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Phylogenetic position of the Tardigrada based on the 18S ribosomal RNA gene sequences
Zoological Journal of the Linnean Society (1996), 116: 61–69. With 2 figures
Tardigrade biology. Edited by S. J. McInnes and D. B. Norman
Phylogenetic position of the Tardigrada based on the 18S ribosomal RNA gene sequences SEUNG YEO MOON AND WON KIM Department of Molecular Biology, Seoul National University, Seoul 151-742, Korea
The phylogenetic position of the Tardigrada remains uncertain. This is due to the limited information available, and the uncertainty of whether some characters are homologous or analogous with other taxa. Based on some morphological characters, current discussion centres on whether the taxon branches from the annelid-arthropod lineage, or lies within the arthropod complex. The molecular data presented here from an analysis of the 18S rRNA gene sequences are used to test the validity of these two hypotheses. Phylogenetic inference by the maximum parsimony and distance (neighbour-joining) methods suggests that the Tardigrada is a sister group of the major protostome eucoelomate assemblage that emerged before the arthropods, annelids, molluscs, and sipunculids evolved. The tardigrade clade also appears as an independent lineage separate from the nematode clade, thus supporting the current idea that tardigrades do not have a close aschelminth relationship. The molecular data also imply that several morphological features, considered significant in determining the phylogenetic relationships of tardigrades, are not synapomorphic characters. ©1996 The Linnean Society of London
ADDITIONAL KEY WORDS: — molecular phylogeny – 18S rDNA. CONTENTS Introduction . . . . . . . . . . . . Material and methods . . . . . . . . . Taxa compared . . . . . . . . . DNA extraction, PCR amplification, cloning Phylogenetic analysis . . . . . . . . Results and discussion . . . . . . . . . Acknowledgements . . . . . . . . . . References . . . . . . . . . . . . .
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61 62 62 62 63 63 65 65
INTRODUCTION
Microsopic size (50 µm–1200 µm) and a small number of distinctive morphological characters have led to relatively few taxonomic works on tardigrades (for recent reviews, see Schuster et al., 1980; Pilato, 1969, 1975, 1982; Bertolani, 1981, 1990, 1992; Kristensen, 1987; Nelson & Higgins, 1990; Nelson, 1982, 1991). Although they were recognized as a phylum by Ramazzotti (1962), their taxonomic rank and phylogenetic affinity remain unresolved. They are considered as enigmatic because some of their somatic characters are found also in other taxa, such as the 0024–4082/96/010061 + 09 $18.00/0
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©1996 The Linnean Society of London
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Nematoda and the protostome coelomates (Annelida, Onychophora, and Arthropoda). Moreover, the absence of clear fossil records and detailed embryological data for tardigrades has increased the difficulty of deciding whether these characters are homologous or analogous with other taxa. Current discussion has focused on tardigrades either stemming from the annelid-arthropod line or being located with the arthropod complex, with a particular affinity to the Onychophora (see Marcus, 1936; Greven, 1982; Hickman, Roberts & Hickman, 1984; Brusca & Brusca, 1990; Kozloff, 1990; Meglitsch & Schram, 1991; Ballard et al., 1992; Kinchin, 1992; Fortey & Thomas, 1993). However, the molecular information presented here from an analysis of the 18S rRNA gene sequences offers a different perspective on the phylogenetic position of the Tardigrada.
