CNIDARIA: ANTHOZOA: CORALLIMORPHARIA): UTILITY of the 5.8S and INTERNAL TRANSCRIBED SPACER (ITS) REGIONS of the Rrna TRANSCRIPTION UNIT

BULLETIN OF MARINE SCIENCE. 59( I): 196--208. 1996 CORAL REEF PAPER

SYSTEMATIC RELATIONSHIPS BETWEEN TROPICAL CORALLIMORPHARIANS (CNIDARIA: ANTHOZOA: CORALLIMORPHARIA): UTILITY OF THE 5.8S AND INTERNAL TRANSCRIBED SPACER (ITS) REGIONS OF THE rRNA TRANSCRIPTION UNIT

Chao/un A. Chen, Bette L. Willis and David J. Miller

ABSTRACT The nucleotide sequences of a segment of the rRNA transcription unit spanning the 3'- end of the 18S rDNA to the 5'-end of the 28S rDNA were determined for the tropical corallimorpharians (Cnidaria: Anthozoa), Rhodactis howesii, R. mussoides, Amplexidiscus fenestrafer, Actinodiscus nummiformis, A. unguja and an undescribed species. Comparison of the 5.8S rDNA sequences indicated a close relationship between the genera Rhodactis and Amplexidiscus and a much more distant relationship between these two genera and Actinod- iscus spp. The level of variation detected in this conserved region of the ribosomal transcription unit is not consistent with den Hartog's revision of the family Actinodiscidae to a single genus. Among the range of species studied, there was considerable variation in both length and % (G+C) content in the ITS regions. Both ITS sequences appear to be conserved within genera but highly variable between genera, and can therefore be used for generic assignment. Analysis of both ITS and 5.8S rRNA sequences supports assignment of the undescribed species to the genus Amplexidiscus.

The Corallimorpharia is a small and poorly characterized order within the an- thozoan sub-class Zoanthmia. Although morphologically similar to both the Ac- tiniaria (sea anemones) and Scleractinia (hard corals), the relationships between the Corallimorpharia and these two major orders of Hexacorallia are controversial (den Hartog, 1980; Fautin, 1993; Chen et a!., 1995). Within the order Coralli- morpharia, two distinct groups are recognisable: azooxanthallate genera which are limited to temperate and deeper waters, and zooxanthallate genera which are limited to the tropics (Carlgren, 1949). This division is supported by phylogenetic analyses of the 5'-end of 28S ribosomal DNA (rDNA): in both parsimony and distance analyses, azooxanthallate corallimorpharians consistently clustered with primitive actiniarians, and zooxanthallate corallimorpharians with more advanced actiniarians (Chen et a!., 1995). Both the Corallimorpharia and Actiniaria may therefore be paraphyletic. Corallimorpharians are sometimes major components of tropical shallow water ecosystems (Fishelson, 1970; den Hartog, 1980; England, 1987). Traditionally, tropical corallimorpharians were classified into two families containing a total of six genera: the Actinodiscidae, containing Actinodiscus, Orinia, Paradiscosoma, Rhodactis and Metarhodactis, and the Corallimorphidae, containing the single genus, Ricordea (Carlgren, 1949). Of these genera, only three are known to have widespread distributions: Actinodiscus is restricted to the Red Sea and Indo-Pa- cific, whereas Rhodactis and Ricordea occur in both the Indo-Pacific and Carib- bean (Carlgren 1949). Orinia, Paradiscosoma and Metarhodactis each contain only a single species, the first two genera being restricted to the Caribbean and the last to the western Pacific (Carlgren, 1949). In addition to these, an Indo- Pacific species originally described as Discosoma yuma (Carlgren, 1900) should probably be relocated to the genus Ricordea (Chen, pers. obser.). Many of the criteria used to distinguish the actinodiscid genera, such as ab-

196 CHEN ET AL.: CORAL SYSTEMATICS 197

o 100 200km

15'S

'25'S

150•E

Figure 1. Map of the Great Barrier Reef Marine Park showing collection sites.

sence/presence of a naked marginal zone, cnidom composition and thickness of the mesogloeal ridge in the oral disc, are ambiguous (for review, see den Hartog, 1980). After extensively reviewing the Caribbean corallimorpharian fauna, which includes three species of actinodiscids, den Hartog (1980) recognized that these characters did not reflect generic differences, and proposed that all five actinodiscid genera should be lumped into the single genus, Discosoma. A new genus has been proposed (Dunn and Hamner, 1980) to accommodate the morphologically-distinct Australian species, Amplexidiscus fenestrafer (family Actinodisci- dae). Systematic relationships amongst these tropical corallimorpharians are equivocal, largely because there are few informative morphological characters. 198 BULLETIN OF MARINE SCIENCE. VOL. 59,1"0. I, 1996

Table I. Taxonomic information, collecting locations of the tropical corallimorpharians, and number of individuals (in the brackets) used in this study. Identification was carried out by following the original descriptions and Carlgren's key (1949); thus we did not use Discosoma as synonymous to the original generic nomenclature.

