This document presents the complete small subunit ribosomal RNA (SSU rRNA) sequences of Giardia ardeae, G. muris, G. duodenalis, and the diplomonad Hexamita. The sequences are compared to analyze phylogenetic relationships. The Giardia sequences range from 1432 to 1453 nucleotides in length, while Hexamita's is 1550 nucleotides. Secondary structure analysis reveals both typically eukaryotic and variable regions. A phylogenetic tree groups the Giardia species separately from Hexamita and Vairimorpha, suggesting they represent a separate kingdom within the domain Eucarya.

1. Unique phylogenetic position of Diplomonadida based on the
complete small subunit ribosomal RNA sequence of
Giardia ardeac, G. muris, G. duodenalis and Hexamita sp.
HARRY VAN KEULEN,5’ ROBIN K. GUTELL,t MICHAEL A. GATES SCOTT K. CAMPBELL, SThNLEY
L. ERLANDSEN,1 EDWARD L. JARROLL’ JAROSLAV KULDA,S AND ERNEST A. MEYER11
Departinent of Biolog Cleveland State Universit Cleveland, Ohio 44115, USA; tMolecu!ar, Cellular and
Developmental Biology University of Colorado, Boulder, Colorado 80309, USA; tDepartment of Cdl Biology and
Neuroanatomy, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA; SDepai.tnein of
Parasitolog Charles University, Vinicna, 12844 Prague, Czechoslovakia; and ‘Department of Microbiology and
ImmunoIog Oregon Health Sciences University, Portland, Oregon 97201, USA
0892-6638/9310007-02231$O1.50. © FASEB 223
ABSTRACT Complete small-subunit rRNA (SSU-rRNA)
coding region sequences were determined for two species
of 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) and
various representative organisms from other taxa. Of the
three Giardia sequences analyzed, the SSU-rRNA from G.
muris is the smallest (1432 bases as compared to 1435 and
1453 for G. ardeae and G. duodenalis, respectively) and
has the lowest G + C content (58.9%). The Hexamita
SSU-rRNA is the largest in this group, containing 1550
bases. Because the sizes of the SSU-rRNA are prokaryotic
rather than typically eukaryotic, the secondary structures
of the SSU-rRNAs were constructed. These structures
show a number of typically eukaryotic signature se-
quences. Sequence alignments based on constraints im-
posed by secondary structure were used for construction
of a phylogenetic tree for these four taxa. The results show
that of the four diplomonads represented, the Giardia
species form a distinct group. The other diplomonad
Hexamita and the microsporidium Vairimorpha necatrix
appear 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. Unique
phylogenetic position of Diplomonadida based on the
complete small subunit ribosomal RNA sequence of
Giardia ardeae, G. mw-is, G. duodenalis, and Hexamita
sp. FASEBJ. 7: 223-231; 1993.
Key Words: ribosomal RNA Diplomonadida Giardia
Hexamita p/zylogenetic relationship
ALTHOUGH MORE THAN 40 DIFFERENT SPECIES of the genus
Giardia have been described (for review, see ref 1), presently
only five species can be well defined, based on morphological
and electrokaryotype characteristics. These are Giardia duo-
denalis, G. ardeae, G. muris, G. agilis, and G. psittaci (2-4). All
are 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); the
name G. duodenalis will be used here. This same morphologi-
cal type of Giardia has been reported from nonhuman sources
also,resulting in the establishment of several axenic isolates
from different host species that were previously designated as
unique Ciardia species (1-6). These various isolates of C. duo-
denalis, although similar morphologically, display variations
in 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 Giardia
would benefit greatly if reliable identification criteria were
available.
Adequate ta.xonomic criteria could include electrophoretic
karyotype analysis, DNA-level comparisons by restriction
site analysis, and sequence analysis of selected genes. The
ribosomal 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 conserved
small 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 a
broader database for discussing phylogeny among Giardia
species and to provide useful information for designing
molecular 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 the
Giardia species in a phylogenetic tree based on SSU-rRNA
sequences appears to place them in a group separate from
Hexamita and Vairimorpha. We propose that these organisms
represent a separate kingdom in the domain Eucarya (11).
MATERIALS AND METHODS
DNA sources
The Giardia ardeae and G. daodenalis isolatesused here were
described previously (7). The source of C. muris DNA is from
cysts isolated from CFI mice (7); the original host was a
‘To whom correspondence should be addressed, at: Department
of 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. 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 techniques
Plasmid DNA containing various cloned rDNA from Giardia
(9) was isolated by the alkaline minipreparation procedure
with the modifications described previously (9). Genomic
DNA from Hexamita was isolated in the same manner as
Ciardia 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 gel
electrophoresis. The DNA fragments were purified by the
freeze-phenol procedure (13).