MATERIAL AND METHODS
Taxa compared The taxa compared in the present study were tardigrade (Hypsibius sp.), nematode (Caenorhabditis elegans), arthropod (Eurypelma californica, Artemia salina, Tenebrio molitor), annelid (Chaetopterus sp.), mollusc (Cryptochiton stelleri), sipunculid (Golfingia gouldii) and nemertine (Cerebratulus lacteus). The platyhelminthes Dugesia tigrina was designated as the outgroup. The culture of live tardigrade Hypsibius sp. was obtained from Ward’s Natural Science International Marketing Group. The complete sequence of the 18S rRNA gene of the tardigrade was determined in the present study, and compared with published gene sequences of the other nine taxa (Ellis, Sulston & Coulson, 1986; Field et al., 1988; Hendricks et al., 1988; Turbeville, Field & Raff, 1992). DNA extraction, PCR amplification, cloning and sequencing Live individuals were washed with distilled water to remove all the external debris and then starved to remove gut contents. The individuals were disintegrated in a microtube using a sonicator for a few minutes, with intervals of 30 seconds for cooling the tube on ice. The isolation of genomic DNA from the individuals was performed using the method modified from Blin and Stafford (1976). The 18S rDNA was amplified using PCR (Saiki et al., 1985, 1988) with two primers located at either end of the molecule (5'-CCTGGTTGATCCTGCCAG-3', 5'-TAATGATCCTTCCGCAGGTTA-3'). The thermal cycle parameters were 94°C, 1 min (initial denaturation, 5 min)/52°C, 2 min/72°C, 3 min (final extension, 10 min). The reaction was cycled 30 times. The amplified double-stranded 18S rDNAs were extracted with equal volumes of phenol:chloroform, followed by purification with GENE CLEAN II kit (BIO 101), and diluted in distilled water. For blunt-ended ligation, both ends of the PCR products were modified using T4 kinase and T4 polymerase. The blunt ended 18S rDNAs were purified with the GENE CLEAN II kit, inserted into pUC19 plasmid vector and transformed to DH5-α cell lines. The double-stranded recombinant plasmid DNA was purified by PEG precipitation or QIAGEN plasmid kit (Pharmacia). The DNA sequencing was performed by the dideoxy-termination method (Sanger et al., 1977) using Taq-Track kit (Promega), with two vector primers [M13 universal primer (–40), M13 reverse primer (–24)] and
PHYLOGENETIC POSITION OF TARDIGRADA
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an additional fourteen primers as used by Spears and colleagues (1992). Sequencing reaction mixtures were electrophoresed on buffer-gradient 6% polyacrylamide gels. Phylogenetic analysis Sequence alignment (Appendix) was performed using the CLUSTAL V multiple alignment program (Higgins, Bleasby & Fuchs, 1992) beginning at universally conserved regions, and then checked by eye. To avoid possible systematic errors, regions of ambiguous alignment including positions exhibiting high length variability were excluded from the phylogenetic analyses. Both maximum parsimony and distance (neighbour-joining) methods were used for the analysis. The bootstrapping method of data resampling (Felsenstein, 1985) was applied to evaluate the significance of the result. For maximum parsimony analysis, the BRANCH AND BOUND search option of PAUP, version 3.0s (Swofford, 1990) was used except for bootstrapping. The HEURISTIC search was employed for the bootstrap test by using TBR branch-swapping and CLOSEST stepwise addition. Ten trees were held at each step. Alignment gaps and unknown bases were considered as missing data. Neighbour-joining analysis was applied using PHYLIP, version 3.5c (Felsenstein, 1993). Evolutionary distances were calculated by the DNADIST program, using Kimura two-parameter model with no transition/transversion bias. Bootstrapping was performed with the SEQBOOT program, with a random input order of taxa.
