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Secondary Structure and Phylogenetic Utility of the Ribosomal Internal Transcribed Spacer 2 (ITS2) in Scleractinian Corals

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Secondary Structure and Phylogenetic Utility of the Ribosomal Internal

Transcribed Spacer 2 (ITS2) in Scleractinian Corals

Chaolun Allen Chen1,2,*, Chau-Ching Chang1,2, Nuwei Vivian Wei1,2, Chien-Hsun Chen1,2, Yi-Ting Lein1, Ho-E Lin1, Chang-Feng Dai2and Carden C. Wallace3

1Research Center for Biodiversity, Academia Sinica, Nankang, Taipei, Taiwan 115, R.O.C. 2Institute of Oceanography, National Taiwan University, Taipei, Taiwan 106, R.O.C. 3Museum of North Queensland, Townsville, Queensland Q4810, Australia

(Accepted June 28, 2004)

Chaolun Allen Chen, Chau-Ching Chang, Nuwei Vivian Wei, Chien-Hsun Chen, Yi-Ting Lein, Ho-E Lin, Chang-Feng Dai and Carden C. Wallace (2004) Secondary structure and phylogenetic utility of the ribosomal internal transcribed spacer 2 (ITS2) in scleractinian corals. Zoological Studies 43(4): 759-771. In this study, we examined the nucleotide characteristics, the secondary structure, and phylogenetic utility of the ribosomal internal spacer 2 (ITS2) from 54 species of scleractinian corals, representing 25 genera and 11 families of both the complex and robust clades previously defined through molecular phylogenetic analyses. The lengths and nucleotide contents of the ITS2 were highly variable among corals. The ITS2 of Acropora is significantly short-er than those of othshort-er corals. Dinucleotide or tetranucleotide microsatellites wshort-ere identified in the genshort-era

Acropora, Cyphastrea, Favites, Goniastrea, Hydnophora, Montipora, Madracis, and Porites. Three distinct

types of secondary structures with the smallest free energy values were predicted using the computer software, Mfold. A standard 4 domains were observed in 17 species of corals, while 23 species has a modified 5 domains with domain I divided into 2 subdomains. These 2 types of secondary structures were observed across 11 coral families. The 3rd type, 5 domains with domain III divided into 2 subdomains, was only seen in the genus Acropora. Among the domains, domain II is highly conserved and is flanked by conserved sequence motifs in adjacent stems. The motif, 5,-CRCGGYC-3,, and its compensatory bases were highly conserved in both the complex and robust clades of scleractinian corals. The robust-clade phylogeny constructed using ITS2 data produced a concordant tree to those based on mitochondrial and nuclear genes. The comparative analysis indicated that the extremely high ITS intragenomic divergence of Acropora is an exception rather than the rule for the evolutionary history of scleractinian corals. Despite the atypical and unusual pattern of molecu-lar evolution in the genus Acropora, data of the ITS2 are still applicable, with adequate adjustment of secondary structures, to the primary sequence alignment of different levels of phylogenetic analyses, from populations to genera, in scleractinian corals. http://www.sinica.edu.tw/zool/zoolstud/43.4/759.pdf

Key words: Internal transcribed spacer 2, Secondary structure, Conserved domain, Scleractinian corals, Phylogenetic utility.

D

NA sequences of the 2 internal transcribed spacers (ITS1 and ITS2) of the ribosomal RNA (rRNA) transcription unit have proven useful in resolving phylogenetic relationships of closely related taxa and in distinguishing species in fun-gal, plant, and animal taxa due to their relatively rapid evolution rates (Baldwin 1992, Schlotterer et al. 1994, Mai and Coleman 1997, Weekers et al. 2001, Oliverio et al. 2002). In addition, the

tran-script folding structure of the ITS provides some signals that guide the ribosomal coding regions when they are processed into small, 5.8S, and large ribosomal RNA (van der Sande et al. 1992, van Nues et al. 1995). The potential to predict the folding structure has enhanced the role of ITS in phylogenetic studies, since it is important to guide reliable sequence alignment based on secondary structures (Michot et al. 1999).

759

*To whom correspondence and reprint requests should be addressed. Tel: 886-2-27899549. Fax: 886-2-27858059. E-mail: cac@gate.sinica.edu.tw

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Many methods have been applied to infer the secondary structure of ITS, including electron microscopy (Gonzales et al. 1990), chemical and structure probing (Yeh and Lee 1990), site-directed mutagenesis (van der Sande et al. 1992, van Nues et al. 1995), and very commonly used computer software prediction programs (e.g., Mfold) which utilize minimum free energy values (Zuker and Steigler 1981). Based on those predictions, a sec-ondary structure for the ITS2 with 4 domains (I~IV) has been proposed for green algae, flowering plants, fruit flies (Drosophila spp.), parasitic flat-worms, gastropods, and the mouse (Schlötterer et al. 1994, Mai and Coleman 1997, Morgan and Blair 1998, Joseph et al. 1999, Michot et al. 1999, Coleman and Vacquier 2002, Oliverio et al. 2002, Gottschling and Plötner 2004). A highly conserved sequence is situated around a central loop and at the apex of a long stem in the 3,-half (Joseph et al. 1999).