DNA sequence analysis
Sequence analysis was by the chain termination procedure
(14). The vectors M13mp18 and mpl9 were used with host
strain 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 States
Biochemicals Corp., Cleveland, Ohio). The sequences for
both strands were obtained by using the universal sequenc-
ing primers for the appropriate clones and were sup-
plemented with internally positioned oligonucleotide
primers. The position of the primers are given relative to the
sequence of C. duodenalis SSU-rDNA (10). For C. ardeae,
primer A, reverse, these were positions 301-285,
5’GCTCTCCGGAGTCGAAC3’, for Ha, reverse, positions
1214-1200, 5’GCCGGCTTGGCGGGTCG3, and for C.
muris, primer 1, forward, positions 10-22,
5’GAThCTGCCGGAC3. Primer I is a universal primer
and was also used to complete the C. ardeae sequence. The se-
quence around the 3’ end of the G. duodenalis SSU-rDNA was
determined for five different isolates (MR4, Dl, P1(9), AB,
and CM). In the case of Hexamita rDNA, fragments were
generated with Saa3AI and RsaI to provide fragments small
enough to sequence both strands. Sequence analysis was by
electrophoresis on 8% polyacrylamide sequencing gels with
8 M urea. Areas with extreme band compressions were ana-
lyzed by using dITP with Sequenase or 7-deaza-dGTP with
TAQuence in the reaction labeling mixture. When necessary,
the standard 8% polyacrylamide, 8 M urea sequencing gels
were 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 similarity
values were calculated as similarity = matches/(matches +
mismatches + gaps/2). A gap larger than five nucleotides
was taken as five (16). The similarity values were converted
to distance values (the number of evolutionary changes per
100 positions) (16). These distances were used to construct a
phylogenetic tree by using the neighbor-joining method of
Saitou and Nei (17). The algorithms of Nei and Miller (18)
were used for tree construction.
Secondary structure
The secondary structure model is based on comparative se-
quence analysis (15) and drawn in a format similar to that
of the Escherichia coli 16S rRNA model, which may be
regarded as the standard or prototype structure (19). The
figures were prepared with the assistance of a new RNA
graphics program, XRNA, developed by B. Weiser (unpub-
lished results). Sequences were manually aligned with the
alignment editor AE2, developed by T. Macke (unpublished
results). Identification of the variable regions in V1-V9 and
numbering of some of the stems (E8-1) are as described by
Neefs et al.(20).
RESULTS
The rDNAs of Ciardia duodenalis, G. ardeae, and C. muris were
cloned, and physical and genetic maps were constructed (9).
The SSU-rDNA from Hexamita was identified by Southern
blot 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 in
pUCI8. Subclones that covered relevant regions of the SSU-
rDNA were prepared in M13 DNA by using the available
restriction enzyme recognition sites. The entire SSU-rRNA
genes of C. ardeae, G. muris, and Hexamita were sequenced
from both strands. Although the entire sequence of the C.
duodenalis SSU-rRNA gene has been published (10, 21), some
sections were resequenced due to minor differences ob-
served, especially at the 3’ end of the SSU-rRNA gene; the
differences are included in the presented sequence and are
explained later.
The sequences of the Ciardia and Hexamita SSU-rRNAs
are shown in Fig. 1, where they are aligned with each other
and with that of E. coli. The 5’ and 3’ boundaries of the ma-
ture SSU-rRNA genes were defined by comparison with
previously 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). Three
alterations are included in the C. duodenalis sequence, based
on 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 isolates
of C. duodenalis, instead of TCTA. These alterations were in-
cluded in the alignments and in the construction of the
secondary structure.
This sequence analysis shows that C. muris has the smallest
sized SSU-rRNA of the three Ciardia species, namely, 1432
nucleotides (nt), followed by 1435 for C. ard.eae and 1453 for
C. 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. math
SSU-rRNA is lower, namely, 58.9%; that of Hexamita is
51.4%.
Secondary structures of the three Ciardia and Hexamita
SSU-rRNAs were generated; the one for C. muris is shown
as a representative structure in Fig. 2. The regions that show
the highest degree of variation are identified as V1-V9 (20).
Because these regions are variable in both primary and
secondary structure, the secondary structures of all four
diplomonad rRNA are shown separately in Fig. 3 for com-
parison of similarities and differences in some of the variable
regions.
