Gutell 027.faseb.j.1993.07.0223


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Gutell 027.faseb.j.1993.07.0223

  1. 1. Unique phylogenetic position of Diplomonadida based on thecomplete small subunit ribosomal RNA sequence ofGiardia ardeac, G. muris, G. duodenalis and Hexamita sp.HARRY VAN KEULEN,5’ ROBIN K. GUTELL,t MICHAEL A. GATES SCOTT K. CAMPBELL, SThNLEYL. ERLANDSEN,1 EDWARD L. JARROLL’ JAROSLAV KULDA,S AND ERNEST A. MEYER11Departinent of Biolog Cleveland State Universit Cleveland, Ohio 44115, USA; tMolecu!ar, Cellular andDevelopmental Biology University of Colorado, Boulder, Colorado 80309, USA; tDepartment of Cdl Biology andNeuroanatomy, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA; SDepai.tnein ofParasitolog Charles University, Vinicna, 12844 Prague, Czechoslovakia; and ‘Department of Microbiology andImmunoIog Oregon Health Sciences University, Portland, Oregon 97201, USA0892-6638/9310007-02231$O1.50. © FASEB 223ABSTRACT Complete small-subunit rRNA (SSU-rRNA)coding region sequences were determined for two speciesof the intestinal parasite Giardia: G. ardeae and G. muris,both belonging to the order Diplomonadida, and a free-living member of this order, Hexamita sp. These se-quences were compared to published SSU-rDNA se-quences from a third member of the genus Giardia, G.duodenalis (often called G. intestinalis or G. lamblia) andvarious representative organisms from other taxa. Of thethree Giardia sequences analyzed, the SSU-rRNA from G.muris is the smallest (1432 bases as compared to 1435 and1453 for G. ardeae and G. duodenalis, respectively) andhas the lowest G + C content (58.9%). The HexamitaSSU-rRNA is the largest in this group, containing 1550bases. Because the sizes of the SSU-rRNA are prokaryoticrather than typically eukaryotic, the secondary structuresof the SSU-rRNAs were constructed. These structuresshow a number of typically eukaryotic signature se-quences. Sequence alignments based on constraints im-posed by secondary structure were used for constructionof a phylogenetic tree for these four taxa. The results showthat of the four diplomonads represented, the Giardiaspecies form a distinct group. The other diplomonadHexamita and the microsporidium Vairimorpha necatrixappear to be distinct from Giardia.-van Keulen, H.,Gutell, R. R., Gates, M. A., Campbell, S. K., Erlandsen,S. L., Jarroll, E. L., Kulda, J., Meyer, E. A. Uniquephylogenetic position of Diplomonadida based on thecomplete small subunit ribosomal RNA sequence ofGiardia ardeae, G. mw-is, G. duodenalis, and Hexamitasp. FASEBJ. 7: 223-231; 1993.Key Words: ribosomal RNA Diplomonadida GiardiaHexamita p/zylogenetic relationshipALTHOUGH MORE THAN 40 DIFFERENT SPECIES of the genusGiardia have been described (for review, see ref 1), presentlyonly five species can be well defined, based on morphologicaland electrokaryotype characteristics. These are Giardia duo-denalis, G. ardeae, G. muris, G. agilis, and G. psittaci (2-4). Allare cosmopolitan intestinal parasitic protozoa of several ver-tebrate classes (1). In humans, Giardia duodenalis (syn. G. lam-blia or C. intestinalis) can cause a chronic enteritis (1); thename G. duodenalis will be used here. This same morphologi-cal type of Giardia has been reported from nonhuman sourcesalso,resulting in the establishment of several axenic isolatesfrom different host species that were previously designated asunique Ciardia species (1-6). These various isolates of C. duo-denalis, although similar morphologically, display variationsin their karyotypes (7), and this has engendered some con-troversy about their taxonomic status (1). Clearly, both sys-tematic and epidemiological studies of the genus Giardiawould benefit greatly if reliable identification criteria wereavailable.Adequate ta.xonomic criteria could include electrophoretickaryotype analysis, DNA-level comparisons by restrictionsite analysis, and sequence analysis of selected genes. Theribosomal RNA (rRNA)2 genes are conserved in function,organization, and sequence in all organisms, yet their intrin-sic variability permits detailed analysis of sequence similar-ity. Therefore, these genes, especially the highly conservedsmall subunit (SSU) rRNA genes, have been used exten-sively in analyzing phylogenetic relationships (8).G. duodenalis, G. math, and G. ardeae differ in the organiza-tion of their rRNA genes (9). This study presents the SSU-rDNA sequences of C. muris and G. ardeae to provide abroader database for discussing phylogeny among Giardiaspecies and to provide useful information for designingmolecular probes to identify individual Giardia species.These SSU-rDNA sequences are compared with each other,with those of C. duodenalis (syn. C. lamblia (10)) and the free-living diplomonad, Hexamita sp., and with several represen-tative pro- and eukaryotic SSU-rRNAs. Clustering of theGiardia species in a phylogenetic tree based on SSU-rRNAsequences appears to place them in a group separate fromHexamita and Vairimorpha. We propose that these organismsrepresent a separate kingdom in the domain Eucarya (11).MATERIALS AND METHODSDNA sourcesThe Giardia ardeae and G. daodenalis isolatesused here weredescribed previously (7). The source of C. muris DNA is fromcysts isolated from CFI mice (7); the original host was a‘To whom correspondence should be addressed, at: Departmentof Biology, Cleveland State University, 1983 E. 24th St., Cleveland,OH 44115, USA.2Abbreviations: rRNA, ribosomal RNA; SSU, small subunit; kbp,kilobase pair; nt, nucleotides.
