11ttlrrbittIttgiu.l05: 37 43. I995. I-.R.(i. Cutrunr (ed.), Biology ol and sorne Related Flatvtorms. 31 i,;1995 Klutter Acadenic Publislters. Printed in Belgium.

55 rRNA sequences'of 12 species of : implications for the phylogeny of the Platyhelminthes

B. I. Joffer, K. M. Valiejo Roman2, V. Ya. Birstein3 & A. V. Troitsky2 1 Zoological Institute RAN, St-Petersburg 199034, Russia 1 A. N. Belozersky Institute of Physico-Chemical Biology, Moscot state University, Moscow 119899, Russia t N. K. Koltzov Institute of Developmental Biology RAN, Moscow I 17331, Russia

Key'words:5S rRNA, Platyhelminthes, Turbellaria, parasitic flatworms, phylogeny

Abstract

55 rRNAs fiom 12 species of free living and parasitic platyhelminthes were sequenced. In the phylogenetic analysis, attention was focused on the statistical estimates of the trees corresponding to existing phylogenetic hypotheses. The available 55 rRNA data agree well with widely accepted views on the relationships between the Acoela, Polycladida, Tricladida, and Neorhabdocoela; our analysis of the published l8S rRNA sequences also demonstrated good correspondence between these views and molecular data. With available 55 rRNA data the hypothesis that the dalyellioid turbellarians is the sister group of the Neodermata is less convincing than the hypotheses proposing the Neodermata as the sister gr<.,up of the Neorhaodocoela, or of the Seriata, or of the branch uniting them. A relatively low rate of base replacement in parasitic flatworms, probably, accounts for the uncertain position of the Neodermata, while a relatively high rate in planarians may explain a relatively too early divergence of the Tricladida in several published phylogenetic trees constructed from various rRNA data.