An inventory of specific nucleotides of all Ciardia species
and Hexamita was made by using the domain-specific bases
and base pairs originally identified by Winker and Woese
(22). Based on the E. coli 16S rRNA positions, these were
scored for the presence of bases that represent each of the
three domains Bacteria, Archaea, Eucarya, or combinations
of these. Because SSU-rRNA of the microsporidium
Vairimorpha necatrix resembles that of diplomonads (see be-
low), Vairimorpha was included in this analysis. The results

3. 104
79
77
GG 76
GG 76
GA $2
213
-- 161
139
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I 13$
)- 16$
273
233
251
236
236
233
391
371
370
374
374
404
-- 497
99 390
. 389
9c 390
9c. 390
434
376
507
506
303
503
556
Lcd GCACGCAGGCGIGUUUGUUAAC GkAAkUCCCGG( ( CCGGMCCMUGMJACUGC ACC<- - 651
Oa*e cGGcGucG I ------- .-------------. ( . <#{231} q#{231} igg I N99C5 99C9 09UU99 567
G.iu,.s G5CcGGA;cCI---- ------------------ 4....... g#{231} ugg#{231} iguqagua 566
GJ.rmair..lls GccCGUAcUZI-------- .------------. (--. l---------------- <c .c ca iqg - uc 564
GJ.b1a GCCCGUAGUl ------- .-- I ---j 2 56
He.ita GcUCGUAGcCGI--------- -------------. (----. I ---------------------------ttati 616
Lcd! - - ---------- ----------------------,u G GG #{149}90CC 4 1?. 713
o ccaccqcoc 9051 5qog------- --. i.qq------------- 64$
Oi, gu a sac- ----.uqt.qq------->--- 651
GJ.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 m
a_I, :j 773
0 IJ 799
GJ.b3a Ii
KisUa G6 VUUCAACAGC u I6
cc I--. 39 IC USAC) Al _ - .CIC GGGIGU&CGGC 3GGCC #{149} OC IGW 2C5U 952
9ucq- -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 1034
G 1 #{163}GCG SC?. XUCCl 9 X c GG n CACC .C OGGU.C)Gc 701 ‘ - 970
Gc k xcc s a wcc cci : -cacs - -- 976
s I ccacc-- - - 1033
s ccG -GG > 1034
25 I GC9 - ------- - .---- 1063
icc 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 1049
1’ 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 1244
cacac (-ccqc) ququgg>- . I 1141
qg)qg). .-------. 7 1147
cgcqg(-gcq-) oogcgg>-. . 7 1179
o9o9g(-qcq-)coqc- . 7 1190
.-- 6AGcGcc I GcccwAGAcAcxuooGcAcG03w.cw.cA 1243
cAuAcMAG?AAocG&cclx 0 < - -i - )GA #{163}334
03C .- <qc.qcgo#{231}CGCGCG0t GCIS4GICCGG9cg 9co aq) -0i9uwJwawG5u..C -C)OC #{163}224
Gos ----- --. uqwac1 XUa .#{248} -- 2G 1220
OGG------------. .- <cc.q-. )2AC goq aqc> -Cc’-w??-1-w2MN-wCvwC -C)OC 1245
GG- -- . - ----. ;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}346
a ai GGAA z - #{163}342
,aA.zn.,tMAd,.cGMa=nwunn 1363
ccccGcGcAccAGGakcGcGccos (cCcCCAc) #{163}366
(UW.WAC) 1450
C- (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 1454
9’, (cqcq-)qcaugcacqaaquuCtcGAAcCCCAAtUGGAGGAACCAG?.AGUCkUMCAAGGCU-GCUGUAGG (GGAA) CCUGCAGcCGGAUC?.----UU&GCCI 1550
Figure 1. Alignment of the nucleotide sequences of Giardia and Hexamita with E. coli SSU-rRNA. The entire nucleotide sequences are
given for Giardia ardeae, C. muris, and G. duodenalis, divided here into C. lamblia, based on Sogin et al. (10), and for G. intestinalis, based
on 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. uc
U U
AUOA 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 aQuA
U U I c-a IA CAQ Qi
0-0 #{149}Oa A
j ( Vs AiIuu iibA A0 UUA #{149}U-A
AA:A_ U A 0-0
U S A C a o
C A A A$00-a #{149}A ,cL AA0-C a /
0-0 U,0, U #{149}#{149}141U OU
0AQ’JAC0 U-A A #{149} a ,C0 U #{149} AU-A
#{149} 0l A-U A U
A A I A....UA 0 A.