  2. 2. 224 Vol. 7 January 1993 The FASEBJournal VAN KEULEN ET AL.naturally infected golden hamster (12). Hexamila sp. was iso-lated from a lake in Czechoslovakia.DNA techniquesPlasmid DNA containing various cloned rDNA from Giardia(9) was isolated by the alkaline minipreparation procedurewith the modifications described previously (9). GenomicDNA from Hexamita was isolated in the same manner asCiardia genomic DNA (7). All cloned DNA used for subclon-ing in M13 vectors was digested with the appropriate restric-tion enzymes and separated from vector DNA by agarose gelelectrophoresis. The DNA fragments were purified by thefreeze-phenol procedure (13).DNA sequence analysisSequence analysis was by the chain termination procedure(14). The vectors M13mp18 and mpl9 were used with hoststrain JM 109. Isolation of single-stranded DNA and the se-quencing reactions were as described in the protocols sup-plied with the Sequenase and TAQuence kits (United StatesBiochemicals Corp., Cleveland, Ohio). The sequences forboth strands were obtained by using the universal sequenc-ing primers for the appropriate clones and were sup-plemented with internally positioned oligonucleotideprimers. The position of the primers are given relative to thesequence of C. duodenalis SSU-rDNA (10). For C. ardeae,primer A, reverse, these were positions 301-285,5’GCTCTCCGGAGTCGAAC3’, for Ha, reverse, positions1214-1200, 5’GCCGGCTTGGCGGGTCG3, and for C.muris, primer 1, forward, positions 10-22,5’GAThCTGCCGGAC3. Primer I is a universal primerand was also used to complete the C. ardeae sequence. The se-quence around the 3’ end of the G. duodenalis SSU-rDNA wasdetermined for five different isolates (MR4, Dl, P1(9), AB,and CM). In the case of Hexamita rDNA, fragments weregenerated with Saa3AI and RsaI to provide fragments smallenough to sequence both strands. Sequence analysis was byelectrophoresis on 8% polyacrylamide sequencing gels with8 M urea. Areas with extreme band compressions were ana-lyzed by using dITP with Sequenase or 7-deaza-dGTP withTAQuence in the reaction labeling mixture. When necessary,the standard 8% polyacrylamide, 8 M urea sequencing gelswere substituted by 8% polyacrylamide, 8 M urea, 20% for-mamide gels.The resulting nucleotide sequences were aligned and ana-lyzed with the DNASTAR programs (DNASTAR Inc.,Madison, Wis.). Values for total sequence similarity calcula-tions were obtained from global alignments over 3411 nucleo-tides according to Woese et al. (15). The sequence similarityvalues were calculated as similarity = matches/(matches +mismatches + gaps/2). A gap larger than five nucleotideswas taken as five (16). The similarity values were convertedto distance values (the number of evolutionary changes per100 positions) (16). These distances were used to construct aphylogenetic tree by using the neighbor-joining method ofSaitou and Nei (17). The algorithms of Nei and Miller (18)were used for tree construction.Secondary structureThe secondary structure model is based on comparative se-quence analysis (15) and drawn in a format similar to thatof the Escherichia coli 16S rRNA model, which may beregarded as the standard or prototype structure (19). Thefigures were prepared with the assistance of a new RNAgraphics program, XRNA, developed by B. Weiser (unpub-lished results). Sequences were manually aligned with thealignment editor AE2, developed by T. Macke (unpublishedresults). Identification of the variable regions in V1-V9 andnumbering of some of the stems (E8-1) are as described byNeefs et al.