Collecting sites Taxon (from the Great Barrier Reef) Identification references* Rhodactis howesii Arthur Bay, Magnetic Island (4) Carlgren, 1949, 1950 Rhodactis mussoides Geoffrey Bay, Magnetic Island Saville-Kent, 1893; Haddon, (2) 1898; Carlgren, 1949 Amplexidiscus fenestrafer Swain Reef (2) Dunn and Hanmer, 1980 Undescribed species North Reef, Orpheus Island (2) Actinodiscus nummiformis Geoffrey Bay, Magnetic Island Carlgren, 1943 (1) Actinodiscus unguja Swain Reef (1) Carlgren, 1900

This paper reexamines den Hartog's proposal in the light of a revision of Indo- Pacific corallimorpharians (Chen, in prep.). The paucity of conserved morphological characters has led us to evaluate molecular criteria for the estimation of systematic relationships between tropical corallimorpharians. The ribosomal RNA transcription unit has proved to be one of the most informative regions for this purpose (reviewed by Hillis and Dixon, 1991). In eukaryotes, two internal transcribed spacers (ITS I and ITS2) separate the 18S, 5.8S and 28S coding sequences, and an external transcribed spacer (ETS) is located 5'- of the 18S gene. These transcribed spacers contain signals for pro- cessing the rDNA transcript. Adjacent ribosomal RNA transcription units are sep- arated by a nontranscribed spacer (NTS) or intergenic spacer (IGS). This region contains subrepeating elements which enhance transcription (For review, see Hillis and Dixon, 1991). The coding regions of the rRNA transcription unit have been extensively used to investigate phylogenetic relationships from the phylum to the genus level (reviewed by Hillis and Dixon, 1991; Wainright et aI., 1993; Sidow and Thomas, 1994; Chen et aI., 1995); the transcribed spacers, on the other hand, are rapidly evolving regions that have been used to resolve relationships among closely-related animal (Maden et aI., 1983; Michot et aI., 1983; Goldman et aI., 1983; Chambers et aI., 1986; Gonzalez et aI., 1990; Wesson et aI., 1992; Anderson and Adlard, 1994) and plant (Torres et aI., 1990; Baldwin, 1992; Lee and Taylor, 1992; Saunders and Druehl, 1993, van Oppen, 1993; Zechman et aI., 1994) taxa. The aim of the present study was to explore the potential of ITS and 5.8S sequence data for estimating relationships among tropical corallimorpharian genera. For these purposes, we selected five species from the family Actinodiscidae and an undescribed species provisionally placed in this family (Chen, unpubI.).

MATERIALS AND METHODS

Material.-The six species of tropical corallimorpharians were collected from various sites on the Great Barrier Reef (Fig. I). Table 1 summarizes taxonomic information, collecting locations, and number of individuals used in the present study. DNA Extraction.-Genomic DNA was extracted as described in Chen et al. (1995). Small pieces (approximately 125 mm3) of tissue were ground into powder under liquid nitrogen, homogenised in 2-ml aliquots of lysis buffer (250 mM Tris-borate buffer, containing 50 mM EDTA, 0.5 M NaCI and 2% (w/v) SDS), and then incubated at 65°C for 30 min. After this time, proteinase K was added to 0.5 mg·ml-I final concentration, and the solutions incubated at 50°C until clear (4-48 h). The Iysates were then extracted two to three times with phenol-chloroform-isoamyl alcohol (25:24: I), and the DNA precipitated with isopropylalcohol and resuspended in sterile double-distilled water. CHEN ET AL.: CORAL SYSTEMATICS 199

188 5.88 288

15~ A7~ 55 ~ -I--CJ---I

BD1 ~ ~A4 .....,-J'_~2SSBD2

Figure 2. Schematized ribosomal DNA repeat unit showing the positions of the ITS and coding regions. Positions and orientations of primers used for PCR and DNA sequencing are indicated by arrows. Primer sequences are given in Table 2.