The ITS region and 5.8S gene have been extensively used to reconstruct phylogenetic rela-tionships among scleractinian corals (Hunter et al. 1997, Lopez and Knowlton 1997, Odorico and Miller 1997, Medina et al. 1999, van Oppen et al. 2000 2002, Diekmann et al. 2001, Forsman et al. 2003, Lam and Morton 2003, Marquez et al. 2003, Fukami et al. 2004). The results indicate that indi-vidual coral colonies host a high degree of intrage-nomic variation, and coral ITS phylogenies in sev-eral cases are polyphyletic among closely related congeners (Odorico and Miller 1997, Medina et al. 1999, van Oppen et al. 2000 2002, Diekmann et al. 2001, Marquez et al. 2003, but see Forsman et al. 2003, Lam and Morton 2003, Vollmer and Palumbi 2004). A high degree of intragenomic variation may result in unreliable sequence align-ments that can generate incorrect phylogenies (Li 1997). Using the secondary structure to guide alignment of ITS DNA sequences may assist in reducing errors in phylogenetic constructions. For example, the variation in ITS2 was estimated to be as high as 40% (p-distance) at the interspecific level for Acropora spp. (Odorico and Miller 1997, van Oppen et al. 2001 2002), which is beyond the level which can be used to produce a reliable alignment (Li 1997). In contrast, the interspecific variation was relatively small (< 8%) among species of Madracis spp. in the Caribbean (Diekmann et al. 2001). Both studies lead to the conclusion that evolutionary patterns of potentially hybridizing corals are consistent with reticulation (Odorico and Miller 1997, Medina et al. 1999, van Oppen et al. 2000 2002, Diekmann et al. 2001,

Marquez et al. 2003). However, this conclusion should be viewed with caution, since the ITS can be either highly or only slightly variable among dif-ferent lineages of scleractinian corals and since mechanisms between maintaining and homogeniz-ing ITS variations are complicated (Hillis and Dixon 1991, Vollmer and Palumbi 2004). Recent analy-ses of ITS regions have revealed that the phyloge-netic signature of recent introgressive hybridization is obscured in the Caribbean Acropora because they share ancient rDNA lineages that predate the divergence of the species (Vollmer and Palumbi 2004).

Family-level phylogenies among scleractinian corals have been inferred using mitochondrial ribo-somal RNA genes, and results indicated that 2 major clades, i.e.,“robust”and“complex”, can be defined according to the skeletal morphology (Romano and Palumbi 1997, Romano and Cairns 2000, Chen et al. 2002). Fukami et al. (2004) examined 3 mitochondrial and nuclear protein-cod-ing genes and demonstrated that several major families in the robust clade, such as the Faviidae, Mussidae, and Merulindae, do not form mono-phylies, suggesting a deep divergence between Pacific and Atlantic coral faunas. Whether a rapid-ly evolving region such as ITS2 rDNA also exhibits a similar phylogeny as seen in the robust clade deserves further investigation. In this study, we examined the secondary structure and phylogenet-ic utility of ITS2 DNA sequences from 54 species of scleractinian corals, representing 25 genera and 11 families. We addressed the following ques-tions: (1) Does the ITS2 secondary structure in scleractinian corals fit the canonical“4-domain model”as seen in other eukaryotes? (2) Are there conserved regions of the secondary structures that can be identified between the 2 clades of sclerac-tinian corals? (3) What is the improvement in the DNA sequence alignment based on guidance by the ITS2 secondary structure? (4) And, what is the resolution of ITS2 DNA sequences in inferring the higher-level phylogeny of scleractinian corals?

MATERIALS AND METHODS Coral samples and ITS2 DNA sequences retrieved from the database

ITS2 DNA sequences were obtained from 2 sources: (1) DNA sequencing of coral collected from reefs in Taiwan and Togian I., Sulawesi, Indonesia by the authors (C.A.C. and C.C.W.),

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respectively, and (2) available sequences from GenBank. Taxonomic information, collection sites, clades, and GenBank retrieval information are list-ed in table 1.