(20).RESULTSThe rDNAs of Ciardia duodenalis, G. ardeae, and C. muris werecloned, and physical and genetic maps were constructed (9).The SSU-rDNA from Hexamita was identified by Southernblot analysis of genomic DNA digested with various restric-tion enzymes and probed with specific Giardia rDNA probes(data not shown). A 1.7-kilobase pair (kbp) PstI fragment ap-peared to contain the entire SSU-rDNA and was cloned inpUCI8. Subclones that covered relevant regions of the SSU-rDNA were prepared in M13 DNA by using the availablerestriction enzyme recognition sites. The entire SSU-rRNAgenes of C. ardeae, G. muris, and Hexamita were sequencedfrom both strands. Although the entire sequence of the C.duodenalis SSU-rRNA gene has been published (10, 21), somesections were resequenced due to minor differences ob-served, especially at the 3’ end of the SSU-rRNA gene; thedifferences are included in the presented sequence and areexplained later.The sequences of the Ciardia and Hexamita SSU-rRNAsare shown in Fig. 1, where they are aligned with each otherand with that of E. coli. The 5’ and 3’ boundaries of the ma-ture SSU-rRNA genes were defined by comparison withpreviously reported sequences (10, 21).The sequences of C. duodenalis were taken from 50gm et al.(10) (C. lamblia) and Healey et al.(21) (C. inlestinalis). Threealterations are included in the C. duodenalis sequence, basedon sequence analysis of the SSU-rDNA of five different C.duotfrnalis isolates: at position 1241, an extra G was found-around position 1275, three Cs instead of two were found;and the last four bases were CTCG in five different isolatesof C. duodenalis, instead of TCTA. These alterations were in-cluded in the alignments and in the construction of thesecondary structure.This sequence analysis shows that C. muris has the smallestsized SSU-rRNA of the three Ciardia species, namely, 1432nucleotides (nt), followed by 1435 for C. ard.eae and 1453 forC. duodenalis. The Hexamita rRNA is the largest, with 1550 nt.The G + C contents of C. duodenalis and C. ardeae are similar,74.8 and 71.2%. However, the G + C content of G. mathSSU-rRNA is lower, namely, 58.9%; that of Hexamita is51.4%.Secondary structures of the three Ciardia and HexamitaSSU-rRNAs were generated; the one for C. muris is shownas a representative structure in Fig. 2. The regions that showthe highest degree of variation are identified as V1-V9 (20).Because these regions are variable in both primary andsecondary structure, the secondary structures of all fourdiplomonad rRNA are shown separately in Fig. 3 for com-parison of similarities and differences in some of the variableregions.An inventory of specific nucleotides of all Ciardia speciesand Hexamita was made by using the domain-specific basesand base pairs originally identified by Winker and Woese(22). Based on the E. coli 16S rRNA positions, these werescored for the presence of bases that represent each of thethree domains Bacteria, Archaea, Eucarya, or combinationsof these. Because SSU-rRNA of the microsporidiumVairimorpha necatrix resembles that of diplomonads (see be-low), Vairimorpha was included in this analysis. The results