RESULTS AND DISCUSSION
Maximum parsimony analysis generated a single minimum-length tree of 666 steps with 180 phylogenetically informative sites (Fig. 1A). The overall consistency index is 0.748, with a consistency index excluding uninformative characters of 0.609. The tree topology is largely concordant with the previous results based on the 18S rRNA gene sequence data in assemblages of the protostomes, including the position of the nemertine Cerebratulus lacteus (see Turbeville et al., 1991, 1992). The tardigrade Hypsibius sp. appears as a sister group of the protostome assemblage which includes representative species of arthropods, annelids, molluscs, sipunculids and nemertines. The tardigrade clade also represents a discrete lineage apart from the nematode clade (Fig. 1A). This proposal is supported by bootstrap analysis (Fig. 1B). Measurement of the reliability of the maximum parsimony phylogeny, by considering all trees within 1% of the length of the shortest tree, also supports the significance of the separation of the tardigrade from the major protostome assemblage (Fig. 1C). As the nematode branch is faster evolving, and the maximum parsimony analysis may generate an erroneous tree(s) when the rates of evolution vary with the evolutionary lineage (Felsenstein, 1978; Nei, 1991), this parsimonious solution was compared with that of the distance (neighbour-joining) method, which is more efficient when the rate of nucleotide substitution varies with lineages (Nei, 1991; Olsen & Woese, 1993). The neighbour-joining analysis, with bootstrapping, was also consistent with that of the maximum parsimony analysis, except for a little variation in the placement of the sipunculid Golfingia gouldii and the mollusc
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Cryptochiton stelleri within the protostome assemblage (Fig. 2). This variation may be explained by the rapid diversification of members in the protostomes, and shows that these 18S rRNA gene sequences are not useful for determining the positions of the sipunculid and the mollusc (Turbeville et al., 1991, 1992). The molecular data presented here propose that tardigrades originated before the advent of the protostomes, and dismisses the hypothesis that they have affinities with both or either of the annelids and arthropods. These data also imply that several morphological features such as cuticle, muscle attachment, cephalic appendages, and/or excretory osmoregulatory system (see Baccetti & Rosati, 1971; Crowe et al., 1971; Bussers & Jeuniaux, 1973; Shaw 1974;, Greven & Groh´e, 1975; Walz, 1979; Kristensen, 1978; Dewel & Dewel, 1979; Greven, 1979; Kristensen, 1981; Greven & Peters, 1986) between tardigrades and arthropods are not synapomorphies, but are either plesiomorphies or results of convergent evolution (Fig. 1A, B; Fig. 2).
Figure 1. Phylogenetic relationships generated from maximum parsimony analysis using PAUP. A, Minimum-length phylogenetic tree. Numbers indicate the branch lengths at each node. B, Bootstrap 50% majority-rule consensus tree. Numbers at nodes represent the bootstrap percentages from 1000 samples. C, 50% majority-rule consensus tree of 101 trees lying within 1% of the length of the shortest tree. Numbers at nodes represent the frequency with which clades descending from nodes were found among the 101 trees saved.
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Figure 2. Phylogenetic relationship generated from neighbour-joining analysis. Numbers at nodes represent the bootstrap percentages from 1000 samples. Average number of substitutions per sequence position is indicated by numbers at the top of the scale bar.
Tardigrades are coelomic animals (Marcus, 1929), and more detailed study on the origin of the mesoderm could provide a clue for finding the synapomorphic character(s) shared by tardigrades and the other major protostomes. The tardigrade clade is also separated from the nematode clade, which supports the current idea that morphological and physiological similarities (e.g. cellular eutely of epidermis, structure of anterior foregut, pharynx and oesophagus, reduction of coelom, absence of circulatory and gas exchange organs, and/or cryptobiosis) between tardigrades and some aschelminthes have been acquired due to their similar life styles in similar habitats (Marcus, 1929; Brusca & Brusca, 1990). However, whether tardigrades simply retained the aschelminth characteristics (although this seems more parsimonious given the trees shown in Figs 1 and 2) or acquired them through a separate evolutionary track remains uncertain. Future comparative analysis of the 18S rRNA gene and other molecules could provide additional evidence for resolving the origins of these characters. Despite the molecular data from this study and the few known morphological characters, the data require further morphological and in particular embryological support.
ACKNOWLEDGEMENTS
We thank Drs G. S. Min for technical assistance in sequencing the 18S rDNA, C. B. Kim for discussions, and S. R. Gelder for providing helpful suggestions that improved the manuscript. This work was supported by grants from KOSEF in 1991–1994, SRC (94–4–2) and the Ministry of Education of Korea (Institute for Molecular Biology and Genetics) in 1994.
REFERENCES Baccetti B, Rosati F. 1971. Electron microscopy on tardigrades. III. The integument. Journal of Ultrastructure Research 34: 214–243.