Molecular methods

DNA extraction, PCR, cloning, and DNA sequencing are described in our previous work (Chen et al. 2000 2002 2003 2004). Target seg-ments containing the ITS1-5.8S-ITS2 region were amplified using the“anthozoan-universal”primer pairs, 1S: 5, -GGTACCCTTTGTACACACCGC-CCGTCGCT-3’and 2SS: 5, -GCTTTGGGCGGC-AGTCCCAAGCAACCCGACTC-3,, as described in Odorico and Miller (1997). Nucleotide sequence analysis concentrated on the ITS2 region, as this region is more typically used in the secondary structure prediction than is the ITS1 region. In addition, the ITS1 region in several scleractinian corals has shown great differences in length and thus cannot be used to produce a reliable align-ment for further analysis (Odorico and Miller 1997). PCR was performed in a PC-9606 thermal sequencer (Corbett Research, Sydney, NSW, Australia) using the following thermal cycle: 1 cycle of 95

°

C for 4 min; 4 cycles of 94

°

C for 30 s, 50

°

C

for 1 min, and 72

°

C for 2 min; followed by 30

cycles of 94

°

C for 30 s, 55

°

C for 1 min, and 72

°

C

for 2 min. The amplification reaction used 50~200 ng of template and BRL Taq polymerase in a 50-µl reaction volume, using the buffer supplied with the enzyme, under conditions recommended by the manufacturer. The PCR products were elec-trophoresed in a 1% agarose (FMC Bioproduct, Rockland, ME, USA) gel in 1X TAE buffer to assess the yield. Amplified DNA was extracted once with chloroform, precipitated with ethanol at -20

°

C, and resuspended in TE buffer. PCR

prod-ucts were cloned using the pGEM-T system (Promega, Madison, MI, USA) under conditions recommended by the manufacturer. Nucleotide sequences were determined for complementary strands of at least 2 clones from each sample using an ABI 377 Genetic Analyzer. The se-quences obtained were submitted to GenBank under the accession numbers listed in table 1.

Sequence alignment, folding of sequences into a putative secondary structure, and phyloge-netic analysis

DNA sequences were initially aligned using CLUSTAL X (Thompson et al. 1994), and default

gap and extension penalties were used followed by manual editing with SeqApp 1.9 (Gilbert 1994). Alignments were then adjusted by eye following the guidance of the predicted secondary structure (see below). Sequences were folded with the RNA secondary structure prediction subroutine, Mfold (Zuker 2003), in SeqWeb vers. 2.1 (Wisconsin package, Wisconsin, USA). Default values were used to fold the ITS2 rDNA. About 20 bases of flanking sequences (5.8S and 28S rRNA) were included because these 2 coding regions have been shown to be important for the folding of ITS2 sequences. Structures inferred by Mfold were examined for common stems, loops, and bulges. Uncorrected pairwise p-distances (Li 1997) were calculated for the alignments from the default options of CLUSTAL X and after guidance of ITS2 secondary structures for the genus Acropora and the families of the Faviidae, Mussidae, and Merulinidae, respectively. Student,s t-test was used to examine the statistical significances of the p-distance matrices before and after readjustment. In order to assess the phylogenetic utility of ITS2, the p-distances at the interspecific level were cal-culated for those genera or subgenera, including Acropora, Isopora, Montipora, Monastrea, Goniastrea, and Madracis, for which ITS2 sequences were available for more than 3 species. Phylogenetic analyses based on the maximum-parsimony (MP), neighbor-joining (NJ), and maxi-mum likelihood (ML) algorithms were performed for the robust clade using PAUP* 4.0b10 (Swofford 2002).

RESULTS ITS2 rDNA in scleractinian corals

In the scleractinian corals surveyed, the ITS2 region varied in length from 104 bp in Acropora longicyanthus to 369 bp in Pocillopora damicornis, whereas the GC content ranged from 44% in Porites lutea to 74% in Astreopora myriophthalma (Table 1). Short dinucleotide simple sequence repeats (microsatellites) were identified in several coral species: (GA)2-6 and (GT)2-7 in Acropora (Isopora) brueggmanni, A. cuneata, A. palifera, and A. togianesis; (AG)5 in Anacropora sp. and Montipora spp.; (CA)3-6 in Madracis spp.; (GC)5in Goniastrea spp. and Hydnophora exesa; and (TG)4 in Cyphastrea japonica and Favites abdita. The tetranucleotide repeats of (CCAT)4 and (AGCA)5-7were respectively identified in A. humilis

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Table 1.

