A test of monophyly of the gutless Phallodrilinae (Oligochaeta, Tubificidae) and the use of a 573-bp region of the mitochondrial cytochrome oxidase I gene in analysis of annelid phylogeny JOHAN A. A. NYLANDER,CHRISTER ERSEÂ US &MARI KAÈ LLERSJOÈ Accepted 7 Setember 1998 Nylander, J. A. A., ErseÂus, C. & KaÈllersjoÈ , M. (1999) A test of monophyly of the gutless Phallodrilinae (Oligochaeta, Tubificidae) and the use of a 573 bp region of the mitochondrial cytochrome oxidase I sequences in analysis of annelid phylogeny. Ð Zoologica Scripta 28, 305±313. A 573-bp region of the mitochondrial gene cytochrome c oxidase subunit I (COI) of two species of Inanidrilus ErseÂus and four species of Olavius ErseÂus (Phallodrilinae, Tubificidae) is used in a parsimony analysis together with a selection of 35 other annelids (including members of Polychaeta, Pogonophora, Aphanoneura, and the clitellate taxa Tubificidae, Enchytraeidae, Naididae, Lumbriculidae, Haplotaxidae, Lumbricidae, Criodrilidae, Branchiobdellida and Hirudinea), and with two molluscs as outgroups. The data support the monophyly of the Olavius and Inanidrilus group, with a monophyletic Inanidrilus. However, parsimony jackknife analyses show that most of the other groups are unsupported by the data set, thus revealing a large amount of homoplasy in the selected gene region. Practically no information is given of within/between family relationships except for a few, closely related species. This suggests that the analysed COI region is not useful, when used alone, for inferring higher level relationships among the annelids. Johan A. A. Nylander, Department of Systematic Zoology, Evolutionary Biology Center, Uppsala University, NorbyvaÈgen 18D, SE-752 36 Uppsala, Sweden. Christer ErseÂus, Department of Invertebrate Zoology, Swedish Museum of Natural History, Box 50007, SE-104 05 Stockholm, Sweden. E-mail: [email protected] Mari KaÈllersjoÈ, Laboratory of Molecular Systematics, Swedish Museum of Natural History, Box 50007, SE-104 05 Stockholm, Sweden Introduction for this group, identifying two genera within it, Inanidrilus Most of the marine Oligochaeta belong to the family ErseÂus, 1979 and Olavius ErseÂus, 1984. Today, about 70 Tubi®cidae, which is currently subdivided into ®ve subfa- species have been described from, mostly, shallow marine milies: Tubi®cinae, Rhyacodrilinae, Telmatodrilinae, waters of tropical and subtropical areas (Giere et al. 1995; Limnodriloidinae and Phallodrilinae (ErseÂus 1990). ErseÂus ErseÂus & Giere 1995; ErseÂus 1997). pointed out, however, that the Tubi®cidae is likely to be No complete phylogeny of the gutless species and their paraphyletic and should include, at least, the Naididae. closest relatives exists and their position within the Phallo- Within the Phallodrilinae, a group of species completely drilinae is unclear (ErseÂus 1992). Previous phylogenetic lacking an intestinal system has been identi®ed (Giere analyses of oligochaetes were all based on various morpho- 1979, 1981; ErseÂus 1979; Giere et al. 1995). These gutless logical characters (e.g. ErseÂus 1984, 1990, 1992; Ferraguti tubi®cids are nutritionally dependent on symbiotic bacteria & ErseÂus, in press; Jamieson et al. 1987; Brinkhurst 1988, living in the body wall (Giere 1981, 1985; Giere & 1989, 1991, 1994; Jamieson 1988; ErseÂus & Ferraguti Langheld 1987; Giere & Milligan 1989; Giere et al. 1995; 1995). The primary aim of the present study is to test the Richards et al. 1982). It was ®rst believed that some gutless monophyly of the gutless group within the Phallodrilinae phallodrilines had evolved in parallel (ErseÂus 1979, 1981, using mitochondrial DNA (mtDNA) sequence data. 1983), but ErseÂus (1984) proposed a monophyletic origin The gene used, cytochrome c oxidase subunit I (COI), is Q The Norwegian Academy of Science and Letters . Zoologica Scripta, 28, 3±4, October 1999, pp305±313 305 Monophyly of gutless Phallodrilinae . J. A. A. Nylander et al. a protein-coding gene of the mtDNA. At the amino acid- 30) and HCO 2198 (50-TAAACTTCAGGGTGAC- level, it is generally regarded as one of the most conserva- CAAAAAATCA-30) (Folmer et al. 1994), applying the tive genes of the animal mitochondria (Kondo et al. 1993; following PCR pro®le: (948C/30 s ± 498C/30 s ± 728C/ Simon et al. 1994; Boore & Brown 1995; Cummings et al. 2 min) X 32 cycles. A typical PCR-protocol was as follows: 1995). At the same time, the COI gene contains more Template DNA, 5 mL; AmpliTaq Polymerase, 1.25 units; rapidly evolving nucleotide sites, particularly in third Primer concentration, 20 pM; dNTP concentration, codon positions, which make it potentially useful for infer- 200 mM; MgCl2 concentration, 2 mM; and 5 mL 10X PCR ring more recent branching events. It has been used to buffer. Sterile