  3. 3. 1047977GG 76GG 76GA $2213-- 161139)- 13$I 13$)- 16$273233251236236233391371370374374404-- 49799 390. 3899c 3909c. 390434376507506303503556Lcd GCACGCAGGCGIGUUUGUUAAC GkAAkUCCCGG( ( CCGGMCCMUGMJACUGC ACC<- - 651Oa*e cGGcGucG I ------- .-------------. ( . <#{231} q#{231} igg I N99C5 99C9 09UU99 567G.iu,.s G5CcGGA;cCI---- ------------------ 4....... g#{231} ugg#{231} iguqagua 566GJ.rmair..lls GccCGUAcUZI-------- .------------. (--. l---------------- <c .c ca iqg - uc 564GJ.b1a GCCCGUAGUl ------- .-- I ---j 2 56He.ita GcUCGUAGcCGI--------- -------------. (----. I ---------------------------ttati 616Lcd! - - ---------- ----------------------,u G GG #{149}90CC 4 1?. 713o ccaccqcoc 9051 5qog------- --. i.qq------------- 64$Oi, gu a sac- ----.uqt.qq------->--- 651GJ.aIils cc i qcqc ccc 99------->---0.1bHg cc i scgca#{231} ccci rs- ---N ai iaocgcu#{231} ,a j iacaaa iaauacauuqauccocac C aauoaaaauauD- 3Q 00 G Z 90 GQG G _ .A1 “IGG531 XCSCCAM 739#{163}soU WCGGC GMG)GCGGCCccCG&CGA1 SAct .- GGGACW ACCCAMaC GGA03q 33$Ga*..e J ma_I, :j 7730 IJ 799GJ.b3a IiKisUa G6 VUUCAACAGC u I6cc I--. 39 IC USAC) Al _ - .CIC GGGIGU&CGGC 3GGCC #{149} OC IGW 2C5U 9529ucq- -GGCI IGMA) -20C cG t G ( MCRMG0CP 2GGI 1UUGC $84- 014g,... . - .GUcAUCG?J -- (GMA) XCUCI 900 4 GC $90- o9c9o9c ) -. cGccGGccGw ci c 917- -. W2GCUCi c 91$U ---- 3UU- 90C 973- - A 5W3G 1034G 1 #{163}GCG SC?. XUCCl 9 X c GG n CACC .C OGGU.C)Gc 701 ‘ - 970Gc k xcc s a wcc cci : -cacs - -- 976s I ccacc-- - - 1033s ccG -GG > 103425 I GC9 - ------- - .---- 1063icc na GcCA I Ul IAAlCDUOCC osG 034 C- 1139-- 1 1 --cGGGcAlz I C AGI------ .- Cc 1043-- I - I - ----GG0QCAIX I IGC I 1Al-- -. C - Cc 10491’ 4- ----CGGWGUGCAUG GCCGCCklGCGU GGC (GMCCCGUnC (UcCAUU) GcGACAAcCCGCkGGI --- ( <c 1016-- .- I - ------CGGlGWCA I ;ccc IGCI W) ICGACAACGSCCG12ACCGGI - - 4 - Ca 1077- .-- - I GCC 21 I 2GUUUAA ACUCUGC CW UU) ICGUUJ 10 XG1 23CMCCAI -- 4.. ( 113$----->co ;GAAcw.scGl G5CGCkCCC1 1244cacac (-ccqc) ququgg>- . I 1141qg)qg). .-------. 7 1147cgcqg(-gcq-) oogcgg>-. . 7 1179o9o9g(-qcq-)coqc- . 7 1190.-- 6AGcGcc I 1243cAuAcMAG?AAocG&cclx 0 < - -i - )GA #{163}33403C .- <qc.qcgo#{231}CGCGCG0t GCIS4GICCGG9cg 9co aq) -0i9uwJwawG5u..C -C)OC #{163}224Gos ----- --. uqwac1 XUa .#{248} -- 2G 1220OGG------------. .- <cc.q-. )2AC goq aqc> -Cc’-w??-1-w2MN-wCvwC -C)OC 1245GG- -- . - ----. ;a)CACGC .109..- VC> ----------------- .-c)cc 1246‘ ----- -- I <gccaqoc .c --------- U)UU 1322: ai GGAMS GG& 9CVu*C #{163}441:4 Al GGMU a IqC- #{163}346a ai GGAA z - #{163}342,aA.zn.,tMAd,.cGMa=nwunn 1363ccccGcGcAccAGGakcGcGccos (cCcCCAc) #{163}366(UW.WAC) 1450C- (uucg-) (GGAA)CCUGcGGUUGGkUCAcU I 1542-- (ccccq-) ----gu9a*cGGcGkcGAGccc;GcGccuGGAGGAAGGsG.AGUcGuMcsAGGuA-JAGG WGAA)CCUGcGG1OGGMC-CW.G I 1433- (gauga.) ----ccqaaCACCGACGMCCGGAGGCUUGGACGAAGGAAAGUcGUMCAkGGW-1Cn75CG (M)CCUGCGGMGGMC--AG I 1432- (ccgc-) -------q--GGGccGcc’7ccccc0cGccuGGAGGAAcGAGAAUAAcAAGGuM1uccGUAGG (AP.) CCUGcGGMJGGAUCC-----C I 1452- (ogcqc-) ------q*aGGGAocccccGccuGG&GGAAGGAcM.GucGUA&cAAGGU&-4cGUAGG (AA)CCUGCGG&UGGAUCC-----UCW.G I 14549’, (cqcq-)qcaugcacqaaquuCtcGAAcCCCAAtUGGAGGAACCAG?.AGUCkUMCAAGGCU-GCUGUAGG (GGAA) CCUGCAGcCGGAUC?.