Taxonomic information, collection site, clades, length, GC cont

ent, microsatellites, and secondary structure types of the ribo

somal

inter-nal transcribed spacer 2 (ITS2), and GenBank accession numbers

of the scleractinians used in this study

.

a: Complex (C) and robust (R) clades

were assigned by Romano and Cairns (2000) and Chen et al. (2002

);

b: T

ypes of secondary structure, I: four-domain model; II:

five-domain model

with domain I divided into two subdomains; III: five-domain mod

el with domain III divided into two subdomains

Acroporidae Acropora humilis Penghu Is., T aiwan C 131 51 (CCA T) 4 III A Y722741 this study W allace (1999) Acropora muricata Penghu Is., T aiwan C 107 51 III A Y722743 this study W allace (1999) Acropora cytherea Magnetic Is., GBR C 116 53 III ACU82723

Odorico and Miller (1997)

Acropora formosa (= muricata ) Magnetic Is., GBR C 11 1 48 III AFU82730

Odorico and Miller (1997)

Acropora hyacinthus Magnetic Is., GBR C 114 49 III AHU82719

Odorico and Miller (1997)

Acropora longicyathus Magnetic Is., GBR C 104 54 III ALU82733

Odorico and Miller (1997)

Acropora valida Magnetic Is., GBR C 112 52 III A VU82727

Odorico and Miller (1997)

Acropora cervicornis Bonaire, Invisibles C 128 54 III AF239046

van Oppen et al. (2000)

Acropora palmata Bonaire, Redslave C 124 52 III AF239081

van Oppen et al. (2000)

Acropora prolifera Bonaire, Redslave C 129 52 III AF2391 19

van Oppen et al. (2000)

Acropora

(Isopora

) brueggemanni

Togian Is., Sulawesi

C 140 64 (GA) 2-6 , (GT) 2-7 III A Y722737 this study W allace (1999) Acropora (Isopora ) cuneata

Togian Is., Sulawesi

C 136 68 (GA) 2-6 , (GT) 2-7 III A Y722738 this study W allace (1999) Acropora (Isopora ) palifera

Togian Is., Sulawesi

C 136 69 (GA) 2-6 , (GT) 2-7 III A Y722744 this study W allace (1999) Acropora (Isopora ) togianensis

Togian Is., Sulawesi

C 141 67 (GA) 2-6 , (GT) 2-7 III A Y722745 this study W allace (1999) Anacropora sp.

Togian Is., Sulawesi

C 177 53 (AG) 4-5 I A Y722747 this study V eron (2000) Astreopora listera Kenting, T aiwan C 197 71 I A Y722742 this study V eron (2000) Astreopora sp. Kenting, T aiwan C 194 74 I A Y722748 this study V eron (2000) Montipora aequituberculata Y ahliu, T aiwan C 177 51 (AG) 4-5 I A Y722772 this study V eron (2000) Montipora angulata Penghu Is., T aiwan C 181 52 (AG) 4-5 I A Y722773 this study V eron (2000) Montipora molis Penghu Is., T aiwan C 180 51 (AG) 4-5 I A Y722776 this study V eron (2000) Montipora peltiformis Penghu Is., T aiwan C 177 50 (AG) 4-5 I A Y722777 this study V eron (2000) Montipora taiwanensis Penghu Is., T aiwan C 177 53 (AG) 4-5 I A Y722778 this study V eron (2000) Montipora tuberculosa Penghu Is., T aiwan C 177 53 (AG) 4-5 I A Y722779 this study V eron (2000) Montipora venosa Y ehliu, T aiwan C 181 52 (AG) 4-5 I A Y722780 this study V eron (2000) Astrocoeniidae Madracis decactis Curacao, N. Antilles C 207 65 (CA) 3-6 II AF251875 Diekmann et al. (2001) Madracis formosa Curacao, N. Antilles C 21 1 64 (CA) 3-6 II AF251891 Diekmann et al. (2001) Madracis mirabilis Curacao, N. Antilles C 209 65 (CA) 3-6 II AF251849 Diekmann et al. (2001) Madracis pharensis Curacao, N. Antilles C 209 65 (CA) 3-6 II AF251925 Diekmann et al. (2001) Madracis senaria Curacao, N. Antilles C 206 66 (CA) 3-6 II AF251904 Diekmann et al. (2001) Taxon Collection site Clade a Size (bp) GC (%) Microsatellite Secondary Accession no. Reference Ide ntification structure type b