Polymerase Chain Reaction (PCR).-PCR primers were designed from the conserved 185 and 285 regions to amplify the entire ITS and 5.8S (Fig. 2). Internal sequencing primers were designed from conserved 185 and 5.85, 285 regions (Fig. 2). The nucleotide sequences of primers used in this study are shown in Table 2. Amplification was performed using the following protocol: one cycle at 95°C (3 min), 50°C (l min), noc (2 min); four cycles at 94°C (30 s), 50°C (l min), noc (2 min), 25 cycles at 94°C (30 s), 57°C (l min), noc (2 min), and one cycle 75°C (\0 min). Taq polymerase (Promega) was used in the buffer supplied with the enzyme and under the conditions recommended by the manufacturer. PCR products were purified by preparative agarose gel electrophoresis, then extraction with phenol: cWoroform: isoamylalcohol, followed by precipitation using ethanol/sodium acetate. Cloning and DNA Sequencing.-PCR products were cloned into pUC vectors by blunt-end ligation. PCR products were end-filled using two units of Klenow fragment (Promega) prior to ligation into Smal cut pUCI8 (Promega). End-filling reactions were carried out in a total volume of 25 f.L1of I X nick translation buffer with 2 mM each of dNTPs and incubated at 16°C for 2 h. After stopping reactions by addition of I f.L1of 0.5 M EDTA, the PCR products were then phosphorylated using T4 polynucleotide kinase (PNK; 20 units per reaction) under the manufacturer's (Promega) suggested conditions. After phenol extraction followed by ethanol precipitation, PCR products were cloned into pUCI8, and the ligations used to transform NM522 E. coli cells, permitting blue/white selection. Plasmid templates were subjected to DNA sequencing using the chain termination method (Sangf:r et aI., 1977; Hattori and Sakaki, 1986). In all cases, multiple clones were sequenced completely in both directions. Sequence Alignment and Pairwise Distance Matrix Analyses.-DNA sequences were initially aligned using the CLUSTAL V program (Higgins et aI., 1992), followed by manual editing using SeqApp 1.9. The amplified region contained the 3'-end of 185, 5.8S, and 5'-end of 285 rDNA which are sufficiently conserved to permit unambiguous alignments to be made between and among the species. Percent (G+C) values were calculated using the NIP option in the Genetic Computer Group software package version seven through Australian National Genetic Information Service (ANGIS). Phyloge- netic analyses of 5.8S rDNA were carried out in PAUP 3.1.1 (Swofford, 1991) using the exhaustive search option.

RESULTS peR amplification using the IS and 2SS primers generated products of approximately l,OOO-bpfor the range of tropical corallimorpharians. The products correspond to a region of the rRNA transcription unit, including the 3' -end of the l8S coding sequence to the 5'-end of 28S, and containing the entire ITSl, 5.8S,

Table 2. Primer sequences used in this study. 1: Numbering relative to the 18S rDNA of Anemonina sulcata (Genebank accession number: X53498); 2: Numbering relative to the Homo sapiens 5.85 and 28S rONA sequence (Genebank accession number: M27830). *: primers used for sequencing only.

Primer Sequences Location In the rDNA IS 5' -GGTACCCTITGTACACACCGCCCGTCGCT-3' #1616-1645 of 18S' 255 5' -GCTITGGGCTGCAGTCCCAAGCAACCCGACTC-3' #21-45 of 28S2 55 5' -GCCGACCCGCTGAATTCAAGCATAT-3' #278-312 of 28S2* A7 5' -AAGTAGTGTGAATTGCAG-3' #63-80 of 5.852* A4 5' -ACACTCAGACAGACATG-3' #139-155 of 5.852* BDI 5' -GTCGTAACAAGGTITCCGTA-3' #1756-1775 of 185'* BD2 5' -ACCCGCTGAATITAAGCATAT-3' #24-45 of 28S2* 200 BULLETIN OF MARINE SCIENCE. VOL. 59, NO. I, 1996

23130 9416 6557 4361 -/2322 ~2027

- 564

1 2 3 4 5 6 7

Figure 3. PCR products amplified by IS and 2SS primers from the tropical corallimorpharians. Lane (I) Rhodactis howesii, (2) R. mussoides, (3) AmpLexidiscus fenestrafer, (4) undescribed species, (5) Actinodiscus nummiformis, (6) A. unguja and (7) molecular weight standards: A DNA cut by Hind III. Sizes of the DNA standards are indicated in bp. and ITS2. Minor size variations between species were detected on agarose gel electrophoresis (Fig. 3). Intraspecific Variation in the ITS Regions of Rhodactis howesii.-Complete nucleotide sequences of eight individual cloned PCR products were determined for Rhodactis howesii, The origins of these clones were as follows: four clones from one polyp, two from a second polyp and one from each of two additional polyps. These polyps were collected randomly from different regions of an aggregation of R. howesii. Comparison with published sequences allowed unambiguous identification of the 18S, 5.8S and 28S coding regions, which were found to be identical between clones, and the ITS-coding region junctions. The ITS2 region was identical among the eight clones, and 190 bases in length, whereas the ITS 1

Table 3. Intraspecific variation in the ITSI region of Rhodactis howesii. Positions refer to the notional alignment in Fig. 4. A, B, C, and D correspond to different individuals of R. howesii. Absence is indicated by "-".

Clone Sequence Position feature AI A2 BI CI C2 C3 C4 DI 283 A or C A A A C C C C A 296 AorG A A A A A G A A 315,316 GT GT GT GT GT 317,318 GA GA GA 319,320 CG CG CG CG CG CG 368 Tor G T G G T G T G G CHEN ET AL.: CORAL SYSTEMATICS 201

1a~I~TS1

Actinod/scusnummlform/s AACCTGCGGA AGGA TCA TTG CCGCAGCAGC AAACCGTCCG GGCGCTTGGC 50 Act/nod/saJs ungujs ...... Amp/exid/scus fenestrafe, ...... •.. A ... TG .••• ------. A A. A. A····· Undescribed species ...... A ... TG •••• ------. A A. AAA-- - -- Rhodact/s howesl/ ...... A ... TAC··. T A- - - - -G .. A AAAAAG . Rhodactis mussoides ...... A ..• T. C", A . • -. - ••.. A AAAAAG .