Introduction the anaiyzed sequences. Indeed, a tree computed from molecular data is a statistical estimate, and one must The phylogeny of the Platyhelminthes is a promising know whether there are statistically significant reasons field for application of molecular methods because, to prefer it (at least, under the assumptions of the used while some problems have already found reasonable tree constructing method) to some other topology, e.g. solutions and may serve as a 'control' (see E,hlers, one supported by morphological evidence. 1985; Rieger et al., 1990; Rohde, 1990), many others remain unsolved. By now, portions of l8S rRNA have been sequence.l fiom various platyhelminthes (Baver- Material and methods stock e/ aI.,1990; Ruitort et a\.,1992 a,b). Yet, addi- tional sequence data are still necessary, in particular, The species studied, their systematic positions and col- from other molecules. With the 12 new sequences lection sites are listed in Table l. The total RNA was presented here, a total of 14 sequences of 55 rRNA isolated by the hot phenol extraction procedure with may now contribute to this goal of elucidating platy- 4M guanidinium isothiocyanate; 55 rRNA was puri- helminth phylogeny. fied by electrophoresis on 87o polyacrylamide gel and The shortness of the 5S rRNA limits its phylogenet- sequenced by the Peattie's chemical method after 3'- ic usage (Hendnks et a\.,1986; Hori & Osava, 1987). end labelling with (5'-32P)pCp as described earlier Therefore, we focused our attention not on inferring (see Troitsky et al., l99l for the protocol used). phylogenetic trees, but on the statistical estimation of The sequences used fbr rooting the platyhelminth the existing phyiogenetic hypothesis, i. e. , on their com- tree were taken from the 'Berlin RNA databank' patibility with 5S rRNA sequence data. This aspect of (Specht et al., 1990) with the exception of 55 rRNA the problem is important irrespective of the length of of Echinorhynchus gadi which was sequenced by Ttble l. Systematic positions 3.3 package and sites of collection of the I]-IYLIP (Felsenstein, 1990). Maxirnum platyhelminth species used in this study likelihood (ML) method was always implementecl with C. coJ empirical 'Turbellaria' estimation of base fiequencils and a transi_ P.re; tion/transversion ratio N.hu: Archoophora = 2. Distances were calculat_ j ed using the 2-parameter Kimura,s cD, Acoela model, also with D. ti a transition/transversion ratio ConvoIura crnvoluta (Abildgard, I g06) = 2. Lower probabili_ D.7a ty tbr fixation Polycladida of transversions is suggested by a very P.EO high level of conservation Noktp lttna humuli.r (stimpson, I g57) (2) of the seilndary strucrure 14. cr of rRNAs, B. es 'k P larutc era reticuldta (Stimpson, g-5-5) which is always affectecl by I transversions, c. gz while the influence of transitions is moderated by non- P. at Tricladida canonical pairing (GU). The value of 2 (rather than, D. sz Fam. Dugesiidae e.9.. l.-5 or 3 t was empirically chosen based on n series K. s: *Duge.sia (Dugesia) joponlca of preliminary (lchikava et experiments with various rRNA data F.h< Kawakatsu, I964) sets. Computation of the ML trees was repeatecl three D u ges ia ( G irardh ti g rinu (Girard, times ) with difTerent species input order (one starting Fam. Planariidae with the root sequence and two random ones) for each Planuria ton)a O. F. Muller, 1773 (4) species set with glabal rearrangements. Being main_ Cc Fam. Dendrocoelidae ly interested Dr in the statistical eirimates ot.given tree Dendnrnel.uim lacreum ((). F. Muller, 1773) topologies, we ornitecl bootstrapping; rnorelver, r/h Neorhabdocoela with Dj the ML method, branch lengths which are Typhloplanoida not signif_ DT icantly positive themselves indicate the places Bolhromesostoma e.ssenl (Braun, gg-5) where D7 I (s) changes in the Kalyptorhynchia tree are most probable (Felsenstein, PI r 990). MC Mac rorhynchus crot:ea (O. Fabricius, 1826) Dalyellioida Be Gg G raJJtl La graffi Mitin, I 97 0 (6) Pa Results and discussion P.s e udo g ralJilla are nic o l a (Meixner. 1938) 0) Ds Ks Twelve 5S rRNA Di.scocoryle sagitattt (Leuckarr, lg42) (g) sequences of the species studied are Fh presented in Fig. I together with the sequences fiom Khavia sinensis Hsu, two species studied earlier (Oham 1935 (9) a et al'., l9g6; Hori F i,q. l et al.,1988). seque Fasciola heparica Linne, l75g (10) P. trtr Approach long. + Species kt the phl,logenetic. anall,sis -sequencetl by other authors. For the species sequenced by us, the numbers in parentheses indicate the collection sites, as well as The first question the hosts for the parasitic species. we had to answer was whether the seve (.1) Dalnezelenetskaya Inlet, giotogicat Barentz Sea; (i) sta_ tree(s) constructed from 5S tion '^Vostok', rRNA data reproduced the ,.si Sea of Japan; (3) laboratory stock; (4") a pond relations between the main groups near St-Petersburg; (5) a pool in Borok (yaroslavl Disirict, of turbellarians rep- Russia); (6) resented in from the marine gashopod, Neptunea despec_ our material (Fig. 2A,) which had been seqr ta, Biological station ,Karresh., convincingly White Sea; (7) Bioiogical established from morphological evidence station 'Kartesh', White Sea; (g) from the fi,sh Coreg'onus (Ehlers, I 985; Rieger e albulct, Ladoga t al. , 1990;y"t, ,." Rohde, I 990 the lake; (9) from the fish Cyprinu.r ,orpii fish_ farm and present volume for diff'erent in Estonia; (10) frorn cows from a slaughte. house. views _ these views ed are not discussed below because no tree topology suit_ catt able for sraristic estimation has been puUtistreOi. cep In view of the fact that changes in ihe us and will be published elsewhere. set of species lcttr All the platy_ are the most important test for the consistency of the SUS helminth sequences wholly fitted to the alignment tree topology (see, e.g. Wainwright et universally used for 55 rRNA (see at., 1993), we bra Specht er a/., constructed the trees for several sets of species. SPC 1990). 3'-terminal uracil residues were Each n;t used in the of them included all the platyhelminih analysis because l4 sequences nel variation in number of 3/_terminal and diff'ered uracils (2 from the others in the single our-o.orp (w or 3) was observed in practically all stud_ (rooting) sequence. This explicitly showea ied species. Phylogenetic analysis the efTect bu was done using of each outgroup on the tree topology ancl provided a i tl' 39 Maximum 10 20 30 40 rented 50 with C . CONVOTUtA ( CC ) GCCUACGACCAUACCAUGUUGAAUACACCGGUUCUCGUCCCAUCACCCAA-Gt] rd a transi- p.reticuTata* (Pr) .AUAo..U...... C.1AC....A.. ..c.-.. : calculat- N.humuTis (Nh) ..UA..Uc.....Uc.YAc....A.y. also cD.japonica (Dj) .u.c...cu...,.u.e....ce...... c.A. u.....u.c...u.. with D.tigrina (Dt) .U.A.u.c.. Uc....cC.. .A...... U....U. probabiti- D.Tacteum (DL) .U.....GU.....AUCU?..c.A.Uc..U.A. .U....U.A. by a very P.torva (Pt) .U.A.c..U.....USCCG..c.A.U..AU.A ..U....U.A-..C.. structure M.crocea (Mc) .U.Ac. ...C.CCS....A.U. sversions. B.esseni (Be) .tl .A. -.c.....UC..Ac..cCA.. ..U.. ..c.-.. G.graffi (cS) .Ac.e..c.....Uc. c.A.. ..U.. d by non- P.arenicoTa (Pa) .AG.c..C.....uc.c....cCA.. :her than, o.sagittata (Ds) ...Ac. ...u.c.... n a series K.sinensis (Ks) N.cAc. ...U.c..A....GU. NA data F.hepatica (Fh) yc.Cc. ...U.c....c.A.U. ..U.. ted three u iiii ui u uiu i iiiiii iiii i i i ii : starting for each 60 70 80 90 700 770 720 rg main- CC UAAGCAACAWIGGGCCUGCUUAGUACUUGCAUGGGUGACCGCWGGGAACACCGGGUGUUGUAGECWru ven tree Pr .OUG.C.....CA.. ..C...... U..U...... C.CUAC...- 'er, with Nh .CUc.c.A. . .YA. . .U. . . . .C...... U. .U...... A. .UA. . . ._ t Dj .c.c..A... ..C...... A...C.. . .e..uc...... u..cA....G.. signif- Dt ....u...uAAc...UGC. .C.A..._ s where ....AU...... A ..u..ccA...CCA.U.A...- D7 cuAcAAu. ..uAc. u..u. .. . .uA.cc...... u..cuA. . . .c....A..._ enstein. pt cccccAA...UAC. c..A. u.cc. u..cuA.....c.u.A..._ Mc .Cce. UC..A. ...C. ....Ue. .U....A...... CU.A..._ Be .CUc.c.A..C...... C. ....Uc. .U..UAC...C...U.A...U eg c.A..c...... Uc. . ...Ue. .U. .UAc...c. .c.CU. ..U Pa ...e.c.A..ACU. C.... .Uc.. U..A.U...... C.C\J..._ Ds ...c. .C...... C. cC...... U. CU....._ I(s U..G. .c...... c. .cc...... u. cuc.A.._ ied are Fh ...G.c.....c...... C. cC...... U. ccccc.._ s fiom iiiiu ii iuiuiiiiii ii uiiii iiiiiiuii iii ii j; Hori rrq /' 5S rRNA sequences known from the platyhelminths. The sequence of P reticulata (t) was published earlier; the majority consensus sequence for D jultrtrtit tt is compiled from three sequences known from tfuee populations (Ohama et aL. , 19g6: Hori et al., t 9gg). Insertion in P tttrva is proved by presence ofa minor rRNA fraction which was partially sequencecl and has in the same position an inseftion 3 nucleotides long. Dots indicate the nucleotides identical to those in C. rttnttoLLrta. i, invariant position; u, unique stare.

