Members of the Montastraea Annularis Coral Species Complex (Cnidaria, Anthozoa, Scleractinia)
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Coral Reefs (2005) DOI 10.1007/s00338-005-0023-3 REPORT Hironobu Fukami Æ Nancy Knowlton Analysis of complete mitochondrial DNA sequences of three members of the Montastraea annularis coral species complex (Cnidaria, Anthozoa, Scleractinia) Received: 30 December 2004 / Accepted: 17 June 2005 Ó Springer-Verlag 2005 Abstract Complete mitochondrial nucleotide sequences Introduction of two individuals each of Montastraea annularis,Mon- tastraea faveolata, and Montastraea franksi were deter- Members of the Montastraea annularis complex (M. mined. Gene composition and order differed annularis, Montastraea faveolata, and Montastraea substantially from the sea anemone Metridium senile, but franksi) are dominant reef-builders in the Caribbean were identical to that of the phylogenetically distant coral whose species status has been disputed for many years genus Acropora. However, characteristics of the non- (e.g., Knowlton et al. 1992, 1997; van Veghel and Bak coding regions differed between the two scleractinian 1993, 1994; Weil and Knowlton 1994; Szmant et al. genera. Among members of the M. annularis complex, 1997; Medina et al. 1999; Fukami et al. 2004a; Levitan only 25 of 16,134 base pair positions were variable. Six- et al. 2004). The fossil record suggests that M. franksi teen of these occurred in one colony of M. franksi, which most closely resembles the morphology of the ancestral (together with additional data) indicates the existence of lineage, that M. faveolata diverged from this ancestral multiple divergent mitochondrial lineages in this species. lineage about 3–4 million years ago (mya), and that M. Overall, rates of evolution for these mitochondrial ge- annularis and M. franksi diverged much more recently nomes were extremely slow (0.03–0.04% per million years (0.5 mya; Pandolfi et al. 2002). based on the fossil record of the M. annularis complex). Genetic studies have played a central role in the At higher taxonomic levels, patterns of genetic divergence analysis of this complex (Fukami et al. 2004a and and synonymous/nonsynonymous substitutions suggest references therein), but progress has been hindered by non-neutral and unequal rates of evolution between the the poorly understood genetics of corals and anth- two lineages to which Montastraea and Acropora belong. ozoans generally, and by low levels of mitochondrial genetic variability (Medina et al. 1999; van Oppen Keywords Montastraea Æ Species et al. 1999; Shearer et al. 2002). For example, M. complex Æ Coral Æ Mitochondrion Æ Cnidaria cavernosa and the M. annularis complex have been isolated for over 30 million years (recent studies indi- Communicated by Biological Editor H.R. Lasker cate that they are not in fact congeneric; Fukami et al. 2004b), but differ by only 2.4% in partial DNA se- H. Fukami Æ N. Knowlton (&) quences of the cytochrome oxidase subunit I (cox1) Center for Marine Biodiversity and Conservation, Scripps Institution of Oceanography, (Medina et al. 1999). University of California San Diego, La Jolla, To provide a comprehensive picture of mitochondrial CA, 92093-0202 USA DNA in this species complex, we determined entire E-mail: [email protected] mitochondrial genome sequences for two individuals of Tel.: +1-858-822-2486 each of the three members of the group, thereby allow- Fax: +1-858-822-1267 ing both intra- and interspecific analyses of variation N. Knowlton and divergence within this closely related assemblage. Smithsonian Tropical Research Institute, We also compare these DNA sequences with those of Balboa Ancon Box 2072, Republic of Panama Acropora tenuis (van Oppen et al. 2002) [a distantly re- Present address: H. Fukami lated coral (Romano and Palumbi 1996, 1997; Romano Seto Marine Biological Laboratory, Field Science Education and Research Center, and Cairns 2000; Veron 2000; Chen et al. 2002)], the sea Kyoto University, Shirahama, Wakayama, anemone Metridium senile (Beagley et al. 1998), and 649-2211 Japan other cnidarians. Metridium senile (Pont-Kingdon et al. 1994; Beagley Material and methods et al. 1998). Secondary structure of tRNA was estimated using tRNAscan-SE (http://lowelab.ucsc.edu/tRNA- DNA extraction, PCR amplification, and sequencing scan-SE/). Base composition bias, numbers of transi- tions (Ts) and transversions (Tv), and pair-wise Total DNA was extracted from eggs stored in guanidine nucleotide divergences based on the Kimura-2 parame- solution (see Fukami et al. 2004a) by conventional ter model (Kimura 1980) were calculated using PAUP* phenol/chloroform extraction and ethanol precipitation 4.05 (Swofford 2002). The number of non-synonymous methods. The corals used were a subset of Panamanian substitutions per non-synonymous site (dN) and the corals previously analyzed (samples Pa01-23, M01-4, number of synonymous substitutions per synonymous Pa00-3, Pa01-5, Pa00-9, Pa01-28 of