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Table 1. (Cont.) Stylocoeniella guentheri Kenting, T aiwan C 234 60 II A Y722791 this study V eron (2000) Dendrophylliidae Tubastrea aurea Penghu Is., T aiwan C 21 1 56 II A Y722796 this study V eron (2000) Fungiacyathidae Fungiacyathus sp. Ilan, T aiwan C 187 60 II A Y722757 this study V eron (2000) Oculinidae Galaxea fascularis Penghu Is., T aiwan C 248 71 I A Y722764 this study V eron (2000) Poritidae Alveopora sp. Kenting, T aiwan C 165 64 I A Y722746 this study V eron (2000) Porites lutea Penghu Is., T aiwan C 233 44 (AGCA) 5-7 I A Y722755 this study V eron (2000) Faviidae Cladocora sp. Kaohsiung, T aiwan R 196 59 II A Y722752 this study V eron (2000) Cyphastrea japonica Penghu Is., T aiwan R 208 63 (TG) 4 II A Y722749 this study V eron (2000) Favites abdita Penghu Is., T aiwan R 232 63 I A Y722755 this study V eron (2000) Goniastrea aspera Penghu Is., T aiwan R 226 68 II A Y722759 this study V eron (2000) Goniastrea sp. Penghu Is., T aiwan R 228 65 II A Y722762 this study V eron (2000) Goniastrea palaunesis Penghu Is., T aiwan R 230 67 II A Y722766 this study V eron (2000) Montastrea annularis

Key Largo, Florida

R 209 62 II AF013731 Medina et al. (1999) Montastrea faveolata

Key Largo, Florida

R 209 63 II AF013733 Medina et al. (1999) Montastrea franksi

Key Largo, Florida

R 209 64 II AF013734 Medina et al. (1999) Montastrea curta Penghu Is., T aiwan R 252 64 II A Y722774 this study V eron (2000) Oulastrea crispata Penghu Is., T aiwan R 191 60 II A Y722781 this study V eron (2000) Platygyra sinensis

Hong Kong, China

R

222

68

II

AF481901

Lam and Morton (2003)

Merulinidae Hydnophora exesa Penghu Is., T aiwan R 246 64 II A Y722769 this study V eron (2000) Mussidae Acanthastrea echinata Penghu Is., T aiwan R 228 67 II A Y722739 this study V eron (2000) Pocilloporidae Seriatopora hystrix Kenting, T aiwan R 236 55 II A Y722794 this study V eron (2000) Pocillopora damicornis Penghu Is., T aiwan R 369 61 I A Y722785 this study V eron (2000) Stylophora pistillata Penghu Is., T aiwan R 226 58 II A Y722795 this study V eron (2000) Siderastreidae Psammocora contigua Penghu Is., T aiwan R 197 61 II A Y722782 this study V eron (2000) Pseudosiderastraea tayami Kaohsiung, T aiwan R 197 64 I A Y722789 this s tudy V eron (2000) Taxon Collection site Clade a Size (bp) GC (%) Microsatellite Secondary Accession no. Reference Ide ntification structure type b

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Fig. 1. Representation of the proposed secondary structure of scleractinian corals. (A) Type I, Anacropora sp. with 4 putative domains; (B) type II, Acantherastrea echinata with 5 putative domains, with domain I divided into 2 subdomains (Ia and Ib); and (C) type III,

Acropora cervicornis with 5 putative domains, with domain III divided into 2 subdomains (IIIaand IIIb). Minimum free energies (∆G) are indicated.

(B)Acantherastrea echinata

3,-end 5.8S 5,-end 28S

∆G=-148 kcal/mole

(C)Acropora cervicornis ∆G=-67.4 kcal/mole (A)Anacropora sp. 3,-end 5.8S 5,-end 28S 3,-end 5.8S 5,-end 28S ∆G=-94.4 kcal/mole III IV III II IV IIIb IIIa IV I II Ia Ib I II

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(A)Robust clade

Faviidae/Merulinidae/Mussidae

Cyphastrea Favites Platygyra Oulastrea Cladocora Goniastrea Montastrea

Pocilloporidae

Siderastreidae

Pseudosiderastrea Psammocora

Stylophora Seriatopora Pocillopora

Poritidae Acroporidae

(B)Complex clade

Acropora

Madracis Styloceneiella Galaxea Tubastrea Fungiacyathus

Astrocoeniidae Oculinidae Dendrophylliidae Fungiacyathidae

Isopora Anacropora Montipora Astreopora Alveopora Porites Hydnophora Acantherastrea

Fig. 2. Structures of domain II representing scleractinian corals of (A) the robust and (B) complex clades. Conserved pyrimidine-pyrim-idine pairings are marked by a dashed-line box.

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and Porites lutea (Table 1).

Putative secondary structures, conserved motifs, and improvements in the ITS2 align-ment

The putative secondary structures of the 54 species from the 25 genera and 12 families of scleractinian corals could be categorized into 3 types: type I, the standard 4-domain model; type II, a 5-domain model with domain I divided into 2 sub-domains (Iaand Ib); and type III, a 5-domain model with domain III divided into 2 subdomains (IIIaand IIIb) (Fig. 1). The 20 bp of the 5.8S and 28S rDNA that was immediately adjacent to the 5’-end and 3’-end of the ITS2 apparently forms canonical bonds with each other. Among the 54 ITS2 sec-ondary structures constructed, 17 belonged to type I, 23 were of type II, and 14 were of type III (Table 1). Type I and II structures were observed in all 12 families of scleractinian corals. In contrast, type III was only seen in the genus Acropora. Among the domains, while stem-loop IV was the most-variable domain, stem II was highly conserved and was flanking by a conserved sequence motif in the adjacent stems I and III. The motif, 5’-CRCG-GYC-3,, and its compensatory bases in stem II were highly conserved in corals of both the robust and complex clades (Fig. 2).