er the severe test for its consistency, especially, in the root between the outgroup and platyhelminth sequences ed the re-9ion. (0.090-0.597 K,,.) varied within the same range as the s rep- provide To maximal homogeneity of the set of distances between the platyhelminth sequences them- been sequences in the analysis (see, e.g., Felsenstein, 1990), selves (0.093 -A.625 K... ). lence we used for rooting all the sequences known from 1990 the anirnals generally considered more closely relat- Phylogenetic ,iews trees ed to the Platyhelminthes, that is Brachionus pli- suit- cdrills (Rotatoria), Echinorhl,nchus gadi (Acantho- Figure 2 shows that six of the eight used rooting cephala), Emplectonema gracile, and Lineus genicu- sequences inferred maximum likelihood (ML) trees :cies /arrzs (Nemertini). In addition, the majority consen- (Figs 28,C) which correspond well to recent views on f the sus sequences fbr fbur more distant lower inverte- the phylogeny of the Turbellaria (Fig. 2A) and ,we differ brate groups were composed and used: cPorifera (3 only in the position of the parasitic flatworms. lach The species), cCoelenterata (6), cNematoda (3), and cAn- seventh tree was very similar, though Echinorhynchus nces nelida (3). The rooting sequences were rather diverse 'branched 'oup offtogether with polyclads' instead ofroot_ (with distances between them of 0.071-0.348 K.,") , ing the platyhelminth tree (Fig. 28, arrowhead). The 1-ect but did not seem to markedly increase the heterogene- tree rooted with Lineus was very different (Fig. 2D), :da ity of the analyzed data sets - at least, the distances though this sequence in itself did not seem peculiar 40