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Ballard JW, Olsen GJ, Faith DP, Odgers WA, Rowell DM, Atkinson PW. 1992. Evidence from 12S ribosomal RNA sequences that onychophorans are modified arthropods. Science 258: 1345–1348. Bertolani R. 1981. The taxonomic position of some eutardigrades. Bollettino di Zoologia 48: 197–203. Bertolani R. 1990. Tardigrada. In: Adiyodi KG, Adiyodi RG, eds. Reproductive Biology of Invertebrates, IV, B. Chichester: John Wiley, 49–60. Bertolani R. 1992. Tardigrada. In: Adiyodi KG, Adiyodi RG, eds. Reproductive Biology of Invertebrates, V. Chichester: John Wiley, 255–266. Blin N, Stafford DW. 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Research 3: 2303. Brusca RC, Brusca GJ. 1990. Invertebrates. Sunderland, MA: Sinauer Associates. Bussers JC, Jeuniaux C. 1973. Chitinous cuticle and systematic position of Tardigrada. Biochemical Systematics 1: 77–78. Crowe JH, Newell IM, Thomas WW. 1971. Fine structure and chemical composition of the cuticle of the tardigrade, Macrobiotus areolatus Murray. Journal de Microscopie 11: 107–120. Dewel RA, Dewel WC. 1979. Studies on the tardigrades. IV. Fine structure of hindgut of Milnesium tardigradum Doy`ere. Journal of Morphology 161: 79–110. Ellis LE, Sulston JE, Coulson AR. 1986. The rDNA of C. elegans: sequence and structure. Nucleic Acid Research 14: 2345–2364. Felsenstein J. 1978. Cases in which parsimony or compatibility methods will be positively misleading. Systematic Zoology 27: 401–410. Felsenstein J. 1985. Confidence limits on the phylogenies: an approach using the bootstrap. Evolution 39: 783–791. Felsenstein J. 1993. PHYLIP (Phylogeny Inference Package), Version 3.5c, Department of Genetics, University of Washington, Seattle. Field KG, Olsen GJ, Lane DJ, Giovannoni SJ, Ghiselen MT, Raff EC, Pace NR, Raff RA. 1988. Molecular phylogeny of the animal kingdom. Science 239: 748–753. Forty RA, Thomas RH. 1993. The case of the velvet worm. Nature 361: 205–206. Greven H. 1979. Notes on the structure of vasa Malpighii in the eutardigrade Isohypsibius augusti (Murray, 1907). In: Weglarska B, ed. Second international symposium on tardigrades, Krakow, Poland, July 28-30, 1977. Zeszyty Naukowe Uniwersytetu Jagiello´nskiego, Prace Zoologiczne 25: 87–95. Greven H. 1982. Homologous or analogous? A survey of some structural patterns in the Tardigrada. In: Nelson DR, ed. Proceedings of the third international symposium on the Tardigrada, August 3-6, 1980. Johnson City, Tennessee, USA. East Tennessee State University Press, 55–76. Greven H, Peters W. 1986. Localization of chitin in the cuticle of Tardigrada using wheat germ agglutinin gold conjugate as a specific electron dense marker. Tissue and Cell 18: 297–304. Greven H, Groh´e G. 1975. Die Feinstruktur des Integumentes und der Muskelansatzstellen von Echiniscoides sigmundi (Heterotardigrada). Helgolander Wissenschaftliche Meeresuntersuchungen 27: 450–460. Hendricks L, van Broekhoven C, Vandenberghe A, van de Peer Y, de Wachter R. 1988. Primary and secondary structure of the 18S ribosomal RNA of the bird spider Eurypelma californica and evolutionary relationships among eukaryotic phyla. European Journal of Biochemistry 177: 15–20. Hickman CP Jr, Roberts LS, Hickman FM. 1984. Intergrated principles of zoology. Mosby College Publishing. Higgins DG, Bleasby AJ, Fuchs R. 1992. Clustal V-improved software for multiple alignment. CABIOS 8: 189–191. Kinchin IM. 1992. What is a tardigrade? Microscopy 36: 628–634. Kozloff EN. 1990. Invertebrates. New York: Saunders College Publishing. Kristensen RM. 1978. On the structure of Batillipes norrevangi Kristensen 1976. 