----UU&GCCI 1550Figure 1. Alignment of the nucleotide sequences of Giardia and Hexamita with E. coli SSU-rRNA. The entire nucleotide sequences aregiven for Giardia ardeae, C. muris, and G. duodenalis, divided here into C. lamblia, based on Sogin et al. (10), and for G. intestinalis, basedon Healey et al. (21). The names are as they appear in GenBank. The sequence of E. coli l6S rRNA is GenBank number V00348. Dashes(-) indicate adjustments of the sequence to allow for optimal alignment.PHYLOGENETIC POSITION OF DIPLOMONADIDA 225
  4. 4. ucU UAUOA S50 -0-00-0 1050U .AA0 0C*A00AAAeA%0 e U A.0AS U OOUAA #{216}#{216}U0#{149}1 II 11111.111 #{149}11 a iii ii ji.i ii oVi;UOOoCA.o4OoUOOUUAU0.OA 00 0A 0-a U00000 aaeouo aQuAU U I c-a IA CAQ Qi0-0 #{149}Oa Aj ( Vs AiIuu iibA A0 UUA #{149}U-AAA:A_ U A 0-0U S A C a oC A A A$00-a #{149}A ,cL AA0-C a /0-0 U,0, U #{149}#{149}141U OU0AQ’JAC0 U-A A #{149} a ,C0 U #{149} AU-A#{149} 0l A-U A UA A I A....UA 0 A.GA-U 0A0/ A COU00 AO 0 00 CCCOO 0-0 0#{149} A A0 c#{193}O #{176}0....* 000.U Uo V4 11111 I. U a-C / U UAO CU IAACC io / V5 ,A0#{149} A0.AO:#{149} 0A A ( GAO #{176}CAaAO’50_A U SU A A/00O00U 4 U0. #{149}a-Cuu0OA SC0 a O”A .0-0A a 00A %“UA 0 0U#{176}%A -A 000A000O a Au 000 AA 0 caUuUCAC A l555110_#{149} 0 -450_A U 500 A-U 0 A 0 -0-0 A0-0 0 0A A00A #{149}0AU0 Co A I - AA 0 U 150 VsU .. #{149}U UA A 50 U#{149} 000AOUAU00000 UA I 0a-C-U C 0-C C A-UIC A AC UAa00’,#{176}0, : S50-t Asa iUUOA U000A 00#{149}A0A#{149} A#{149}a I #{149}%A A 0-0 Iull.lulSI.I % Ua,,A0 C CIeos::1 AUUOAOOA0000UA A #{149}U 0CA o A #{149} U400A u.s #{149} ACUQUUOOCCOU 0AAUOQQA Al C 0 U* sum A ts#{176},%*u 0 C oJ_0.12oo,OUACOOU A .a U0 ,. .I 0 AA Co U 0 A #{149}a0,% %_aAGaCa050.eAa A 1300-c 0 A aI U _oA #{149}1400#{149}a,cCaUCH 11111 C A U’AUC00UAa0U uA,iI.A 0-a .SIIul#{149}IulAC a A350 #{149} A U-A aUA0000UOOA”#{149}A c-a 0 #{149} A*O- - 0:a Oa-CA 5I -4 _ S0-0 -*0 A-U 0SA a I c-a I00d’,AO0CAOC S #{149}A IA#{149}0 I‘AC’ A I Aa.u u_O #{149}C CA -U.S oAaUOOa hi II II 0 C-a-#{149} i 0CA ca #{149}UAUO U-AA 00 0U0* o oAa,,O0A.A .../ C 0 C A#{149} a A VgU-. _Aa 0o Oa0 0-0A #{149}u_C_S c-s0-0 U.a250 Q:oA _,‘.ioo a-CAa #{149}A p - a-c0 0 #{149}A0 A A#{149}0O A..U0 a-o1350a-c 0-0U U a A#{149}SU%t1 - Es -1 AUaAU0 a-C /5:: *00200 0_GA #{176}ICO COCUGOCIS 111111A o*#{176} 000A0000 U- 0AACV2 150C UA CO Ao AA A00Figure 2. Secondary structure of SSU-rRNA of Giardia muris. Every 10th base is marked and every 50th is numbered in the sequencebeginning at the 5’ end. The numbering of variable regions as V1-V9 and the eukaryotic specific stem E 8-1 are as described by Neefset a!. (20). Region V6 is absent in eukaryotes but is shown here to indicate where its prokaryotic equivalent appears. The stem structureis eukaryotic stem 35 (20). Regions V2 and V4 are left unstructured.226 Vol. 7 January 1993 The FASEB Journal VAN KEULEN ET AL.
  5. 5. VivsrvsGiardia duodenalis Ci ardi a ardeaeviGiardia murisV.Hexami ta spPHYLOGENETIC POSITION OF DIPLOMONADIDA 227Figure 3. Secondary structure model of diplomonad rRNAs. Line drawings of SSU-rRNA of all four diplomonad rRNAs are given. Thenames of the organisms are shown in the figures. The regions VI to V9 are indicated as in Fig. 2.