Act/nod/scus nummlform/s TCGAGAC TTT GACTTTGACG TGCTCGCTCT GTGAACCGGT ACCTTGGTCG 100 ActJnod/saJs unguja ...... Amp/ex/discus fenestrafe, A .•. A - •• - - • -. ·TA. CT. Undescribed species A ... A ------TA. CT. Rhodact/s howesll A ..•. - -. ·TA. CT. RhodactJs musso/des A. . . . -. - - - • -. ·TA. CT.

Actinod/saJs numm/form/s CGAAGGA AGG AAGGAAGGAA GGACGGACGG CCGCTGTCTA GGTCGGTCGG 150 Act/nodiscus ungujs T -. GC ...... A. .. . . Amp/exJd/saJs fenestrafe, - • • • • • ------... TGC AAC .. A .••• Undescribed species •••••••••• . .. TGC AAC .• A .••• Rhodactlshowesli • - - - - - ••• - ---- ... TTG AAC.AA.T-- Rhodsctls musso/des - - - - - •• - - - - • - •... TTG AAC. AA. T - -

Actinod~cusnummlform~TCGGTCGCTA GGTCGGTCGG A TGTCGAGCG GATGTTTAGG GGGTCGGTGG 200 Act/nod/scus unguja .... c.. .. Amp/ex/discus fenestrafe, - - ••••• - •. .-.- - •• - •••• -- ••••••• -.- .G. Undescribed species • - •••• - •• Rhodsct/s howesl/ - •••••• - RhodactJs musso/des • - - - - - ••

Act/nod/scus nummlform/s A TGGA TGACG CCACCGTTAA ATGGCTGTTT AGGCGCTCCC GGCCTGCCTC 250 Actinod/scus ungujs ...... Amp/exJd/scus fenestrafe, - c.. G .. ------. T Undescribed species -C .. GC. •. -. T Rhodactfs howesi/ • C.. -G . ... T·· . - . - ·T .• - oCT Rhodactfs mussoldes - C.. - -G . • ·T. -- oCT

Actinod~aJsnummlformisCGCTGCCGCC ACTGCCGTCG CACCAGCCGC CGCCGTCGAC GAAGCTGGCC 30Ci Actlnodlscus ungula ------Amp/exidiscus fenestrafe, G.. CAG. C. - · .... TT-. T TTT. -c ... A · C. AA- - - -- Undescribed species G.. CAG. C.• · . A .. TT-. T TTT. ·C .•. A · .. AA····· Rhodactls howesll . CG. T. G .. G. - -. A .. GGAGG. A - .. . AG. -C. C .. A. C. A- - - -- Rhodactis mussoides ... CG. T. G · AG. -'. A .. GGAGG. A· .. ·AG. ·C. C.. A. C. A· - - ••

Actinodiscus nummiformis GTCCGTGC TT GCCAGTGTAC TACCTCTGAG ACCTGTGTGA CTAAGATGAA 350 Actinodiscus unguia •• - - • - - ••• . A Amp/exJd/scus fenestrafe' - - -. . T - - · . A ... AA. G A. AG· - -A. A GTT. ACT. AG AATGA. G..• Undescribed species . . T· . •. A •. CAA. G A. AG·· ·A. A GTT. ACT. AG AATGA. G . Rhodsctls howes// - - - T. . . T .. · TG .... ACG C. AA - - - A. C GTG. T .•. TT GCTTA. A . Rhodactis musso/des . . T .. · TG··. ·ACG C. AA·· ·A. A GCG. T ... TT GCTTA. A . 1~5.as

ActJnodiscus numm/formis - GACAAAAAA AACAAAAGGT TGACAACTTT GGACGGTGG - A TCTCTCGCG 400 Actinod/scus unguja A. A G . Amp/exid/scus fenestrafe,' CCG . · G. G. G.. A· T . . T. G. Undescribed species . CCG...... · G. G. G.. A- T T - T. GC Rhodactis howesll - - G... T. C .. A. G A· T...... • . T. GC Rhodsctis mussoldes •• G. . . G. C .. A. G A- T T ..... T. TC

Actinod/scus nummiform/s TCGCGCA TCG ATGAAGAACG CAGCCAAGTG CGATAACTAG TGTGAATTGC 450 Actinod/scus unguia Amp/ex/d/scus fenestrafe, ...... · ..... GC ...... G . Undescribed species ...... GC ...... G . Rhodact/s howesil • ...•. GC .. . .. G . Rhodsctis musso/des ....•. GC ...... G . 202 BULLETIN OF MARINE SCIENCE, VOL. 59, NO.1, 1996