Alignment of the ITS2 primary sequences was considerably improved based on adjustment of the secondary structure. Conserved stems identified in the secondary structural domains provided

con-sistent bases for correcting the alignment of vari-able loop regions for the phylogenetic analysis (data not shown). The ITS2 uncorrected p-dis-tances guided by the secondary structure were significantly smaller than those derived from the default options of CLUSTAL X. In the complex clade, the mean p-distance was 0.2717 ± 0.09756 before manual readjustment, which was signifi-cantly larger than that (0.2353 ± 0.09583) after guidance by the secondary structure for species in the subgenus Acropora (paired t-test = 5.688, p < 0.001). However, a reliable alignment could not be obtained among the lineages within the complex clade due to the extremely high divergence between Acropora and other corals (see below). In the robust clade, a similar trend could be observed in the families of the Faviidae, Merilinidae, and Mussidae which means that p-dis-tances (0.27569 ± 0.07609) were significantly decreased after readjustment using the secondary structure (0.24848 ± 0.06634, paired t-test = 9.438, p < 0.001). However, attempts to align the ITS2 between the robust and complex clades were unsuccessful due to the extremely high divergence among clades (see below).

Phylogenetic utility of ITS2 in scleractinian corals

The interspecific p-distances of ITS2 varied considerably in different genera and subgenera of scleractinian corals (Fig. 3). In the 6 genera exam-ined, the highest value was observed in the sub-genus Acropora with a mean p-distance of 0.2353. This value is close to the level of intergeneric vari-ation within the robust clade. Varivari-ation within the genus Madracis was the smallest with a mean p-distance of 0.0134. p-p-distances among Acropora and other corals of the complex clade were so high (> 0.6) that intergeneric alignment was not reliable for phylogenetic reconstruction.

In order to assess the phylogenetic utility of ITS2, we focused only on an analysis of the robust clade, since it was not possible to produce a reli-able alignment within the complex clade or between the 2 clades. Taxa were selected based on the phylogenetic trees published in Romano and Cairns (2000) and Chen et al. (2002). Two species of the complex, Tubastrea aurea and Fungiacyathus sp., were used as outgroups. Of the 334 characters in the ITS2 alignment, 207 (61.98%) were variable and 138 (41.32%) were parsimony-informative for the robust clade. Phylogenetic construction using the MP, NJ, and

Acropora Isopora Montipora Montastrea Goniastrea Madracis

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 Genus Uncorrected P -distance

Fig. 3. Interspecific variability (uncorrected p-distances) in dif-ferent genera of scleractinian corals. Means and standard deviations are indicated by closed dots and bars, respectively.

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ML algorithms produced identical topologies (Fig. 4). Removal of the large insertions and deletions (indels) at the hypervariable domains (e.g., domain IV) did not affect the topology of the phylogenetic tree (data not shown). Parsimony analysis revealed a single MP tree with a tree length of 564, a consistency index of 0.628, a retention index of 0.53, and a rescaled consistency index of 0.33. The ML analysis of ITS2 yielded a topology of a -ln likelihood of 3345.17. The tree showed that the generic relationship based on the ITS2 did not cor-respond with the family tree based on skeletal morphology (Veron 2000), except for the astron-coneid, Styloceneiella guentheri, and Madracis for-mosa. Using Tubastrea aurea and Fungiacyathus sp. as outgroups, genera of the Faviidae (except for Oulastrea and Cladocora) were grouped with Hydnophora (Merulinidae) and Acantherastrea (Mussidae) with high bootstrapping support (Fig. 4).

DISCUSSION

While the ITS2 region presents a dramatic range of length variations among scleractinian corals, its size remains relatively homogenous within each of the major groups. Acropora has the shortest ITS2 not only among scleractinian corals but also in metazoans and eukaryotes (Odorico and Miller 1997). This is an atypical but unique feature of Acropora, since lengths of the ITS2 from other genera in the Acroporidae are comparable to those of other scleractinians. Nevertheless, some species of Acropora, such as A. humilis, A. togia-nensis (this study), A. aspera, A. pulchra, A. florida (Marquez et al. 2003), A. cervicornis, A. palmata, and A. prolifera (van Oppen et al. 2000, Vollmer and Palumbi 2004), as well as the subgenus Isopora possess significantly longer ITS2 regions than others due to the occurrence of dinucleotide or tetranucleotide simple sequence repeats (i.e., microsatellites). Different compositions of

0.05 substitutions/site

Fig. 4. Phylogenetic analysis of the robust-clade ITS2 derived from the Neighbor-joining (NJ), maximum parsimony (MP), and maxi-mum likelihood (ML) methods. The 3 algorithms produced the same tree topology. The tree is presented with bootstrapping support labeled above (NJ) and below (MP) the branches (< 50% not shown). Scleractinian families are coded by bars with different shading.