  6. 6. E. coliPosition’C. muris G. duodenatis G. ardeae HexainitaTypeVairimorphaBase pair TypeBase pair Type Base pair8 Ub ar(eu)b C eu C eu9:25 C:G eu+ar C:G eu+ar C:G eu+ar10:24 C:G ar U:A eu A:U ba # S33:551 A:U eu+ba A:U eu+ba A:U eu#{247}ba52:359 G:C eu+ar G:C eu+ar C:G (ba) # S53:358 C:G eu+ar C:G eu+ar C:G eu+ar113:314 C:G eu+ar C:G eu+ar C:G eu+ar!21 A eu A eu U (ba) # S292:308 A:U eu A:U eu U:U u # S307 C eu (ba) C eu (ba) U (eu) # S335 C ba+ar C ba+ar A eu #S338 A eu+ba A eu+ba A eu+ba339:350 C:G eu + ba G:G u A:U u # 5341:348 U:A eu U:G u U:A eu 5361 C eu+ar C eu+ar C eu+ar365 A eu + ar A eu + ar A eu +367 U eu+ba U eu+ba U eu+ba377:386 C:G eu+ar U:A (eu) #{149} G:C (ba) #$393 A eu+ba A eu+ba A eu+ba500:545 G:C ba+ar U:A eu U:A eu514:537 G:C eu+ar G:C eu+ar A:U u#5549 C eu+ba C eu+ba U ar #S558 G ba A eu* U ar#5569:881 G:C eu G:C eu G:C eu585:756 U:A eu C:G ar* U:A eu+ar$675 U eu+ar U eu+ar U eu+ar684:706 G:C eu+ar G:C eu+ar G:C eu+ar716 C eu+ar C eu+ar C eu+ar867 C eu + ar C eu + ar U eu + ar# S880 U eu U eu U eu884 G eu G eu G eu923:1393 A:U eu+ba A:U eu+ba A:U eu+ba928:1389 G:C ba+ar A:U eu A:U eu930:1387 G:C eu G:C eu A:U ar # 5931:1386 G:C eu+ar A:U u #{149} A:U u933:1384 A:U eu+ar A:U eu+ar A:U eu+ar962:973 U:G eu U:G eu U:G eu966 U eu + ar U eu + ar U eu + ar974 A ba C ba A ba C ba A ba (5)1098 G eu + ar G eu + ar A u # 51109 A eu+ar A eu+ar A eu#{247}ar1110 G eu+ar G eu+ar G eu+ar1194 A eu G ar A eu G ar A eu (5)1201 U eu U eu C ba+ar# 51211 U eu+ba C eu C eu U eu+ba C eu (5)1212 A eu+ar A eu+ar A eu+ar1381 C eu+ar C eu+ar C eu+ar1487 A eu A eu G ba+ar# 51516 U eu G ar (ba) A ba # 5‘The positions are numbered according to Escherichia co/i 16S rRNA; the signatures are indicated as being like the signature sequence of Bacteria(ba), Archaea (ar), or Eucarya (eu) or a combination of two of these, based on the data from Winker and Woese (22). Unique sequences are indicatedas U. The signatures in parentheses indicate minor forms of a signature feature. .6The signatures that are shared among all Giardia species are shownin the columns marked with superscript b; the ones that differ are indicated slightly to the left and right of these two columns. Indicates signatures thatdiffer between all 46 shared Giardia signatures and Hexamita; # indicates where the Vairimorpha signatures differ from the 46 Giardia signatures, and Sindicates where Vairimorp/za differs from Hexamita in the same 46 signatures. The (5) indicates the difference between Vairimorpha and Hexamita in the threesignatures that vary in the Giardia group.228 Vol.7 January1993 The FASEBJournal VAN KEULEN FT AL.are presented in Table 1 and further analyzed in Table 2. InGiardia SSU-rRNA, two positions are typical for the Archaea(three in the case of C. duodenalis), two for the Bacteria, andthree for both Archaea and Bacteria. In contrast, 20 posi-tions are like Archaea plus Eucarya, and 14 to 15 are uniquefor Eucarya. For Hexamita these numbers are 3, 1, 18, and 15,respectively. The sequence of Hexamita shows three uniqueTABLE 1. Comparison of 16S-lik.e rRNA sequence signaturessignature features. Note that of the signature sequencesdescribed by Winker and Woese (22), 22 are shared betweenArchaea and Eucarya, 6 between Bacteria and Eucarya, 12between Archaea and Bacteria, and 9 are unique among thethree groups. Of the 49 positions, all Giardia species have 46in common. The signatures ofHexamita differ in 11 positionsfrom the ones that all Giardia species have in common. The
  7. 7. Oxytricha nova- Tetrahymena thensophiia0.01TABLE 2. Distribution of signature sequencesPHYLOGENETIC POSITION OF DIPLOMONADIDA 229Eu Eu + Ar Eu + Ba Ar + Ba Ar Ba uniqueGiardiamuris 14(15) 20 8 3 2(1) 2(3) 0Giardiaduodenalis 14(15) 20 7 3 3(2) 2(3) 0Giardiaardea 15(16) 20 7 3 2(1) 2(3) 0Hexamita 15(16) 18 7 1 3(2) 1(3) 3Vairimorpha 12(13) 15 5 2 3 3(6) 5The number of domain-specific signatures is indicated. The abbreviations are as in Table 1. The number in parentheses indicates minor forms ofa number of signature differencesbetween Hexamita andC. muris is 13,whereas these totalsare 14 with C. ard#{128}aeand12 with C. duodenalis, corresponding to 26.5, 28.5, and24.4%, respectively.The differencein signature sequencesamong the three Giardiaspecies is between 2 and 6% (see Ta-ble 1 and Table 2). Comparison with Vairimorpha shows alarger number of different signatures, a total of 20 of theshared 46 of Ciardia (43%).Structural similarity values and distance data were gener-ated for the SSU-rRNA of the three Ciardia species, Hexam-ita, and a number of representative organisms from eubac-terial (Bacteria), archaebacteria! (Archaea), and eukaryotic(Eucarya) origin. The entire SSU-rRNAs sequences werealigned on the basis of primary and secondary structure con-servation of the 16S-like rRNA (19; and R. R. Gutell, un-published analysis). A phylogenetic tree was constructed us-ing the neighbor-joining method (Fig. 4). The Ciardia clustertogether, leaving