2. The muscle-attachments and the true cross-striated muscles, Zoologischer Anzeiger 200: 173–184. Kristensen RM. 1981. Sense organs of two marine Arthrotardigrades (Heterotardigrada, Tardigrada). Acta Zoologica 62: 27–41. Kristensen RM. 1987. Generic revision of the Echiniscidae (Heterotardigrada), with a discussion of the origin of the family. In: Bertolani R, ed. Biology of the tardigrades. Selected symposia and monographs U. Z. I., 1. Modena, Italy: Mucchi, 261–335. Marcus E. 1929. Tardigrada. In: Bronn HG, ed. Klassen und Ordnungen des Tierreichs, 4. Akademische Verlagsgesellschaft, Leipzig, 1–608. Marcus E. 1936. Tardigrada. In: Schultze F, ed. Das Tierreich. Walter de Gruyter, Berlin. Meglitsch PA, Schram FR. 1991. Invertebrate zoology Oxford: Oxford University Press. Nei M. 1991. Relative efficiencies of different tree-making methods for molecular data. In: Miyamoto MM, Cracraft J, eds. Phylogenetic analysis of DNA sequences. New York: Oxford University Press, 90–128. Nelson DR. 1982. Developmental biology of the Tardigrada. In: Harrison FW, Cowden RR, eds. Developmental biology of freshwater invertebrates. New York: Alan R Liss, 363–368. Nelson DR. 1991. Tardigrada. In: Thorp JH, Covich AP, eds. Ecology and classification of North American freshwater invertebrates London: Academic Press, 501–521. Nelson DR, Higgins RP. 1990. Tardigrada. In: Dindal D, ed. Soil biology guide. New York: Wiley, 393–419. Olsen GJ, Woese CE. 1993. Ribosomal RNA: a key to phylogeny. The FASEB journal, 7: 113–123. Pilato G. 1969. Evoluzione e nuova sistemazione degli Eutardigrada. Bollettino di Zoologia. 36 3: 327–345.
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Pilato G. 1975. On the taxonomic criteria of the Eutardigrada. First international symposium on Tardigrada, Pallanza, Italy, June 17-19, 1974. Memorie dell’ Istituto Italiano di Idrobiologia 32: Suppl.: 277–303. Pilato G. 1982. The systematics of Eutardigrada. A comment. Zeitschift f¨ur zoologische Systematik und Evolutionsforschung 20, 4: 271–284. Ramazzotti G. 1962. Il phylum Tardigrada. Memorie dell’ Istituto Italiano di Idrobiologia 16: 1–595. Saiki R, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. 1988. Primerdirected enzyme amplification of DNA with a thermostable DNA polymerase. Science 239: 487–489. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. 1985. Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350–1354. Sanger F, Nickeln S, Coulson AR. 1977. DNA sequencing with chain terminating inhibitors. Proceedings of the National Academy of Science (USA) 74: 5463–5467. Schuster RO, Nelson DR, Grigarick AA, Christenberry D. 1980. Systematic criteria of the Eutardigrada. Transactions of the American Microscopical Society 99: 284–303. Shaw K. 1974. The fine structure of muscle cells and their attachments in the Tardigrade Macrobiotus hufelandi. Tissue and Cell 6: 431–445. Spears T, Abele LG, Kim W. 1992. The monophyly of brachyuran crabs: a phylogenetic study based on 18S rRNA. Systematic Biology 41: 446–461. Swofford DL. 1990. PAUP: Phylogenetic Analysis Using Parsimony, Version 3.0s, Illinois Natural History Survey, Champagne. Turbeville JM, Pfeifer DM, Field KG, Raff RA. 1991. The phylogenetic status of arthropods, as inferred from 18S rRNA sequences. Molecular Biology and Evolution 8: 669–686. Turbeville JM, Field KG, Raff RA. 1992. Phylogenetic position of phylum Nemertini, inferred from 18S rRNA sequences: molecular data as a test of morphological character homology. Molecular Biology and Evolution 9: 235–249. Walz B. 1979. Cephalic sense organs of Tardigrada. Current results and problems. In: Weglarska B, ed. Second international symposium on tardigrades, Krakow, Poland, July 28-30, 1977. Zeszyty Naukowe Uniwersytetu Jagiello´nskiego, Prace Zoologiczne 25: 161–168.