Outgroups Platygyra sinensis 91 95 87 76 73 83 91 76 82 98 100 Goniastrea aspera Hydnophora exesa Montastrea curta Favites abdita Cyphastrea japonica Montastrea annularis Acantherastrea echinata Styloceneiella guentheri Madracis formosa Oulastrea crispata Psammocora contigua Cladocora sp. Pseudosiderastrea tayami Fungiacyathus sp. Tubastrea aurea Faviidae Mussidae Merulinidae Siderastreidae Astroncoeniidae

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microsatellites were also observed in the genera, Cyphastrea, Favites, Goniastrea, Hydnophora, Madracis, Montipora, and Porites, indicating that ITS2 microsatellites evolved independently in the different lineages of scleractinian corals.

The occurrence of microsatellites in the ITS region has had great impacts on phylogenetic studies of corals. Short repeated sequences even-tually increase the difficulties of aligning homolo-gous regions and subsequently influence phyloge-netic reconstructions. For example, a study on the species boundaries of 5 Caribbean Madracis corals, M. mirabilis, M. senaria, M. decactis, M. for-mosa, and M. pharensis, using the ITS1-5.8S-ITS2 region indicated that M. senaria and M. mirabilis formed monophyletic groups, while the other 3 formed paraphyletic groups (Diekmann et al. 2001). Those results suggested a reticulate speci-ation through repeated introgressive hybridizspeci-ations in Madracis. Nevertheless, this conclusion should be accepted with extreme caution, since careful alignment and inclusion/exclusion of CA repeats (Table 1) at the 3’-end of ITS2 can generate very different phylogenetic relationships. Madracis senaria and M. mirabilis no longer formed a mono-phyly as seen in previous reports (data not shown). Similar concerns about ITS microsatel-lites for phylogenetic analyses were also dis-cussed in freshwater crayfish (Harris and Crandall 2000) and ants of the genus Strumingenys (Hung et al. 2004).

The secondary structure of coral ITS2 does not always conform to the 4-domain model as pre-dicted in green algae, flowering plants, fruit flies (Drosophila spp.), parasitic flatworms, gastropods, and the mouse (Michot et al. 1999, Schlötterer et al. 1994, Mai and Coleman 1997, Morgan and Blair 1998, Joseph et al. 1999, Coleman and Vacquier 2002, Oliverio et al. 2002, Gottschling and Plötner 2004). The difficulties of arriving at a common ITS2 secondary structure for scleractinian corals is probably due to limitations of the comput-er program, Mfold. The Mfold program gencomput-erates multiple free-energy diagrams, and a“consensus” model is inferred from the structural features com-mon to all. It has been argued that even closely related taxa with very similar primary sequences can result in/exhibit vastly different structures (Hershkovitz and Zimmer 1996). More significant-ly, experimentally derived RNA structures frequent-ly exhibit suboptimal free-energy conformations (Gutell et al. 1994). Identifying covariation sites is recognized to improve the stability of the sec-ondary structure. Covaration analysis based on a

large-scale comparison of 340 sequences of Asteraceae ITS suggested that 20% of ITS1 and 38% of ITS2 nucleotide position are involved in base pairing to form helices (Goertzen et al. 2003). Interestingly, the ITS2 secondary structure model of Asteraceae based on covariation analysis gen-erally agree with structural features generated by thermodynamic criteria (Goertzen et al. 2003), indi-cating that even without considering covariations the computer-based secondary structure model should still be regarded as a reliable“backbone” for further readjustment of primary alignment in phylogenetic analysis. A future study examining ITS sequences from a large data set of closely related taxa may be suitable for examining the covariations of ITS2 in more detail for scleractinian corals. In addition, the most-conserved feature of the scleractinian ITS2 in domain II and the adja-cent regions of domains I and III found in all of the scleractinian corals studies indicate that these con-served sequences are likely to play an important role in folding the secondary structure of coral ITS2. Similar conservation was also observed among different genera of hard ticks which have a 5-domain ITS2 secondary structure (Hlinka et al. 2002).