the other diplomonad Hexamita and themicrosporidium Vairimorpha to group with other eukaryotes.DISCUSSIONSequence similarities of the SSU-rRNA genes within a genuscan be as close as 98.6 to 100% (23). However, within the ge-nus Giardia, the sequence similarity is much lower. Thesimilarities of Ciardia muris with C. ardeae and C. duodenalis areonly 80 and 76%, respectively. Within the suborderDiplomonadida a comparable result is obtained when Hex-amita sp., a free-livingrepresentative of this group, is in-cluded (approximately 63% similarity with all Ciardia). Allfour organisms have a SSU-rRNA that shows a relatively lowdegree of sequence similarity, which results in long branchlines in the estimated phylogenetic tree (see below).However, the diplomonads also share a number of charac-teristic features. First of all, they have the smallest rDNAs se-quenced so far, with the exception of the SSU-rRNA gene ofVairimorpha necatriy, which is only 1244 nt in size. The SSU-rRNA of Ciardiamuris is the next shortest, with 1432 nt; allthese rRNAs are much smaller, for instance, than 16S rRNAIe.nOpus laevlBZea mayaI__I chlamydoinonaa reinharcftiiI SaCCharOmYCCS ce.revislaeCracllariopsls op.Plasmodium malarlaeLlictyOBtelium discoideumrrypan050ma brucel- crith1da fascicuiata- Euglena grac.iLiaValrlmorpba neca trixHexamlta op.Glardia ardeaeGiardia duodezza1.jGiardla murioIIHalobacteriimt vol canil- Suit olobua soltataricusThezmotcga maritimaHacberlch.ia coilFigure 4. Phylogenetic dendrogram constructed for SSU-rRNA. The tree was constructed using the neighbor-joining method. Thehorizontal component of separation represents the evolutionary distance between organisms, measured in units of the average numberof base changes per sequence position.Sequences are quoted by accession number in Genbank and EMBL nucleotide sequence libraries:Xenopus laevis X02995; Zea mays K02202; Chiamydomonas reinhardtii M32703; Saccharomyces cerevisiaeM27607; Gracilariopsissp. M33639; Ox,-tricha nova X03948; Tetrahyinena therinophila M10932; Plasmodium malariae M54897; Dictyostelium discoideum K02641; Trypanosoma brucei M12676;Critlzidiafasciculata X03450; Euglena gracilis M12677; Vairimorpha necatrixY00266; Giardia duodenalis M19500; C. mum X65063; HalobacteriumK00421; Sulfolobus solfataricus X03235; Therinotoga rnaritima M21774; Escheric/zia coli V00348; Giardia ardeag Z17210; Hexamita sp.Z17224.
  8. 8. 230 Vol. 7 January 1993 The FASEB Journal VAN KEULEN FT AL.from E. coli (1542 nt). With this small size in mind, it istempting to attribute prokaryotic features to the Hexamitaand Giardia rRNA. However, close inspection of the secon-dary structure reveals the presence of typical eukaryotic sig-natures in these rRNAs, as shown in Table 1. The presenceof these signatures is independent of the G + C content, as46 of the 49 are shared among the three Ciardia. Other typi-cal eukaryotic features are that the eukaryote-specific loop E8-1 around position 120, though small, is present in alldiplomonads. Also, variable region 2 is not split into twostems as in all eubacteria, and the more problematic V4region is unlike the typical eubacterial stem. However, it isalso unlike the V4 region of many eukaryotes, irrespective ofwhich folding model is used (20, 24). Region V6 has the typi-cal stem structure found in archaebacterial and eukaryoticSSU-rRNA (22). However, a number of “difficult” regionsare present in all the diplomonad rRNAs, namely, V2 andV4, for which no phylogenetically conserved structure can bepresented. Note that the V4 region of G. duodenalis differsfrom all other diplomonad rRNA structures (Fig. 3). The se-quence that makes up region V7 in all other SSU-rRNAstructures, with the exception of Vairimorpha, is the mosttruncated in the diplomonads (20). Other individual nt or ntpairs show a strong resemblance with the domain-specificsignatures of Eucarya (Table 1). Finally, the bulge at position810 (ACG; Fig. 2) is a typical eukaryotic feature (R. R.Gutell, unpublished results).For phylogenetic tree construction, a number of optionsare possible. The entire sequence can be used or a selectioncan be made disregarding “difficult” regions. Second, severaltree construction procedures are available, given an estimateof the sequence distance matrix. To get a simple overall pic-ture, the entire SSU-rRNA sequences of all taxa were usedfor alignment and the resulting distance matrix was analyzedonly by the neighbor-joining method (Fig. 4).The resulting dendrogram shows that, of thediplomonads, all those representing the genus Giardia forma distinct group. As observed by others (10), the Ciardia spe-cies form the first branching in the eukaryotic line when theneighbor-joining procedure is used. After this come Hexamitaand Vairimorpha, in that order. However, no definitive state-ment on grouping can or should be made here, due to thelack of additional diplomonad species and microsporidianrepresentatives.A more selective