GA-U 0A0/ A COU00 A
O 0 0
0 CCCOO 0-0 0#{149} A A0 c#{193}O #{176}0....* 000.U U
o V4 11111 I. U a-C / U UA
O CU IAACC io / V5 ,A0#{149} A0.AO:
#{149} 0
A A ( GAO #{176}CAaAO’
50_A U SU A A/00O00U 4 U0. #{149}a-C
uu0OA SC0 a O”A .0-0
A a 00A %“UA 0 0U#{176}%A -
A 000A000
O 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 Vs
U .. #{149}
U UA A 50 U#{149} 000AOUAU00000 UA I 0a-C-U C 0-C C A-UI
C 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 % U
a,,A0 C CIeos::1 AUUOAOOA0000UA A #{149}U 0
CA o A #{149} U400A u.s #{149} ACUQUUOOCCOU 0
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A 0-a .SIIul#{149}IulAC a A
350 #{149} A U-A aUA0000UOOA”
#{149}A c-a 0 #{149} A*O
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A a I c-a I
00d’,AO0CAOC S #{149}A IA#{149}0 I
‘AC’ A I Aa.u u_O #{149}C CA -U.S o
AaUOOa hi II II 0 C-a-
#{149} i 0CA ca #{149}UAUO U-A
A 00 0U0* o o
Aa,,O0A.A ...
/ C 0 C A
#{149} a A Vg
U-. _Aa 0
o O
a0 0-0A #{149}u_C_S c-s0-0 U.a
250 Q:oA _,‘.ioo a-CAa #{149}A p - a-c0 0 #{149}A
0 A A#{149}0
O A..U0 a-o1350
a-c 0-0
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#{149}SU%t1 - Es -1 AUaA
U0 a-C /
5:: *00
200 0_GA #{176}I
CO COCUGOC
IS 111111
A o*#{176} 000A000
0 U- 0AAC
V2 150
C U
A C
O A
o A
A A0
0
Figure 2. Secondary structure of SSU-rRNA of Giardia muris. Every 10th base is marked and every 50th is numbered in the sequence
beginning at the 5’ end. The numbering of variable regions as V1-V9 and the eukaryotic specific stem E 8-1 are as described by Neefs
et a!. (20). Region V6 is absent in eukaryotes but is shown here to indicate where its prokaryotic equivalent appears. The stem structure
is eukaryotic stem 35 (20). Regions V2 and V4 are left unstructured.
226 Vol. 7 January 1993 The FASEB Journal VAN KEULEN ET AL.

5. Vi
vs
r
vs
Giardia duodenalis Ci ardi a ardeae
vi
Giardia muris
V.
Hexami ta sp
PHYLOGENETIC POSITION OF DIPLOMONADIDA 227
Figure 3. Secondary structure model of diplomonad rRNAs. Line drawings of SSU-rRNA of all four diplomonad rRNAs are given. The
names of the organisms are shown in the figures. The regions VI to V9 are indicated as in Fig. 2.

6. E. coli
Position’
C. muris G. duodenatis G. ardeae Hexainita
Type
Vairimorpha
Base pair TypeBase pair Type Base pair
8 Ub ar(eu)b C eu C eu
9:25 C:G eu+ar C:G eu+ar C:G eu+ar
10:24 C:G ar U:A eu A:U ba # S
33:551 A:U eu+ba A:U eu+ba A:U eu#{247}ba
52:359 G:C eu+ar G:C eu+ar C:G (ba) # S
53:358 C:G eu+ar C:G eu+ar C:G eu+ar
113:314 C:G eu+ar C:G eu+ar C:G eu+ar
!21 A eu A eu U (ba) # S
292:308 A:U eu A:U eu U:U u # S
307 C eu (ba) C eu (ba) U (eu) # S
335 C ba+ar C ba+ar A eu #S
338 A eu+ba A eu+ba A eu+ba
339:350 C:G eu + ba G:G u A:U u # 5
341:348 U:A eu U:G u U:A eu 5
361 C eu+ar C eu+ar C eu+ar
365 A eu + ar A eu + ar A eu +
367 U eu+ba U eu+ba U eu+ba
377:386 C:G eu+ar U:A (eu) #{149} G:C (ba) #$
393 A eu+ba A eu+ba A eu+ba
500:545 G:C ba+ar U:A eu U:A eu
514:537 G:C eu+ar G:C eu+ar A:U u#5
549 C eu+ba C eu+ba U ar #S
558 G ba A eu* U ar#5
569:881 G:C eu G:C eu G:C eu
585:756 U:A eu C:G ar* U:A eu+ar$
675 U eu+ar U eu+ar U eu+ar
684:706 G:C eu+ar G:C eu+ar G:C eu+ar
716 C eu+ar C eu+ar C eu+ar
867 C eu + ar C eu + ar U eu + ar# S
880 U eu U eu U eu
884 G eu G eu G eu
923:1393 A:U eu+ba A:U eu+ba A:U eu+ba
928:1389 G:C ba+ar A:U eu A:U eu
930:1387 G:C eu G:C eu A:U ar # 5
931:1386 G:C eu+ar A:U u #{149} A:U u
933:1384 A:U eu+ar A:U eu+ar A:U eu+ar
962:973 U:G eu U:G eu U:G eu
966 U eu + ar U eu + ar U eu + ar
974 A ba C ba A ba C ba A ba (5)
1098 G eu + ar G eu + ar A u # 5
