organization between the mt genomes are shown for only the Scleractinia. Note the relatively small number of rearrangements required to account for genome organization between the scleractinians and Corallimorphus compared with the large number of rearrangements that appear to have occurred in the corallimorpharians.

Fig. 3.—

Fig. 3.—

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mt gene order phylogeny of anthozoans. The trees shown are majority-rule cladograms generated using the CONSENSE program in PHYLIP (Felsenstein 1989). The numbers shown at the nodes indicate the percentages of 1,000 jackknife analyses supporting the topology shown in breakpoint and DCJ analyses, respectively. Numbers of species exhibiting the gene arrangement shown are indicated in parentheses. (A) Gene order phylogeny with Lophelia included. (B) Gene order phylogeny with Lophelia excluded. Note the weak support for the Lophelia/Corallimorphus clade in (A).

Among metazoans, one unique characteristic of the mt genomes of hexacorallians is the presence of a self-splicing intron within the ND5 gene that contains a number of complete genes. In the case of the Zoanthidea, Antipatharia, and Actiniaria for which data are available, only two genes, ND1 and ND3, are contained in the ND5 intron, whereas in the Type CII, all of the genes (including trnM, but excluding trnW) are contained in the ND5 intron. In the Type CI pattern, nine protein-encoding genes are located in the ND5 intron, whereas in Types CIII, SII, and SIII, the same ten protein-coding genes and rns are contained in the ND5 intron. In Type SI, the number of genes within the ND5 intron is reduced to 8 due to a rearrangement event between Type SI and these two types of mt genomes in the scleractinians (fig. 2).

Discussion

The most surprising finding of this study was that the mt genome of the deep-sea corallimorpharian, C. profundus, more closely resembles scleractinians in gene organization than it does other corallimorpharians (fig. 3A and B). Although molecular phylogenetic analyses based on nucleotide or amino acid sequence data for mt proteins yield fundamentally different results with respect to the relationship between the “complex” and “robust” scleractinian clades, there is no disagreement concerning the monophyly of the Corallimorpharia nor about the early divergence of Corallimorphus within that clade (fig. 1; Kitahara et al. 2014). On morphological grounds, Corallimorphus is also considered the most coral like of corallimorpharians (Moseley 1877; den Hartog 1980; Riemann-Zürneck and Iken 2003). Several authors (den Hartog 1980; Owens 1984; Cairns 1989, 1990; Fautin and Lowenstein 1992) have pointed out the level of similarity between Corallimorphus and members of the scleractinian family Micrabaciidae, which are characterized by a reduced skeleton, the fleshy polyp totally investing the rudimentary corallum. Molecular clock estimates imply that the micrabaciids and gardineriids diverged from the scleractinian lineage in the mid-Paleozoic, well prior to the Robusta/Complexa split (Stolarski et al. 2011). The similarity between the earliest diverging members of both the Scleractinia and Corallimorpharia in terms of both morphology and mt genome architecture (fig. 2) implies that Corallimorphus occupies a key position in the corallimorpharian <-> scleractinian transition. Corallimorphus therefore diverged either close to the point of the scleractinian/corallimorpharian divergence (under scleractinian monophyly) or at the point of skeleton loss (under the “naked coral” scenario).

If we accept that the organization of the mt genome in Corallimorphus most closely reflects the ancestral pattern (figs. 1 and 4), then extensive reorganizations are required to generate the consensus corallimorpharian architecture (CII in fig. 2) and that seen in Corynactis; in contrast, the rearrangements documented to date within Scleractinia require far fewer steps. In the case of Lophelia, the presence of a 67 bp direct repeat comprising the 3′-end of the ND1 and 5′-end of COB genes (Emblem et al. 2011) implies that the likely mechanism of reorganization was tandem duplication and random loss (Moritz et al. 1987; Zhang 2003), which may also account for the COII–COIII inversion seen in Madrepora (Lin et al. 2012). We were unable to identify signatures of duplication-mediated rearrangement in corallimorpharians; however, neither are there obvious examples of inversion of segments of the mt genome in this Order. Rather, extensive segmental reorganization without inversion has occurred within Corallimorpharia, possibly facilitated by the less compact nature of the mt genomes (reviewed in Boore and Brown 1998). This contrasts markedly with the situation in octocorals, where many successive inversion events explain the observed diversity of mt gene organization (Brockman and McFadden 2012).

Fig. 4.—

Fig. 4.—

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Hypothetical scheme for the evolution of mt genome architecture in the Scleractinia and Corallimorpharia. The scheme is based on the phylogenetic tree shown as figure 5 in Kitahara et al. (2014), with patterns of gene organization (numbered as in fig. 2) indicated in green boxes.