Sequences of 18S rRNA genes used in the analysis. The position number is the nucleotide numbering of A. salina.
APPENDIX
68 S. YEO MOON AND W. KIM
APPENDIX (continued)
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Tardigrade biology. Edited by S. J. McInnes and D. B. Norman
Phylogenetic position of the Tardigrada based on the 18S ribosomal RNA gene sequences SEUNG YEO MOON AND WON KIM Department of Molecular Biology, Seoul National University, Seoul 151-742, Korea
The phylogenetic position of the Tardigrada remains uncertain. This is due to the limited information available, and the uncertainty of whether some characters are homologous or analogous with other taxa. Based on some morphological characters, current discussion centres on whether the taxon branches from the annelid-arthropod lineage, or lies within the arthropod complex. The molecular data presented here from an analysis of the 18S rRNA gene sequences are used to test the validity of these two hypotheses. Phylogenetic inference by the maximum parsimony and distance (neighbour-joining) methods suggests that the Tardigrada is a sister group of the major protostome eucoelomate assemblage that emerged before the arthropods, annelids, molluscs, and sipunculids evolved. The tardigrade clade also appears as an independent lineage separate from the nematode clade, thus supporting the current idea that tardigrades do not have a close aschelminth relationship. The molecular data also imply that several morphological features, considered significant in determining the phylogenetic relationships of tardigrades, are not synapomorphic characters. ©1996 The Linnean Society of London
ADDITIONAL KEY WORDS: — molecular phylogeny – 18S rDNA. CONTENTS Introduction . . . . . . . . . . . . Material and methods . . . . . . . . . Taxa compared . . . . . . . . . DNA extraction, PCR amplification, cloning Phylogenetic analysis . . . . . . . . Results and discussion . . . . . . . . . Acknowledgements . . . . . . . . . . References . . . . . . . . . . . . .
. . . . . . . . . . . . . . . and sequencing . . . . . . . . . . . . . . . . . . . .
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. . . . . . . .
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61 62 62 62 63 63 65 65
INTRODUCTION
Microsopic size (50 µm–1200 µm) and a small number of distinctive morphological characters have led to relatively few taxonomic works on tardigrades (for recent reviews, see Schuster et al., 1980; Pilato, 1969, 1975, 1982; Bertolani, 1981, 1990, 1992; Kristensen, 1987; Nelson & Higgins, 1990; Nelson, 1982, 1991). Although they were recognized as a phylum by Ramazzotti (1962), their taxonomic rank and phylogenetic affinity remain unresolved. They are considered as enigmatic because some of their somatic characters are found also in other taxa, such as the 0024–4082/96/010061 + 09 $18.00/0
61
©1996 The Linnean Society of London
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S. YEO MOON AND W. KIM
Nematoda and the protostome coelomates (Annelida, Onychophora, and Arthropoda). Moreover, the absence of clear fossil records and detailed embryological data for tardigrades has increased the difficulty of deciding whether these characters are homologous or analogous with other taxa. Current discussion has focused on tardigrades either stemming from the annelid-arthropod line or being located with the arthropod complex, with a particular affinity to the Onychophora (see Marcus, 1936; Greven, 1982; Hickman, Roberts & Hickman, 1984; Brusca & Brusca, 1990; Kozloff, 1990; Meglitsch & Schram, 1991; Ballard et al., 1992; Kinchin, 1992; Fortey & Thomas, 1993). However, the molecular information presented here from an analysis of the 18S rRNA gene sequences offers a different perspective on the phylogenetic position of the Tardigrada.