water was added to give a total volume of assess relationships at practically all systematic levels, from 50 mL. PCR products were puri®ed using the QIAquick phyla (Folmer et al. 1994; Hoeh et al. 1996) to within spin PCR Puri®cation Kit (QIAGEN, Santa Clarita, CA species (Sperling & Hickey 1994; WuÈ ster et al. 1995). This USA) following the protocol from the manufacturer. study therefore has a secondary aim: to see whether COI- sequence data are useful also at different levels of annelid Sequencing relationships. Species investigated are representatives from Puri®ed DNA was sequenced by the dideoxy method a wide range of taxa. They include species believed to be (Sanger et al. 1977) using both of the ampli®cation primers closely related within a genus, but we also do comparisons and three additional, internal primers: C1-J-1751 (50- between taxa of much higher ranks. GGATCACCTGATATAGCATTCCC-30) of Simon et al. (1994); COI-355, alias LumberJack (50-GGAACAGGAT- Material and methods GAACAGTTTACCC-30), a modi®cation of the C1-J- The COI-sequence data analysed in this paper come from 1859 primer of Simon et al. (1994); and COI-355R, alias a variety of sources. Data for the six species of gutless Reversed LumberJack (50-GGGTAAACTGTT- Phallodrilinae and ®ve other oligochaete species (see CATCCTGTTCC-30). The COI-355R primer is the Table 1 and below) were sequenced by the ®rst author reverse-complement of the COI-355 primer and both were following the procedures outlined below. Other sequence developed at the Laboratory of Molecular Systematics, data were kindly provided by Professor Bent Christensen, Swedish Museum of Natural History. The number (355) Copenhagen (Christensen & Theisen, 1998; B. Christen- refers to the position of the D. yakuba 50 nucleotide (Clary sen, unpublished; data available upon request) or were & Wolstenholme 1985). For cycle sequencing, the extracted from Genbank (See Table 1). Two molluscs Thermo Sequenase cycle sequencing with ¯uorescent 1 Katharina tunicata (Polyplacophora) and Oliva sayana dye CY5-primer labelling protocol from Amersham Phar- (Gastropoda) were used as outgroup taxa in the parsimony macia Biotech (Uppsala, Sweden) was used, except that the analyses. samples were not ethanol precipitated after thermocycling. For each sequencing reaction 2 pmol primer (1.5 pmol/ Specimens mL) and 5 mL PCR product were used with a cycle pro®le Single specimens of the gutless phallodrilinae species of 958C for 2 min, then (958C/30 s ± 488C/30 s ± 728C/ Olavius albidus, O. prodigus, O. tantulus, O. imperfectus, Inan- 60 s) X 30 cycles. Electrophoresis was carried out on a 6% idrilus reginae, I. leukodermatus, two other phallodrilines acrylamide gel using an ALF-Express DNA sequencer (Aktedrilus arcticus, Pirodrilus minutus), two rhyacodrilines from Amersham Pharmacia Biotech. (Heronidrilus heronae, Heterodrilus paucifascis) and one enchytraeid (Fridericia tuberosa), were used for sequencing. Alignments Specimens were collected by Dr Emilia Rota, Siena, Italy Sequences were aligned with the sequence of Lumbricus (F. tuberosa) and the second author (all other taxa) and terrestris (Boore & Brown 1995) using AssemblyLIGN v preserved in 80±96% ethanol. 1.0.7 (Oxford Molecular Group Inc., Campbell, CA, USA). Final alignment adjustments were done manually, Extraction and PCR amplification comparing both nucleotides and amino acids. Nucleotides Genomic DNA was extracted following a general extrac- were translated into amino acid sequences using the L. tion method as described by Palumbi et al. (1991). In brief, terrestris genetic code (Boore & Brown 1995) for the anne- single alcohol-preserved specimens were ground in homo- lids, and the Invertebrate Mitochondrial Code (Hoffmann genizing buffer and incubated overnight with proteinase K. et al. 1992) for the molluscs. DNA was then isolated by phenol-chloroform extraction followed by ethanol precipitation. A 710-bp fragment of Sequence analyses the COI gene was ampli®ed using the ¯anking primers PAUP v 3.1.1 (Swofford 1991) was used for ®nding the LCO 1490 (50-GGTCAACAAATCATAAAGATATTGG- most parsimonious trees. Heuristic searches with 100 306 Zoologica Scripta, 28, 3±4, October 1999, pp305±313 . Q The Norwegian Academy of Science and Letters J. A. A. Nylander et al. Monophyly of gutless Phallodrilinae Table 1 Table of included species, with references to COI gene sequences. Sequence sources: 1 = Boore & Brown (1994); 2 = Harasewych, M. G., Adamkewicz, S. L., Blake, J. A., Saudek, D. M., Spriggs, T. & Bult, C. J. direct GenBank submission; 3 = present study; 4 = Christensen & Theisen (1998); 5 = Christensen unpublished; 6 = Black, M. B., Hoeh, W. R., Hashimoto, J., Debruyeres, D., Lutz, R. A. & Vrijenhoek, R. C. direct GenBank submission; 7 = Boore & Brown