procedure that restricts the number ofnucleotide positions might give different results, as mightother methods of phylogenetic tree analysis. These variationsare not reported in this study in view of the overall robust-ness of the tree procedure selected (25-27). However, thepositioning of the Ciardia species as presented here agreeswith other information: all Giardia and the single Hexainitaappear to have the smallest SSU-rRNAs known, with onlyVairimorpha as an exception. All have a smaller than usual,but free, 5.8S rRNA (9; unpublished data). The positioningof Hexamita with respect to Ciardiais supported by ultrastruc-tural characteristics, which place Giardia the furthest awayfrom a hypothetical common diplomonad ancestor (28).Analysis of the sequence signatures shows that Hexamitadiffers from Giardiain 11 of the 46 common Ciardiapositions.The Hexamita signatures include three positions that areunique. Vairimorpha,which also has a unique position in thetree, shows a large difference in signatures with both Ciardiaand Hexamita. It differs in 20 of the 46 positions with Ciardiaand in 19 of the same 46 positions with Hexamita (a total of22 ifall 49 positions are compared).With respect to the three Ciardia species, the unique posi-tion of Ciardia muris and the closer relationship between C.ardeae and C. duodenalis are also not unexpected. As observedpreviously (9), the rDNA operon of C. muris differs from theother two Ciardia rDNAs in a number of ways: the distancebetween the SSU-rDNA and LSU-rDNA is shorter in C.muris; the spacer is longer than in C. duodenalis and is heter-ogeneous. The SSU-rRNA is the shortest of the threeCiardia, and it has the lowest G + C content. These sug-gested affinities are sustained by morphological evidence.The morphological features of C. muris are unique whencompared with those of the other two Ciardia; it is rounderin shape, and has a different median body morphology anda different ultrastructure for the ventrolateral flange (29).Also, the host specificity of C. muris is different and more res-tricted (rodents) than that of C. duodenalis (many differentmammals and birds). However, C. ardeae is also, as far as isknown, restricted to a few avian hosts (wading birds, e.g.,great blue heron, egret, and green heron). Although theSSU-rRNA genes of G. duodenalis and C. ardeae show agreater degree of similarity when compared with each otherthan when either one is compared with C. muris, it is clearfrom these data that C. ardeae is sufficiently different in itsribosomal genes to be recognized as a distinct species. Mor-phological differences between C. duodenalis and C. ard.eae aresubtle, so that the present analysis, together with the previ-ously described karyotypic evidence (7), supports the propo-sition that C. ardeae is a distinct species (4).Taken together, the position of the parasites Vairimorphaand Giardia and of the free-living diplomonad Hexamita in thephylogenetic trees and their overall eukaryotic, but alsounique signatures (the unresolved V2 and V4 regions), ap-pear to place these organisms in a unique systematic realm.These organisms could be arranged in a separate kingdomwithin the domain of Eucarya, as suggested by Woese et al.(11). Cavalier-Smith (30) has suggested the name Archezoafor the kingdom that includes phyla with organisms that lackmitochondria and peroxisomes. The size and organization ofthe rRNA genes substantiates this division. However, thenew data presented here have complicated this view; nowtwo new Ciardiaspecies and another diplomonad are added.The tree shows that the three species of Ciardiaconsistentlygroup together, leaving both Hexamita and Vairimorpha out-side this group, which is in agreement with the differences insignature sequences. A superkingdom of Archezoa, whichcan then be divided into individual kingdoms, appears to bea more appropriate classification. One of these kingdomscould comprise the three species of the genus Ciardia. To es-tablish several kingdoms as indicated and to solve theproblems of species identification within the genus Ciardia,more information is needed from a greater number ofDiplomonadida, both parasitic and free living.This work was supported by grants from the Ohio Boards of Re-gents Academic and Research Challenge Programs (H.V.K.,E.L.J.), the U.S. Environmental Protection Agency cooperativeagreement CR-816637-01-0 to the University of Minnesota (S.L.E.)and Cleveland State University (E.L.J.), and National Institutes ofHealth grant GM 48207 (R.R.G.). We acknowledge the technicalassistance of Mrs. J. M. Robles-Resto, and we are grateful to Dr.M. Nei for providingcomputer programs for the neighbor-joiningalgorithm. We would also like to thank the W. M. Keck foundationfor its generous support of RNA science on the Boulder campus.R.R.G. is an associate in the Evolutionary Biology Program of theCanadian InstituteforAdvanced Research.
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