MATERIAL AND METHODS
Taxa compared The taxa compared in the present study were tardigrade (Hypsibius sp.), nematode (Caenorhabditis elegans), arthropod (Eurypelma californica, Artemia salina, Tenebrio molitor), annelid (Chaetopterus sp.), mollusc (Cryptochiton stelleri), sipunculid (Golfingia gouldii) and nemertine (Cerebratulus lacteus). The platyhelminthes Dugesia tigrina was designated as the outgroup. The culture of live tardigrade Hypsibius sp. was obtained from Ward’s Natural Science International Marketing Group. The complete sequence of the 18S rRNA gene of the tardigrade was determined in the present study, and compared with published gene sequences of the other nine taxa (Ellis, Sulston & Coulson, 1986; Field et al., 1988; Hendricks et al., 1988; Turbeville, Field & Raff, 1992). DNA extraction, PCR amplification, cloning and sequencing Live individuals were washed with distilled water to remove all the external debris and then starved to remove gut contents. The individuals were disintegrated in a microtube using a sonicator for a few minutes, with intervals of 30 seconds for cooling the tube on ice. The isolation of genomic DNA from the individuals was performed using the method modified from Blin and Stafford (1976). The 18S rDNA was amplified using PCR (Saiki et al., 1985, 1988) with two primers located at either end of the molecule (5'-CCTGGTTGATCCTGCCAG-3', 5'-TAATGATCCTTCCGCAGGTTA-3'). The thermal cycle parameters were 94°C, 1 min (initial denaturation, 5 min)/52°C, 2 min/72°C, 3 min (final extension, 10 min). The reaction was cycled 30 times. The amplified double-stranded 18S rDNAs were extracted with equal volumes of phenol:chloroform, followed by purification with GENE CLEAN II kit (BIO 101), and diluted in distilled water. For blunt-ended ligation, both ends of the PCR products were modified using T4 kinase and T4 polymerase. The blunt ended 18S rDNAs were purified with the GENE CLEAN II kit, inserted into pUC19 plasmid vector and transformed to DH5-α cell lines. The double-stranded recombinant plasmid DNA was purified by PEG precipitation or QIAGEN plasmid kit (Pharmacia). The DNA sequencing was performed by the dideoxy-termination method (Sanger et al., 1977) using Taq-Track kit (Promega), with two vector primers [M13 universal primer (–40), M13 reverse primer (–24)] and
PHYLOGENETIC POSITION OF TARDIGRADA
63
an additional fourteen primers as used by Spears and colleagues (1992). Sequencing reaction mixtures were electrophoresed on buffer-gradient 6% polyacrylamide gels. Phylogenetic analysis Sequence alignment (Appendix) was performed using the CLUSTAL V multiple alignment program (Higgins, Bleasby & Fuchs, 1992) beginning at universally conserved regions, and then checked by eye. To avoid possible systematic errors, regions of ambiguous alignment including positions exhibiting high length variability were excluded from the phylogenetic analyses. Both maximum parsimony and distance (neighbour-joining) methods were used for the analysis. The bootstrapping method of data resampling (Felsenstein, 1985) was applied to evaluate the significance of the result. For maximum parsimony analysis, the BRANCH AND BOUND search option of PAUP, version 3.0s (Swofford, 1990) was used except for bootstrapping. The HEURISTIC search was employed for the bootstrap test by using TBR branch-swapping and CLOSEST stepwise addition. Ten trees were held at each step. Alignment gaps and unknown bases were considered as missing data. Neighbour-joining analysis was applied using PHYLIP, version 3.5c (Felsenstein, 1993). Evolutionary distances were calculated by the DNADIST program, using Kimura two-parameter model with no transition/transversion bias. Bootstrapping was performed with the SEQBOOT program, with a random input order of taxa.