Actinodlscus nummlfonnisAGAACTCAGT GAATCATCAA GTCTTTGAAC GCAAA TTGCG CTCCTGGGGT 500 ActJnodlscus unguja . . • ...... G •••• Amp/eKidiscus fenestrafe,. . • . T. , ...... G. . •.•.• G•.• ..T T. Undesctfbed species . . .. T . . ... , ... G. . ...•. G •.• ,. T T. Rhodact1s howesll .... T . . •...... G...... G..• .. T ..•. T. Rhodact1s musso/des . . . . T . . G. .. T .... T. I~TS2

ActJnodIscus nummlfonnis GTCCCGGG~G CA TGTCTGTC TG~GCGTCCG • TTTC • TTTT TACGCAAGCG 5SO ActJnodlscus ungula . • • , . Amplex/dlscus fenestrafe, C. . . . A...... T G. A AG.C- - .. TT.TTA . Undescribed species C .... A . .... T G. A AG.C- Rhodactishowesll C •••. A . .... T G. A AG.C. ------RhodactJs mussoides C .••. A ..•. .... T G. A AGCC. -- -- ••••

Actinodlscus nummlfO/111lsGCGGCGTCCG ACCCCCGCCG ACGGGTTTGA GGCTTTGTCG CGCTAGGTGG 600 Actlnodlscus ungula ...... •. · .. T ..... T . Amplex/dlscus fenestrafe, • AA TGAAGGA · A.. ". AAA · AAA. CGA .• · C. GGAA·G. -G .. C .. Undescribed species - AACGC - • - • ·T.··· .. G. G.••. G. T. · .. GGAA-G. · . , ·G .. C.. Rhodactls howesn - AACGC· ••• ·T. -- - - . G. C- · TTG .. G-. A · .. -G .. C.. Rhodactls mussoides • AACGC· - •• -T.--· .... .G·C· · TTG ... '. A · .. ·G. ·C ..

ActJnodiscus nummiformls CAGGAAGGCG CGTCCCTCTA AGTGACA TCC GCCACGGCTG GCCGGTCGGC 650 Actlnodlscus unguja . AmplexJdlscus fenestrafe,. TCTG T. TCA.G.A.GG GA. CTGTCTG T. T .. CTAC. · . GT CCT Undescribed species . TCTG T. TCA.G.A.GG GA. CTGTCTG T. T .. CTAC. · . GT CCT Rhodactis howesl; . GTTG T. TCA. GAA.GG ·A. CT. CCAG · AT-. CAAG. C. GT CCT Rhodactis musso/des . GTTGG .. T. TCA.GAA. GG -A.CT.CCAG · AT·. CAAG. C. GT CCT

ActJnodlscus nummlformls TGCGGTTCAC AGCGAGCCCC CGGAGGCGGC TCCGTCT·CG TTCCGCAACG 700 ActJnodiscus ungula ...... AA .. Amp/exldlscus fenestrafe,C. AA .. G.. A .. TAG··T. G T.CG A. G -- .. A. GGTA · GT .. G. GA. Undescribed species c. AA .. G.• A · GTAG--T. G T. CG A. G - - .. G. GGTC · GT .. G. GA. RhodactJs howesii C. AA .. G·. A · C .•• -- .. G A. A .. A.A. G - - - -A. GG.. CG· .. GG· .. RhodactJs musso/des C. AA .. G·. A · C. -- -- .. G A. A.. A.A. G - - - -A. GG. A CGA .. GGG ..

Actinod~cusnummlform~ACGCCGC·CG CGGCACGCCA GACGCGGGCA CCACAGCCGG TGACGCCGGC 750 ActJnodlscus unguja - .. A... G•...... T . ...•...... •.•..• G•....•.•.•.•. Amp/exidlscus fenestrafe' G. - - TAAA TA AA. A.A C CTTTTC. A.. .. GTC. AG. A .. TGT. T. T. Undescribed species G. •• TAAA TA AA.A. A•.. C ·TTTTC.A GTC.AG-A .. TGT. T. T. Rhodact1showesll GT· -. TGT .. GA. AGG-.T. A. GAGCA TTTT ... CT · TT .. A . Rhodactis mussoldes GT· '. TGT .. GA. AGGG. T. · . GAGC. A. G A. TTTT .. CT · TT .. A . 1~8S Act1nodlscus numm/formls TTGTCGA TCG ACCGACCGAC CACGGGAGAC CTCAGA TCAG GCAA 794 Actlnodlscus ungula ...... Amp/ex/d~cus fenestrafe' C '. CGCG. G. -AT.TTGTC. T. GGCTT. .. Undescribed species C '. CGCG. G. -AT.TTGTc. T. GGCTT. .. RhodactJs howesn C -. C. CCCGC CG. A.. ACCG · .• ,CTT. .. RhodactJs mussoides C -. C. CCCGC CG.A .. ACCG · . - -C TT. .. Figure 4. Alignment of the DNA sequences from 6 tropical corallimorpharians in the region from the 3'-end of 18S to the 5'-end of 28S rDNA. The ends of the 18S, ITS!, 5.8S, ITS2, and 28S are indicated by arrows at the top of the alignment. Dots indicate invariant positions among 6 species of corallimorpharians; dashes indicate gaps, which were introduced by the defaults options of Clustal V program and manual alignment. sequences had an average similarity of 97.5%, and the size of this region varied from 197 to 201 bases. Table 3 summarizes the differences among the eight clones. The length differences between clones were caused by deletions/insertions occurring at positions 315-318 and 317-321 (numbers refer to the consensus shown as Fig. 4). With a single exception, all of the remaining variations in this region were transversions (Table 3). CHEN ET AL.: CORAL SYSTEMATICS 203