Fukami et al. site (dS) of the translated sequences were calculated 2004a). using Nei and Gojobori’s method (Nei and Gojobori Initially, mitochondrial DNA from M. annularis was 1986) in MEGA. A Z test was used to determine whether amplified by the long-PCR method using four primers: dN and dS differed significantly among M. annularis, 5¢ GAC AGA GAA ACT TTC GTG ACA CCA TTC A. tenuis and Metridium senile. Gap regions were ATA 3¢ (MTLNF3) and 5¢ GTA AGA CGA GAA GTC excluded from analyses except for calculations of base CCC ATG GAG CTT TAC 3¢ (MTLNR3) [designed composition. from partial DNA sequences of the large-subunit ribo- somal RNA (rnl)ofAcropora nasuta (Fukami et al. unpublished data)], and 5¢ GTC CCA ATT AGA CCT Results and discussion GCT CCA ACA CC 3¢ (COI-LOF) and 5¢CAA CGA TTT TCA ACA TGC GAG CCC CTGG 3¢ (COI-LOR) Overall structure of the Montastraea annularis [designed from partial DNA sequences of cox1 of A. complex mitochondrial genome nasuta (Fukami et al. unpublished data)]. Long-PCR bands (more than 5 kb) were obtained using the fol- The mitochondrial DNA of the three members of the M. lowing primer combinations: MTLNF3·MTLNR3, annularis complex is a circular molecule, as is typical of COI-LOF·MTLNF3, and COI-LOF·COI-LOR. The anthozoans but not other cnidarians (Bridge et al. 1992; PCR protocol was 10 cycles at 94°C for 15 s, 60°C for van Oppen et al. 2002). Sequences were 16,137 or 30 s, 68°C for 5 min, followed by 20 cycles at 94°C for 16,138 bp in length (Table 1), and thus slightly shorter 15 s, 60°C for 30 s, 68°C for 5 min with 20 s increments than those of the four other anthozoans whose mtDNAs in the extension times for each cycle using Taq poly- have been completely sequenced: two in the subclass merase (SIGMA). Generally, the amplified fragments Alcyonaria or Octocorallia [the soft coral Sarcophyton were purified using GeneClean (Promega) from pieces of glaucum, 18,453 bp (Beaton et al. 1998); the sea pen gel cut after observing the PCR products in a 1.0% Renilla kolikeri, 18,911 bp (Beagley et al. 1995)] and two agarose/TAE gel. Alternatively, the amplified fragments in the subclass Zoantharia or Hexacorallia [the sea separated by electrophoresis on 0.7% low melting-point anemone Metridium senile, 17,443 bp (Beagley et al. agarose/TAE gel were cut from the gel and treated by 1998); the scleractinian coral A. tenuis, 18,338 bp (van GELase (Biocompare) at 42°C overnight to digest the Oppen et al. 2002)]. The AT content of the whole agarose. The recovered fragments were digested with mitochondrial genome of the M. annularis complex EcoRV and SmaI, cloned in pGEM-T Easy Vector (66%) was slightly higher than that of other cnidarians (Promega) after being treated at 72°C for 10 min by Taq (e.g., 62% for A. tenuis) both including and excluding polymerase to add adenine to make the fragments insert non-coding regions. As in Acropora, cytosine was the easily into the vector, and sequenced using an ABI least common and thymine the most common nucleotide automated sequencer. in Montastraea mitochondrial DNA (25.0% A, 13.2% Based on the nucleotide sequences obtained, the C, 20.5% G, 41.3% T). Base frequency for specific genes remainder of the mitochondrial DNA sequence of M. differed significantly (P<0.005) for one or more pair- annularis was determined using primer walking methods. wise comparisons in ATP synthase subunit 6 (atp6), Mitochondrial sequences from M. franksi and M. fave- ATP synthase subunit 8 (atp8), cytochrome oxidase b olata were subsequently determined using several prim- (cob), cox1, cytochrome oxidase subunit 3 (cox3), ers designed based on the complete mitochondrial NADH hydrogenase subunit 2 (nad2), NADH hydrog- nucleotide sequence of M. annularis. All sequences are enase subunit 4 (nad4), NADH hydrogenase subunit 5 available in DDBJ (accession numbers (nad5), NADH hydrogenase subunit 6 (nad6), short- AP008973-AP008978). Open Reading Frames (ORFs) subunit ribosomal RNA (rns), and rnl (Table 1). were translated in DNASIS version 2 (Hitachi Software As in other cnidarian mitochondrial genes, ATG was Engineering, Inc.) and MEGA Program version 2.1 the start codon, and both TAA and TAG were stop (Kumar et al. 2001) using the A. tenuis genetic code (van codons. ATA and GTG also acted as start codons for Oppen et al. 2002). Genes and their start and stop co- some genes, as is the case in Acropora (van Oppen et al. dons were identified by comparison with mitochondrial 2002). There were five cases of gene overlap (indicated DNA sequences of A. tenuis (van Oppen et al. 2002) and by negative values for intergenic regions in Table 1) Table 1 Comparison of the whole mitochondrial genome among the scleractinian corals Montastraea annularis complex (Ma) and Acropora tenuis (Ac) (van Oppen et al. 2002) and the sea anemone Metridium senile (Ms) (Beagley et al. 1998) Length (bp) Genetic distance (%) Region Ma At Ms Ma-At Ma-Ms At-Ms trnM 72 71 71 42.9 81.8 36.8 rnl 1,973 2,261 2,220 32.5* 38.9* 25.1* IGR 0 102 N.A.