Trefusiidae Are a Subtaxon of Marine Enoplida (Nematoda): Evidence from Primary Structure of Hairpin 35 and 48 Loops of SSU Rrna Gene L
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Molecular Biology, Vol. 35, No. 5, 2001, pp. 778–784. Translated from Molekulyarnaya Biologiya, Vol. 35, No. 5, 2001, pp. 912–919. Original Russian Text Copyright © 2001 by Rusin, Aleshin, Vladychenskaya, Milyutina, Kedrova, Petrov. MISCELLANEOUS UDC 575.85'113:575.86:595.132.1 Trefusiidae Are a Subtaxon of Marine Enoplida (Nematoda): Evidence from Primary Structure of Hairpin 35 and 48 Loops of SSU rRNA Gene L. Yu. Rusin, V. V. Aleshin, N. S. Vladychenskaya, I. A. Milyutina, O. S. Kedrova, and N. B. Petrov Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119899 Russia; E-mail: [email protected]; fax: (095) 939-3181 Received December 22, 2000 Abstract—A rare nucleotide substitution was found in the evolutionarily conserved loop of hairpin 35 of the 18S rRNA gene of marine free-living nematode, Trefusia zostericola (Nematoda: Enoplida). The same substi- tution was found in all the marine Enoplida studied but not in other nematodes. Such a molecular synapomor- phy indicates that marine enoplids are more closely related to T. zostericola than to freshwater Triplonchida. Maximum parsimony, neighbor-joining, and maximum likelihood analyses of complete nucleotide sequences of the gene, with the heterogeneity of nucleotide sites in evolution rates taken into account, support this con- clusion. Hence, the hypothesis of particular primitiveness of Trefusiidae among nematodes should be rejected. Phylogenies based on molecular data support the morphological reduction of metanemes in Trefusiidae. Along- side with the unique change in hairpin 35 loop among marine Enoplida (including T. zostericola), hairpin 48 is also modified by a rare transversion which could be found among Mesorhabditoidea nematodes, in related gen- era Pelodera, Mesorhabditis, Teratorhabditis, Parasitorhabditis, Crustorhabditis, and Distolabrellus, and in 11 orders of Rhodophyta. Rare mutations in hairpins 35 and 48 tend to be fixed correlatively in evolution and could be found in all the Acanthocephala species. X-Ray data show that these regions (H31 and H43, in alter- native nomenclature) are spatially brought together in native ribosomes. The nature and distribution of molec- ular autoapomorphies in phylogenetic trees of high-rank taxa are discussed. Key words: 18S rRNA gene, molecular phylogeny, Nematoda, Adenophorea, Enoplida, Trefusiidae, long- branch attraction (LBA) effect INTRODUCTION todes could help select species for further nucleotide sequence studies. In this respect, free-living marine The breakthrough in Caenorhabditis elegans nematodes of the Enoplida order are of special inter- genome sequencing placed it among organisms most est. Basing on the morphological characters of these profoundly studied by molecular methods, and made animals, a hypothesis has been suggested that this its genome a milestone in comparative studies of group is the most primitive among nematodes [3, 4]. genes of other multicellular animals. In addition, Cladistic analysis has isolated from Enoplida a para- C. elegans is one of the model objects in embryology. phyletic residue that was combined in order Trefusiida The influence of mutations of some genes is intensely [5]. However, no synapomorphies in anatomy and fine studied in relation to programmed cell death during structure of these animals have been revealed [5], embryogenesis, to development of the nervous sys- hence cladistic analysis attributes them to the most tem, and in other aspects. To understand which of primitive nematodes. Previous analyses also placed these properties are typical of C. elegans and which Trefusiidae at the basis on the nematode evolutionary could be found in other animal taxa differently related tree [6–8] because Trefusiidae do not have well-devel- to C. elegans, new taxa are to be studied, especially oped characters of morphological specialization [4–6]. those belonging to Nematoda [1]. Nowadays, large- scale investigations are being carried out on some ani- The aim of the present study was to test the hypoth- mal and plant nematode parasites (Brugia malayi, esis of the ancestral origin of Trefusiidae employing Onchocerca volvulus, Ancylostoma caninum, Meloid- molecular data. Molecular synapomorphies were dis- ogyne incognita) and free-living nematode, Pristion- covered in Trefusia zostericola, as well as in Enoplida chus pacificus. Numerous documents on the genome species. This observation contradicts the hypothesis structure of these species are kept in GenBank. of particular evolutionary primitiveness of Trefusiidae Knowledge of the phylogenetic relatedness of nema- species. 