Table 4. Size (bp) and (G+C)-content of the ITS regions of tropical corallimorpharians

Taxon ITSI (%GC) ITS2 (%GC) Rhodactis howesii 201 (50) 190 (65.1) Rhodactis mussoides 193 (49.4) 191 (65.3) Amplexidiscus fenestrafer 176(48) 205 (61.3) Undescribed species 177 (48.8) 220 (55.7) Actinodiscus nummiformis 350 (60.7) 231 (72.6) Actinodiscus unguja 286 (57.6) 228 (70)

Interspecific Variation in the ITS Regions.-For comparing the interspecific variation of ITS regions among the range of Australian tropical corallimorpharians, the complete nucleotide sequences of at least two cloned PCR products from each of five additional species were individually determined. The length of the ITS I region varied from 176 in Amplexidiscus fenestrafer to 350 bases in Actinodiscus nummiformis, and the (G+C) content varied from 48% in A. fenestrafer to 60.7% in A. nummiformis (Table 4). The length of the ITS2 region of 6 tropical corallimorpharians varied from 190 in Rhodactis howesii to 231 bases in A. nummiformis, and the (G+C) content varied from 55.7% in the undescribed species to 72.6% in A. nummiformis (Table 4). The optimum alignment shown as Figure 4 was generated using the CLUSTAL V program, but this can be regarded as only notional, as variation in both length and sequence in the ITS regions prohibits unambiguous alignment between the taxa, even using the highly conserved coding regions as relative standards. Sequence comparisons show that both of the ITS regions are conserved within genera but vary a great deal between genera (Table 5). The ITS sequences for the undescribed species were found to be sufficiently similar to those of A.fenestrafer that they could be aligned without difficulty (Fig. 5), implying a close relationship between these taxa. Intergeneric Relationships Inferred from 5.BS rDNA Sequence Comparisons.- Sufficient variation was present in the 5.8 rRNA coding sequences to permit phylogenetic analysis at the intergeneric level. Figure 6 shows an unrooted to- pology derived from exhaustive parsimony analysis of these data. The 5.8S sequence of the undescribed species differed at only one position from that of Amplexidiscus fenestrafer (Fig. 6), consistent with a close relationship between these taxa.

Table 5. Pair-wise comparison of ITS sequences, based on the alignment shown as Figure 4. (Values are percent identity.)

Taxon ITSI rrS2 Within genera Actinodiscus 89.5 83.4 Rhodactis 85.1 83.1 Amplexidiscus YS. undescribed species (US) 95.5 80.3 Between genera Mean:!: SE Mean:!: SE

Actinodiscus YS. Rhodactis 25.43 :'::2.33 20.62:!: 1.07 Actinodiscu.f YS. Amplexidiscus 21.79 :'::2.62 20.94:':: 0.6 Rhodactis YS. Amplexidiscus 28.35 :'::0.2 34.83 :'::0.3 US YS. Actinodiscus 21.65 :'::3.22 21.79 US YS. Rhodactis 28.63 :'::0.2 38.89 204 BULLETIN OF MARINE SCIENCE. VOL. 59. NO. ]. ]996

18S I ITSl ~~ Undescribed species AACCTGCGGA AGGATCATTA CCGTGGCAAG AAAACGAATA CCTTGTGAAC 50 Amp/exidiscus fenestrafer...... - ...... G ••.••......

Undescribed species CTGTTGCAAC CGATAGTTGG GGG·CTGGTC GGGCGCACCG TTAAATGGCT 100 Amp/exidiscus fenestra fer ...... G..••.. ••. T .••.•......

Undescribed species TTGGCCAGCC CCGCAACATT GTTTTCCCGA AGAAAACGTG TGCAAGCAAA 150 Amp/exidiscus fenestra fer ...... C...... 5 85 1-+ . Undescribed species GAAAGAAAGT TTACTTAGAA TGAAGGAAAC CGAAAAAAAG CGAGAGAGAC 200 Amp/exidiscus fenestrafer' ......

Undescribed species AACTTTTGAC GGTGGTTCTC TTGGCTCGCG CATCGATGAA GAACGCAGCC 250 Amp/exidiscus fenestra fer ...... •.... A •.•......