The robust clade phylogeny constructed using the ITS2 is concordant with the phylogenies based on mitochondrial and nuclear ribosomal genes and protein-coding genes which show that the Faviidae, Merulindae, and Mussidae are mono-phyletic within the suborder Faviina (except for Oulastrea), but relationships among these families are apparently not monophyletic (Romano and Cairns 2000, Chen et al. 2002, Fukami et al. 2004). For example, both Fukami et al. (2004) and the present study showed that Montastrea annu-laris, a major Caribbean reef builder, is grouped with Cyphastrea japonica and forms a paraphyletic relationship with its Pacific congener, M. curta. Oulastrea crispata is clustered with a siderastreid, Psammocora contigua, and forms a trichotomic relationship with another Faviid, Cladocora sp. The affinity of both Oulastrea and Cladocora to the family Faviidae has been questioned, and place-ment of these 2 genera needs to be re-examined (Romano and Cairns 2000, Chen et al. 2002). Our data did not group Psammocora and Pseudo-siderastrea in a monophyletic group, which indi-cates that generic relationships within the family Siderastreidae should also be reconsidered.

ITS rDNA sequences are the most frequently used DNA markers for studying scleractinian evo-lution (Hunter et al. 1997, Lopez and Knowlton

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1997, Medina et al. 1997, Odorico and Miller 1997, van Oppen et al. 2000 2002, Diekmann et al. 2001, Rodriguez-Lanetty and Hoegh-Guldberg 2002, Vollmer and Palumbi 2004), especially on the topics of reticulate evolution among closely related species. However, the phylogenetic utility of the ITS has been questioned (van Oppen et al. 2002, Vollmer and Palumbi 2004). This is due to the extremely high levels of variability in the ITS which have been observed within and among sev-eral Acropora species (Odorico and Miller 1997, van Oppen et al. 2002, Marquez et al. 2003). Debates on interspecific hybridization versus incomplete lineage sorting as an explanation for the high ITS intragenomic variation have been overwhelmed by evidence from Acropora species (van Oppen et al. 2000 2001 2002, Vollmer and Palumbi 2002 2004, Marquez et al. 2003, Miller and van Oppen et al. 2003). Vollmer and Palumbi (2004) concluded that nuclear rDNA should be abandoned as a species- and population-level phylogenetic marker due to its complicated and undistinguishable characteristics of molecular evo-lution. We argue that this conclusion should be treated with great caution, since Acropora has sev-eral atypical and unusual characteristics that are significantly distinct from other scleractinian corals. First, Acropora has the shortest ITS not only among scleractinian corals but also among meta-zoans (Odorico and Miller 1997). For the other scleractinian corals, the length of the ITS is com-patible among genera. The mechanism by which Acropora species possess such a short sequence is still unknown. Second, even though it has the shortest DNA sequences, Acropora ITS2 forms a unique but stable 5-domain secondary structure, which differs from that of other scleractinian corals. Third, ITS sequence divergence within and among Acropora spp. is the highest observed so far. Except for Acropora, variations in the ITS2 are moderate, and it can reliably be aligned even across different genera of scleractinians to pro-duce robust phylogenies. These characteristics strongly indicate that high ITS intragenomic diver-gence of Acropora may be an exception rather than the rule for the evolutionary history of sclerac-tinian corals. In contrast, our analyses strongly indicate that ITS rDNA in scleractinian corals, with careful readjustment under guidance of the sec-ondary structure, is still applicable to different lev-els of phylogenetic analyses from populations to genera.

Acknowledgments: Many thanks to the staff of

the Penghu Aquarium, a facility of the Taiwan Fishery Research Institute, for providing hospitality during our field trips. We thank members of the Evolution and Ecology Discussion Group, Research Center for Biodiversity (RCBAS), Institute of Zoology, Academia Sinica (IZAS), and 2 anonymous referees for constructive comments. This is Evolution and Ecology Group, IZAS contri-bution no. 32. This work was supported by grants from RCBAS, IZAS, and the National Science Council, Taiwan to the senior author.

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數據

Table 1.Taxonomic information, collection site, clades, length, GC content, microsatellites, and secondary structure types of the ribosomal inter- nal transcribed spacer 2 (ITS2), and GenBank accession numbers of the scleractinians used in this study
Table 1.(Cont.) Stylocoeniella guentheriKenting, TaiwanC23460IIAY722791this studyVeron (2000) Dendrophylliidae Tubastrea aureaPenghu Is., TaiwanC21156IIAY722796this studyVeron (2000) Fungiacyathidae Fungiacyathussp.Ilan, TaiwanC18760IIAY722757this studyVer
Fig. 1. Representation of the proposed secondary structure of scleractinian corals.  (A) Type I, Anacropora sp
Fig. 2. Structures of domain II representing scleractinian corals of (A) the robust and (B) complex clades
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