Can comparisons of mt genome organization resolve the question of coral monophyly? Although the data presented here are consistent with monophyly of the Scleractinia, they do not exclude the possibility of an origin for corallimorpharians within the coral clade. Phylogenetic analyses based on gene order (fig. 3A and B) were ambiguous. Although both AA- and nt-based molecular phylogenetic analyses unambiguously support monophyly of the Corallimorpharia, the gene order analysis (fig. 3A and B) did not. We interpret the grouping of Lophelia and Corallimorphus in this analysis as an artifact resulting from superficial similarities in gene organization in these two organisms; although gene order is similar, the sequences of those genes are highly divergent. The idea that the grouping of L. pertusa with C. profundus is artifactual is supported by the relatively low DCJ and BPD confidence values (58/49) associated with this node (i.e., well below the 85% confidence interval recommended by Shi et al. 2010). When L. pertusa was removed from the analysis, the overall DCJ and BPD statistic performances at the nodes of Corallimorpharia and Scleractinia increased, particularly for the node of C. profundus and Scleractinia/M. oculata, where support increased from 94/75 to 97/82 (fig. 3).

The mt genomes of the Robusta differ from both corallimorpharians and all other corals in several characteristics. First, within the larger Scleractinia/Corallimorpharia clade, the Robusta have the most compact mt genomes (size range 14,853–17,422 bp) as a consequence of having in general shorter intergenic regions and the largest number of overlapping gene pairs (three to six cases of overlaps). In contrast, corallimorpharians have the largest mt genomes (size range 20,092–22,015 bp), longer intergenic regions, and no cases of overlapping genes, with complex corals intermediate in these characteristics (genome sizes 17,887–19,387 bp; 0–2 overlapping gene pairs—most frequently a single case of overlapping genes). Second, the Robusta differ in structural comparisons of the ND5 group I intron (Emblem et al. 2011) as well as in molecular phylogenetics based on this feature. A group I intron interrupts the ND5 gene of all hexacorallians examined to date; these introns typically come and go during evolution but that in hexacorallians contains a variable number of genes and has become an essential feature. The hexacorallian ND5 intron has been “captured” in the sense that it is now dependent on host-derived factors for splicing, as indicated by the substitution of the ωG (the last nucleotide of the intron) by ωA (reviewed in Nielsen and Johansen 2009; Emblem et al. 2011). Although these characteristics are common across the coral-corallimorpharian clade, the ND5 introns of robust corals have a more compact core and overlapping intron and ND5-coding sequences (Emblem et al. 2011). In some robust corals, ωA is replaced by ωC, indicating a higher level of dependency on host factors for processing and thus greater integration of intron and host. These qualitative factors, as well as molecular phylogenetics of the ND5 intron sequences, are most parsimoniously accommodated by scleractinian monophyly (Emblem et al. 2011). Third, of the three lineages, the mt genomes of Robusta have the highest (A+T) content and most constrained codon usage, one obvious consequence of which is that phenylalanine is overrepresented in the proteins that they encode, suggesting that mt DNA repair may be reduced in the Robusta (Kitahara et al. 2014).

The features outlined above, in which the Robusta differ from complex corals and corallimorphs, are derived characteristics—they serve to resolve the robust corals but do not unambiguously identify the sister group. Scleractinian monophyly explains all of the data most parsimoniously, but the alternative cannot yet be ruled out. The mt genome has been exhaustively mined for answers, but these must likely wait for the availability of appropriate nuclear markers.

Supplementary Material

Supplementary table S1 and figures S1 and S2 are available at Genome Biology and Evolution online (http://www.gbe.oxfordjournals.org/).

Supplementary Data
supp_6_5_1086__index.html (982B, html)

Acknowledgments

The authors thank members of the Coral Reef Evolutionary Ecology and Genetics Laboratory (CREEG), Biodiversity Research Center, Academia Sinica (BRCAS), and New Zealand Institute of Water and Atmospheric Research for assistance with sampling and field logistics. We also thank Philippe Bouchet (MNHN) who generously loaned the specimen of Gardineria hawaiiensis from the Paris Museum, and Bertrand Richer de Forges and IRD- Nouméa staff and collaborators for their great effort in collecting and preserving the New Caledonian specimen examined in the present study. The corresponding authors also thank two anonymous reviewers for their comments, which were very helpful in revising the manuscript. M.-F.L. gratefully acknowledges receipt of a James Cook University Postgraduate Research Scholarship (JCUPRS). M.V.K. acknowledges the support of the São Paulo Research Foundation (FAPESP) and São Paulo University Marine Biology Centre (CEBIMar). This study was supported by grants from the National Science Council (NSC) and Academia Sinica (Thematic Grants 2005–2010) to C.A.C., and from the Australian Research Council to D.J.M. Coral samples were collected under appropriate permits. This is the CREEG, RCBAS contribution no. 108.

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