0026-8933/01/3505-0778 $25.00 © 2001 MAIK “Nauka /Interperiodica” SYNAPOMORPHIC SUBSTITUTIONS IN NEMATODE 18S rRNA GENE 779 EXPERIMENTAL Mermithida, and Trichinellida orders), (iii) Triplon- chida (including Tobrilina and Diphtherophorina), DNA was isolated from ethanol-fixed Trefusia zos- and, (iv) Enoplida (including marine representatives tericola specimen collected in the White Sea Bay of of the order) have the highest bootstrap support. The Kandalaksha. PCR-amplified fragments of 18S rRNA composition of these clades differs from the conven- genes were obtained using primers described earlier tional one, but this result is in accord with data [9], purified electrophoretically and cloned in pBlue- obtained earlier in molecular and embryological phy- script KS+ (Stratagene) linearized with EcoRV. logenetic studies [18–20]. The sequence of the 18S Nucleotide sequences of both strands were deter- rRNA gene of Trefusia zostericola with high bootstrap mined using the fmol DNA Sequencing System support at all the trees belongs to the same cluster as (Promega) with universal primers to the vector part of those of other marine Enoplida, except for the MP and plasmid and a set of internal primers complementary NJ trees constructed without correction for the site- to conserved regions of the sequenced gene. The specific heterogeneity of evolution rates (Fig. 1). resulting sequence (GenBank AF329937) was a con- sensus of four completely and two partially sequenced To check the reliability of placement of T. zosteri- clones. The numbers of GenBank sequences used in cola in the Enoplida cluster, the ML method of phylo- phylogenetic analysis are given in the figures. genetic mapping was employed [17]. The same set of Sequences were aligned manually using the model of species excluding outgroups was subdivided into four 18S rRNA secondary structure given in [10]. Align- groups (Enoplida, Triplonchida, Dorylaimia, and ments can be obtained on request (petr@bio- Chromadoria) corresponding to the monophyletic evol.genebee.msu.su). Complete sequences were used clades at the tree (Fig. 1). T. zostericola was taken as in analyses except the V9 hypervariable region (apical a separate group to verify its position at the tree. For part of hairpin 49). MP, NJ (PHYLIP [11]), and ML each of the five possible quartets of taxa, support val- (fastDNAml, version 1.2.2 [12]) methods were ues were calculated for each of the three possible employed to construct phylogenetic trees. Bootstrap topologies using the TN model corrected for the het- analysis (1000 resamplings for MP and NJ trees and erogeneity of sites in the rate of evolution. The results 20 resamplings for ML trees) was used to assess the of this analysis are presented in the table. It is evident statistical validity of internal nodes [13]. For ML that the highest support in all the cases is observed in trees, an alternative approach was used, taking into topologies uniting T. zostericola with Enoplida (67.7, account numerous suboptimal trees which did not dif- 66.3, and 62.8% in different combinations of the taxa fer from the minimal tree by the Kishino–Hasegawa quartets). All the alternative combination of T. zoster- criterion [14]. When phylogenetic trees were con- icola with in other taxa had much lower support. structed with regard to differences in the evolutionary DNAPARS (PHYLIP package) allows deducing rate of site-specific substitution, parameters of stepwise changes in sequences for each node of the gamma-distribution were calculated using PUZZLE maximum parsimony tree constructed with the aid of 4.0 [15] and TN model [16] with six categories of sites this program. The set of the inferred Enoplida apo- differing in the rate of substitution accumulation. The morphies (including T. zostericola and excluding same program and model were employed in four-clus- Triplonchida) contains 55 characters. Comparison of ter mapping [17]. the corresponding regions in an extended set of 18S rRNA sequences shows that 53 of them are located in RESULTS AND DISCUSSION the evolutionarily variable regions of the gene. For 43 characters, variations were observed even among Nucleotide sequences of the 18S rRNA gene of five analyzed sequences of marine Enoplida. In other representatives of all the main evolutionary lineages words, fixation of nucleotide substitutions at these of Nematoda contained in GenBank were analyzed. sites took place repeatedly during evolution. For 11 Among them, the set of Enoplida sequences was traits identical for five Enoplida, frequencies of exhaustive to date. Representatives of aschelminths homoplastic substitutions among representatives of (nematophores, priapulids, gastotrichs, and rotifers) other nematodes could differ. The only A G tran- and more distant evolutionary lineages, annelids and sition (site 1195 in Enoplus brevis sequence) is located cnidarians, were used as outgroups. in the conserved region of the gene. Among nema- todes,