Undescribed species AGCTGCGATA AGTAGTGTGA ATTGCAGAAT TCAGTGAATC A TCGAGTCTT 300 Amp/exidiscus fenestra fer ......

Undescribed species TGAACGCAAA TGGCGCTCTT GGGTTCTCCC AGGAGCATGT CTGTCTGAGT 350 Amp/exidiscus fenestra fer ......

I~T52 Undescribed species GTCGGATTTC AGTCACC- -- - -GAACGC------TCGCGG GC - -GTGTTA 400 Amp/exidiscus fenestra fer ...... '" . TTAT TA ... T.AAG GAAAC .. AAA AAAA. C. AG.

Undescribed species GCCGGAAGGC GCGGGCGGCT CTGAGGTGTC ACGCACGGGA TCTGTCTGTC 450 Amp/exidiscus fenestra fer ......

Undescribed species TACCTACGGC GTGTCCCTCG AAGTGCAAAG TAGTCGTGCG GGCAGGCGGC 500 Amp/exidiscus fenestrafer' ...... •... A.

Undescribed species GGTCTGTCGG AGAGGCTAAA TAAAGAAAGC CC·TTTTCGA CACCGTCGAG 550 Amp/exidiscus fenestra fer . . . A...... C ...... 1-+285 Undescribed species -ATGTGTCTG TCCGCGCGTG GATGATGTCT TTGGCTTGAC CTCAGA TCAG 600 Amp/exidiscus fenestraferG ......

Undescribed species GCAA 604 Amp/exidiscus fenestrafer' ...

Figure 5. A]ignment of the sequences for the region from the 3'-end of ]8S to the 5'-end of 28S rDNA between the undescribed species and Amplexidiscus fenestrafer. Data are presented as in Figure 4.

These analyses also indicate a close relationship between Amplexidiscus fenestrafer and Rhodactis spp., and a much more distant relationship between these genera and Actinodiscus spp.; in the 175 bp 5.8S rDNA, 20 positions (=11.4% of positions) were constant within Actinodiscus spp. and common between the genera Rhodactis and Amplexidiscus but differed between the two groups.

DISCUSSION Intraspecific Variation in the ITS] Region of Rhodactis howesii.-Differences observed between individual cloned ITS sequences may reflect errors arising from CHEN ET AL.: CORAL SYSTEMATICS 205

Amp/eXidiSCUS fenestra fer

[ Actinodiscus nummiformis Undescribed species

Actinodiscus unguja Rhodactis howesii

[ Rhodactis mussoides Figure 6. Unmoted phylogenetic tree for six species of tropical corallimorpharians. This tree was generated from the aligned 5.8S rDNA sequences (consensus length = 173 bp) using the exhaustive search option in PAUP 3.1.1. Tree length = 22. the use of Taq polymerase, or real intraspecific variation (Gonzalez et aI., 1990; Wesson et aI., 1992; Gardes et aI., 1991; Vogler and DeSalle, 1994). The presence of the same variant in clones from different individuals is strong evidence against these differences being artifactual (i.e., PCR errors), and this is the case for all but one of the R. howesii sequence variants. The level of intraspecific variation detected in R. howesii is comparable with other species; for example, the corre- sponding figures for the mosquito, Aedes aegyptii are 1.07% and 1.17% for the ITS 1 and ITS2 regions respectively (Wesson et aI., 1992), and the average variation between six human ITSI clones was 0.34% (range: 0-1.46%; Gonzalez et aI., 1990). Replication slippage, the mechanism responsible for length polymoT'- phisms in microsatellites (SchlOtterer and Tautz, 1992) is also the probable cause of intraspecific length polymorphisms in ITS regions (Stewart et aI., 1983; Gon- zalez et aI., 1990; Gardes et aI., 1991; Wesson et aI., 1992). Interestingly, in R. howesii the majority of the variations are in the 5'-region of ITSl, which in Drosophila has been shown to diverge particularly rapidly (SchlOtterer et aI., 1994) and to account for most intraspecific variants (SchlOtterer and Tautz, 1992). Multigene families, such as the rRNA genes, undergo concerted evolution-they evolve coordinately within a species. A variety of processes effectively homog- enize the individual repeating units within interbreeding species, however, intrach- romosomal homogenization is thought to occur at a much higher rate than inter- chromosomal processes (Schl6tterer et aI., 1994). The observed level of intraspecific variation, and in particular the fact that dinucleotide insertion/deletion events account for a large proportion of this, suggests that R. howesii harbours multiple rRNA loci on different chromosomes, for which there are several precedents (see, for example, Kumar and Rai, 1990). Interspecific Variation of ITS Sequences.-Considerable variation exists in the size of ITS regions amongst eukaryotes. For example, the ITS] is around 1,000 bp in mammals (Michot et aI., 1983; Gonzalez et aI., ]990) whereas in some spruce species it is around 165 to 175 bp (Smith and Klein, ]994). At 176-350 (ITS 1), and 190-231 (ITS2) bp, the ITS regions of tropical coraIlimorpharians are similar in size to those of many other invertebrates. The moderate to high % (G+C) content of the ITS regions in the Australian corallimorpharians (48-60.7% in ITS1 and 55.7-72.6% in ITS2) is also typical, and may be required for pro- cessing of the primary transcript (although the ITS regions of some Drosophila spp. have <20% (G+C); Schl6tterer et aI., 1994). Although it was not possible to unambiguously align the ITS sequences of all six tropical corallimorpharians, these regions are phylogenetically informative. Firstly, the ITS sequences from congeneric species were very similar and cOUlld 206 BULLETIN OF MARINE SCIENCE. VOL. 59. NO. I. 1996