RESULTS AND DISCUSSION
Maximum parsimony analysis generated a single minimum-length tree of 666 steps with 180 phylogenetically informative sites (Fig. 1A). The overall consistency index is 0.748, with a consistency index excluding uninformative characters of 0.609. The tree topology is largely concordant with the previous results based on the 18S rRNA gene sequence data in assemblages of the protostomes, including the position of the nemertine Cerebratulus lacteus (see Turbeville et al., 1991, 1992). The tardigrade Hypsibius sp. appears as a sister group of the protostome assemblage which includes representative species of arthropods, annelids, molluscs, sipunculids and nemertines. The tardigrade clade also represents a discrete lineage apart from the nematode clade (Fig. 1A). This proposal is supported by bootstrap analysis (Fig. 1B). Measurement of the reliability of the maximum parsimony phylogeny, by considering all trees within 1% of the length of the shortest tree, also supports the significance of the separation of the tardigrade from the major protostome assemblage (Fig. 1C). As the nematode branch is faster evolving, and the maximum parsimony analysis may generate an erroneous tree(s) when the rates of evolution vary with the evolutionary lineage (Felsenstein, 1978; Nei, 1991), this parsimonious solution was compared with that of the distance (neighbour-joining) method, which is more efficient when the rate of nucleotide substitution varies with lineages (Nei, 1991; Olsen & Woese, 1993). The neighbour-joining analysis, with bootstrapping, was also consistent with that of the maximum parsimony analysis, except for a little variation in the placement of the sipunculid Golfingia gouldii and the mollusc
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Cryptochiton stelleri within the protostome assemblage (Fig. 2). This variation may be explained by the rapid diversification of members in the protostomes, and shows that these 18S rRNA gene sequences are not useful for determining the positions of the sipunculid and the mollusc (Turbeville et al., 1991, 1992). The molecular data presented here propose that tardigrades originated before the advent of the protostomes, and dismisses the hypothesis that they have affinities with both or either of the annelids and arthropods. These data also imply that several morphological features such as cuticle, muscle attachment, cephalic appendages, and/or excretory osmoregulatory system (see Baccetti & Rosati, 1971; Crowe et al., 1971; Bussers & Jeuniaux, 1973; Shaw 1974;, Greven & Groh´e, 1975; Walz, 1979; Kristensen, 1978; Dewel & Dewel, 1979; Greven, 1979; Kristensen, 1981; Greven & Peters, 1986) between tardigrades and arthropods are not synapomorphies, but are either plesiomorphies or results of convergent evolution (Fig. 1A, B; Fig. 2).
Figure 1. Phylogenetic relationships generated from maximum parsimony analysis using PAUP. A, Minimum-length phylogenetic tree. Numbers indicate the branch lengths at each node. B, Bootstrap 50% majority-rule consensus tree. Numbers at nodes represent the bootstrap percentages from 1000 samples. C, 50% majority-rule consensus tree of 101 trees lying within 1% of the length of the shortest tree. Numbers at nodes represent the frequency with which clades descending from nodes were found among the 101 trees saved.
PHYLOGENETIC POSITION OF TARDIGRADA
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Figure 2. Phylogenetic relationship generated from neighbour-joining analysis. Numbers at nodes represent the bootstrap percentages from 1000 samples. Average number of substitutions per sequence position is indicated by numbers at the top of the scale bar.
Tardigrades are coelomic animals (Marcus, 1929), and more detailed study on the origin of the mesoderm could provide a clue for finding the synapomorphic character(s) shared by tardigrades and the other major protostomes. The tardigrade clade is also separated from the nematode clade, which supports the current idea that morphological and physiological similarities (e.g. cellular eutely of epidermis, structure of anterior foregut, pharynx and oesophagus, reduction of coelom, absence of circulatory and gas exchange organs, and/or cryptobiosis) between tardigrades and some aschelminthes have been acquired due to their similar life styles in similar habitats (Marcus, 1929; Brusca & Brusca, 1990). However, whether tardigrades simply retained the aschelminth characteristics (although this seems more parsimonious given the trees shown in Figs 1 and 2) or acquired them through a separate evolutionary track remains uncertain. Future comparative analysis of the 18S rRNA gene and other molecules could provide additional evidence for resolving the origins of these characters. Despite the molecular data from this study and the few known morphological characters, the data require further morphological and in particular embryological support.
ACKNOWLEDGEMENTS
We thank Drs G. S. Min for technical assistance in sequencing the 18S rDNA, C. B. Kim for discussions, and S. R. Gelder for providing helpful suggestions that improved the manuscript. This work was supported by grants from KOSEF in 1991–1994, SRC (94–4–2) and the Ministry of Education of Korea (Institute for Molecular Biology and Genetics) in 1994.
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Sequences of 18S rRNA genes used in the analysis. The position number is the nucleotide numbering of A. salina.
APPENDIX
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APPENDIX (continued)
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