Table 2' Compatison ofseveral tree topologies applied to eight species sets rooted with differenr outgroup sequences

Tree Rooting sequence topo- Brachionus Emplectoneml cCoelenterata Echinorhynchu.t logy LnL LnL LnL LnL ML - 1038.459 -1044.60 - 1053.06 - 1041.01 C -1045.945 1.06 1051 .56 0.96 - -1060.18 1.00 1057.90 1 .68 D - -1047.508 1.49 1.1'r -1052.96 1.23 1.91 B -t061.75 -i059.54 -1047.020 1.37 1053.20 1.49 - -1061.71 1.49 -t059.82 1.99*

AI -1059.0M 1.66 1.61 -1065.21 -1073.40 1.64 1071.49 2.0'l* A2 1.94 - -1064.366 - 1068.82 t.76 1076.89 1.75 - -1074.84 2.09* A3 -106'7.434 2.13* 2.09* -1071.52 - t 080. 14 2.t3* * 1077.85 2.37* Rooting sequence topo- cAnnelida cPorifera cNematoda Lineus logy LnZ LnL LnL LnL ML - 1026.779 - 1049.8-s -1078.22 - 1047 .73

C 1030.887 0.53 0.63 - -1054.75 - 1083.03 0.62 0.74 D -r058.37 -1032.716 0.73 1.02 -1056.61 1085.00 1.03 1060.05 0.87 B - - -1032.17',l 0.89 1056.66 0.95 - - I 084.80 0.93 - I 059.99 0.85 A1 1.21 1068.74 1.41 -1042.349 - tog7 .17 1.42 1071.06 A2 - - 1.24 - 1046.519 1.39 1073.18 1.59 - -ll02.6s 1.70 *1074.87 1.42 A3 1050.101 1.82 - - 1077.08 2.05* I 105.89 1.89* *1078.69 - 1.70 ol Ln L, logarithm likelihood, , - student's l:t /-ratio for the difference in Ln r between gir"n o;(r";E;;;; ML tree for corresponding data set. * denotes cl that difference is statistically ,ignin.oriif>].c6 for2<0.05).