be aligned unambiguously (Table 5; Fig. 4). Secondly, the ITS sequences of the undescribed species closely resembled those of Amplexidiscus fenestrafer; the degree of similarity (95.5% identity in ITSI and 80.3% in ITS2, based on the alignment shown in Fig. 5) suggests that the undescribed species should be as- signed of to the genus Amplexidiscus. A full taxonomic description of this species is in preparation (Chen, unpubl.); however, preliminary morphological studies, summarized below, also support this conclusion. Although the undescribed species is considerably smaller than Amplexidiscus fenestrafer (oral disc diameters around 60 mm and 280-400 mm respectively), the two are very similar in all other aspects of external morphology : the discal tentacles are small and simple, and the few marginal tentacles are large and barely discernible when the animal is expanded, becoming pronounced only in contraction (Chen, unpubl., ref. to Dunn and Hamner, 1980). The external and internal morphology of A.fenestraferclearly distinguish it from other tropical corallimorpharians (Dunn and Hamner, 1980); detailed internal and anatomical examination of the undescribed species should unambiguously clarify the relationship of the two taxa. Intergeneric Relationships among Tropical Corallimorpharians.-After re-ex- amining the type specimen of the family Actinodiscidae, (Actinodiscus nummiformis) and all of the Caribbean species, den Hartog concluded that the genera were without significant morphological basis, and lumped all of the Actinodis- cidae into the single genus, Discosoma (den Hartog, 1980). When viewed in the context of den Hartog's conclusion, the level of variation in the 5.8S rONA (which is usually considered to be highly-conserved) amongst tropical corallimorpharians is surprizingly high. Although there are no strictly comparable studies, the degree of 5.8S rONA variation between Actinodiscus spp. and Rhodactis/Amplexidiscus is approximately the same as that between the most distantly related higher plants (Suh et aI., 1992). Our data are thus not consistent with the assignment of these corallimorpharians to a single genus. Although analysis of the 5.8S rONA sequences indicates a close relationship between the genera Rhodactis and Amplexidiscus and a distant relationship between these genera and Actinodiscus, this region is unlikely to provide the reso- lution required to evaluate close intergeneric relationships amongst corallimorpharians. To clarify these relationships, we are presently sequencing a large segment (approximately 1,000 bp) of the 28S rONA for a wide range of tropical and temperate corallimorpharians. The Potential of ITS Sequence Comparisons to Resolve Closely-related Taxonom- ic Groups among the Corallimorpharia.-Tropical corallimorpharians clearly re- quire taxonomic revision before evolutionary relationships can be addressed. As in several other invertebrate groups, relationships between sibling species and the assignment of new species are major problems (Knowlton, 1993; Knowlton and Jackson, 1994). In particular, the relationships between geographically-distinct, but morphologically-similar, species such as Rhodactis howesii, R. indosinensis, R. rhodostoma and R. sanctithoma (Chen, in prep.) are unclear. In such cases molecular criteria are potentially particularly useful. The present study indicates that the ITS regions of the ribosomal transcription unit are likely to be useful in assessing intrageneric relationships amongst tropical corallimorpharians.

ACKNOWLEDGMENTS

The authors thank Dr. D. Blair, Dr. R. G. Rowan, Dr. J. E. Veron, Mr. P. Spencer and Mr. D. Odorico for commenting on the manuscript. Special thanks to Mr. B. Stobart for collection and field assistance. This work was supported by grants from the Australian Research Council (DJM) and the Great Barrier CHEN ET AL.: CORAL SYSTEMATICS 207

Reef Marine Park Authority (CAC). CAC is a recipient of OPRA and JCUPA scholarships. This paper is adapted from a thesis by CAC in partial fulfilment of the rcquirement for a PhD degree at James Cook University.

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DATEACCEPTED:May 23, 1995.

ADDRESS: (C.A.C.) Department of Molecular Sciences, and Department of Marine Biology, James Cook University of North Queensland, Townsville, Queensland 4811, Australia; (B.L.W.) Department of Marine Biology, James Cook University of North Queensland, Townsville, Queensland 4811. Aus- tralia; and (D.J.M.)*, Department of Molecular Sciences, James Cook University of North Queensland. Townsville, Queensland 4811, Australia. (e-mail;[email protected]) *To whom all correspon- dence should be addressed.