(distances to the other rooting sequences 0.101_0.244 phylogenetic ri. relationships of the Neodermata remain K,,", those to the platyhelminth species O.l4Z_0.475 controversial. The Neodermata is most K,,"; see often consid_ the previous paragraph). This confirms that ered s( either the sister group for the Dalyellioida, rhabdo- our series of outgroup sequences was an effective rl test coels- with doliiform pharynx (see, e.g., Ehlers, for the consistency l9g5; ( of the dominant tree topology. yet, Brooks, 1989), two or as an earlier branch of rhabdocoels details of this topology are probably -5 erroneus: which retained the less specialized Dugesia rosulate pharynx is def,nitely a monophyletic hx;n, and the (see N Joffe et al.,l98l.,Joffe & Chubrik, lggg; Koriko_ Cestoda is generally considered more closely related va & Joffe, 1988 for review and evidence from the to the Monogenea, than to the Trematoda. ( This ques_ morphology of the pharynx tions and nervous system). Two the reliability of other branching patterns and o other hypotheses which could be tested with our outlines the importance of statistical data estimation of the would consider the Neodermata tree topologies the sister group either (see below). for the S Neorhabdocoela + Seriata 1c.f. Ruitori er al., 1992b), or for the Seriata (note again that the hypothe_ ! On the position of the Neodermata ses suggested by Rohde, (1990 and present volume; (, cannot be discussed here). Parasitic flatworms could be grouped together in the ll Phylip allows a sratistical test of whether the dif- same way in all ML trees, though two branches with I ference between logarithm likelihoods or lengths of zero length are to be taken in account here (Fig. two given trees is significant. We estimated 6 topolo_ 2). Maximum I parsimony (Mp) and Fitch trees near_ gies representing these four hypotheses (Fig. 3) in each ly always support monophyly of the Neodermata. I The of our 8 species sets. Statistical estimates lstudent,s t_ ( 41 Neodermata Tricladida Neorhabdocoela Polycladida Acoela C.convoluta B C D Linaeus C.convoluta C.convoluta G.grafft P.reticulata P.reticulata P.areqicola N.humulis N.humulis .essent G.grafJi G.graffi P.reticulata P.are4icola P.are4icola N.humulis .essem .essent .crocea .crocea M.crocea F.hepatica D.sagittata K.sinensis K.sinensis K.sinensis F.hepatica D.ianonica D.iaoonica , D.tisina D.tierina Plorva , - - PJorva D.lacteum D.lacteum D.lacteum

Fig 2 A Phylogenetic tree representing recent views on the evolution the of Turbellaria and one of widely accepted views on the position of the Neodermata B-D Topologies of maximum likelihood trees for eight sets of species with different outgroup sequences. B. Brachionus, ErrytLetlottenut' ccoelenterata s,nd EChirutrhinc,hu.r; the last occupies a position shown with arrowhead lanj noi in tirr" root;. C. cAnnelida, cPorifera, cNematoda. D. Lineu.r (the branches present in the trees on Figs B and C are shown by the wide lines).

ratio) fbr the trees based on the 'dalyellioid' hypothesis method. :main The trees corresponding to recent views on were 1.15-2 fold higher than fbr three other hypothe_ rnsid- the platyhelminth phylogeny, provided that rhe Neo_ ses, and its variant (Fig. 3,A3) was significantly worse rbdo- dermata were not derived together with the Dalyel_ than ML tree in 6 of 8 data sets (Table 2). Thus, the 1985; lioida, were less than 5Vo longer than Mp trees, and 'dalyellioid' hypothesis fits poorly with the available coels the difference was far from being statistically signifi_ rynx 5S rRNA data compared with other tested assumptions. cant. None of the 3 other hypotheses may be preferred. tiko- In addition, we unired the data published by Ruitort r the et al., 1992a and Baverstock et al., 1991, corrected Compatabilitt, of recent hypotheses on the phl,logeny Iwo alignement and, thus, obtained two data sets includ_ of the Turbellaria with rRNA molecular data data ing 16 and 13 platyhelminrh species with 443 and 536 well ther aligned positions correspondingly. For the sake of Statistical estimates described in the previous para- al., comparison, they were rooted as in Baverstock er al. graph also demonstrated that the tree topologies repre_ (1991). .he- Studied both by ML and Mp methods, these senting widely accepted recent views on the phylogeny ne) data also demonstrated no statistically significant dis_ of the Turbellaria, with Neodermata not branching with agreement with phylogenetic concepts corresponding the Dalyellioida (Figs rif- 2A,3F,, b,c,d), fit well with 55 to the tree shown on Fig. 2A. of rRNA data (Table 2). lo- The maximum parsimony (MP) method returned ch l -1 0 shortest trees, depending of the species set. Esti- t- mates of various hypotheses about the relationships of Neodermata were the same as obtained from ML +LA'

A1 A2 Typhloplanoida l'yphloplanoida a\l)cl lll Kalyptorhynchia rr il rE Kalyptorhynchia J- Kalyptorhynchia .in,l Typhloplanoida \lll()11() Lg Dalyellioida Dalyellioida Dalyellioida { r ctttltrk Neodermata Neodermata Neodermata C Tricladida Neodermata Neorhabdocoela l{tl'cre Neorhabdocoela --t- Tricladida Tricladida Neodermata { Neorhabdocoela Neodermata

B C.cortvoluta 1 P.reticulata B.esseni N.humulis M.crocea outgroup G.graffi P.arenicola G.graffi P.arenicokt B.esseni r B.esseni M.crocea M.crocea G.graffi Djaponica P.arenicola D.tigrina M.crocea F.hepatica B.esseni K.sinensis P.torva Q.sra.ffi D.sagittata D.lacteunt P.aretticolrt

Fig 3' A'D' Trees, representing four hypotheses about the relationships .dalyellioid, ofthe Neodermata with the Turbellaria, the hypotheses is shown in 3 variants' Al-A3 E, topologies of the trees used for the statistical estimation of these hypotheses. To nrake trees col.responding to hypotheses shown in B,C,D the neodermate subtree (bottom, was grafted left) to the base turbellarian tree (bottom, center) at marked the positions with arrowheads b,c,d accordingly. To compose the trees corresponding to A l-A3, the neorhabdocoel subtree (on the right of the dashed line) was replaced by subtrees al-a3 correspondingly, with the neodermate subtree grafted at the positions shown by arrowheads.

The rates of nucleotide base replacement and phylo- stock e/ al., 199\; Ruitort et al., l992a,b) may result, genetic trees correspondingly, from too low and too high rates of base replacement. The influence of the heterogeneity The relative rate of change may be approximated from in the rates of base replacement is important foi it can the lengths of the branches in the trees (ML, Mp, or drastically affect the topology of phylogenetic trees Fitch-Margoliash) or directly from a disrance matrix. independently of the sequence lengths. With any method, the rate of base replacement in 55 rRNA of planarians was estimated as high, and that of parasitic platyhelminths as very low. These results Acknowledgments completely agree with the estimates obtained from lgS rRNA by Ruitort et al. (1992b). high A rate of evo_ We are very grateful to Dr O. N. yunchis (Research lution may cause inappropriate early divergence of a Institute for Lake and River .quick Fishery, St_petersburg) group in the tree or incorrect clustering of the and to Dr T. A. Timofeeva (Zoological Institute, St_ clocks' together (see, e.g., Lake, 1991). Therefore, the Petersburg) who collected for us two parasitic species. uncertainty of the position of the Neodermata and/or a Dr E. N. Grusov from the same Institute let us use his relatively too early position of the planarian branch in computer, though it notably hindered his own work. We various trees constructed from 55 and l8S rRNA data are also very grateful to our colleagues fiom Moscow (see Hendriks et al., 1986 Hori & Osava, 1987; Baver_ State University: to Dr V K. Bobrova for the help in a

43 /nchia c\perimental work, to Miss Katya Drusina for perf.ect tions toida rind willing technical assistance, p.ot. between Tremat and rurbellarial and to e S Perersburg) Parasitologia (st- ..\ntonov for constant 22: 2974;:.a I attention to this work and critical *"1["Jiri;,i;,1,#;,1,""'. re88 on the a re rrrrrks to the manuscript. nervous svsrem or rans. Fortschritte Zool./progress Igl-'1g4. Zool. 36: Lake, A. J., 1999. Origin o.f the eukaryotic )ela rare-invariant nucleus determined by References analyses RNo K. Bremer & :f,.lbo.s:m:l *.r".," l" iilo*rnrrn, H. Jornval 1eds.r" The Science publishers. h";;;;; ;; if.. Err"ui". ^. Amsterdam: g7_ l0l. l]rverstock, P R., R. Fielke, A M. Johnson, Ohama, T., T. Kumazaki. R. A. Bray & I. Bev_ H Hori, S..Osawa & M. cridge, 1991. Conflicting ohylogenetic water plararians Takai, 19g3. Fresh lryp"ri,"r"^iJ. ard a I \iric praryhelminrhs the para_ tested by pa-rrior ,.qr.niinglll in r he is,* * o,i,.,*Tff ItNA. parasirol. ai'.ruorornor .rJilillffi ::11 Int. J. 2t:329_339. Rieger.- R M . S. ryrer. p i:".ll,rj:,Jr; "r. s smiii, l]rooks, D. R., 1999. helminthes: a;:;)].;:.;e0. prary_ The phylogeny of the Cercomeria Turbellaria_ (platy_ ptaryhelminrhes f, e. W. ff*.iror*A i."r. an ,"J,un f"Orl, a gen erat r,ii pr.. and Rhvnchoc,oer" "rlir,ron i'"'Jlljllli r:i:?t 1.?, " "" iiie" ri. ,.0., ",.v'p., "i anaromy ot rnvertebrates.""i. Ehlcrs. U ,^l 995 Das phylogenetishe witey-Liss, New york: ,a System der plathelminthes. I.t;;.."r,. Fisher, Sturrgaft _ New york,3l7 G. - pj, t,' he "i"',I,.T.; c tvhe I m i Feisensrein, pHyLIp n thes w i t J., I990. 3.3. University i : .PI h s peci ar ofCalifornia Herbar_ 3'r1,l,ll :"::J,'^ ium, Berkley, California Rui:::: Mj K" Hendriks, a'ffi i"Ji;l?i.l,iflTll L., E. Huysmanr, Vg den 1992a. Enzyme il,lf ;,g,,iJ. primary Berghe & R. De Wachter, eleclroohoresis. lgS rRNA re86. srrucrure 3' els of phylogeneric sequences. and lev_ jh: ,ib"r";;i RiA; , resoturion pods and "f ls J i **"- among ..u.rrii|".1"..o, applicability of 55 RNA ro rhe water planariaas (plarvhe r."rn ;;;;;;;;;* I m -"-"-'"i n thes, r.:"i;;;,rrrLlduruil' lution. J. Mol, Evol. 2,1: 103-109. "r" J.Zool.tO: l425.14:j9 rf,Ii"",r,.raludlco a_ Hori, H., A. N1uro, S. Osawa. Ruiron. M. Takai,_K._y Lue & M, M., K. G. Field. F I988. Evoturion Kawakatsu, A Rxff & J Bagufla' l992b of turbeilorra r. a"ar..J?ir"is'ltororno, rRNA \equences ,ra on,lipttllogenY l8s R NA seq "ru of Platvhelminthes. uences. Forrschrirte. Zool./progress ;;; syrr. E.;i ;l';;' Biochem. Hori. H. & S. i;, i ia_, u, Osawa. 19g7. Origins anj.""",*,i" Specht, T., J. Wolters & V I deduced fr.ont rRNA rRNA lgg0 Compilation of 55 -5S ser "r"".*ii,rrr., and 55 rRNA n.r" )^-u:1T*':.iequences. ro'e, B r, G s,a;;;;;; &;lTlh#$":'?i,:::ii"l[.'r11i, 2230 Nucl. Acids Res. lg:221-5_ mongenei i ih phylogeneticheskie Troitsky, sviasi , ,r.n.ii*iy"ri. A. V, yu E. Melekhr )nx structure in the ntonor Lpt". theses ;:,x ".*,x[:ffi x,,[;"liifi rnding :l;;,ru,;,:,:i#:llin:,T,#:,,'::[iiii",:;::Jl,,;; x, #: i,i:,]:ilr;i rUous *o .;o;;J.;;;;,J,:":: Jofli, B. I. & G. ,,warnwrighr, - il ,;:l:;:;i,:!:lri:i:i., ashed K. Chubrik,,l9gg. Srroenie p. G. Hinklc. ^, phylogenericheskie glotki trenratorl i M..L. Sogin sviasi mezhrju Monophyletic a S-X. S,l.t.f. fnS:. fr..otoJu l"ir.i"ii*1". origins of the Merazoa: stlucture f ffr" an evotutionarl.lint< of the pharynx in trematocles Fungr. Science with ona pfyog"nlrl. ,.fo_ 260: 140_-.141

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