1109 A eu+ar A eu+ar A eu#{247}ar
1110 G eu+ar G eu+ar G eu+ar
1194 A eu G ar A eu G ar A eu (5)
1201 U eu U eu C ba+ar# 5
1211 U eu+ba C eu C eu U eu+ba C eu (5)
1212 A eu+ar A eu+ar A eu+ar
1381 C eu+ar C eu+ar C eu+ar
1487 A eu A eu G ba+ar# 5
1516 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 indicated
as U. The signatures in parentheses indicate minor forms of a signature feature. .6The signatures that are shared among all Giardia species are shown
in the columns marked with superscript b; the ones that differ are indicated slightly to the left and right of these two columns. Indicates signatures that
differ between all 46 shared Giardia signatures and Hexamita; # indicates where the Vairimorpha signatures differ from the 46 Giardia signatures, and S
indicates where Vairimorp/za differs from Hexamita in the same 46 signatures. The (5) indicates the difference between Vairimorpha and Hexamita in the three
signatures 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. In
Giardia SSU-rRNA, two positions are typical for the Archaea
(three in the case of C. duodenalis), two for the Bacteria, and
three for both Archaea and Bacteria. In contrast, 20 posi-
tions are like Archaea plus Eucarya, and 14 to 15 are unique
for Eucarya. For Hexamita these numbers are 3, 1, 18, and 15,
respectively. The sequence of Hexamita shows three unique
TABLE 1. Comparison of 16S-lik.e rRNA sequence signatures
signature features. Note that of the signature sequences
described by Winker and Woese (22), 22 are shared between
Archaea and Eucarya, 6 between Bacteria and Eucarya, 12
between Archaea and Bacteria, and 9 are unique among the
three groups. Of the 49 positions, all Giardia species have 46
in common. The signatures ofHexamita differ in 11 positions
from the ones that all Giardia species have in common. The

7. Oxytricha nova
- Tetrahymena thensophiia
0.01
TABLE 2. Distribution of signature sequences
PHYLOGENETIC POSITION OF DIPLOMONADIDA 229
Eu Eu + Ar Eu + Ba Ar + Ba Ar Ba unique
Giardiamuris 14(15) 20 8 3 2(1) 2(3) 0
Giardiaduodenalis 14(15) 20 7 3 3(2) 2(3) 0
Giardiaardea 15(16) 20 7 3 2(1) 2(3) 0
Hexamita 15(16) 18 7 1 3(2) 1(3) 3
Vairimorpha 12(13) 15 5 2 3 3(6) 5
The number of domain-specific signatures is indicated. The abbreviations are as in Table 1. The number in parentheses indicates minor forms of
a signaturefeature.
total number of signature differencesbetween Hexamita and
C. muris is 13,whereas these totalsare 14 with C. ard#{128}aeand
12 with C. duodenalis, corresponding to 26.5, 28.5, and
24.4%, respectively.The differencein signature sequences
among the three Giardiaspecies is between 2 and 6% (see Ta-
ble 1 and Table 2). Comparison with Vairimorpha shows a
larger number of different signatures, a total of 20 of the
shared 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 were
aligned 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 cluster
together, leaving the other diplomonad Hexamita and the
microsporidium Vairimorpha to group with other eukaryotes.
DISCUSSION
Sequence similarities of the SSU-rRNA genes within a genus
can be as close as 98.6 to 100% (23). However, within the ge-
nus Giardia, the sequence similarity is much lower. The
similarities of Ciardia muris with C. ardeae and C. duodenalis are
only 80 and 76%, respectively. Within the suborder
Diplomonadida a comparable result is obtained when Hex-
amita sp., a free-livingrepresentative of this group, is in-
cluded (approximately 63% similarity with all Ciardia). All
four organisms have a SSU-rRNA that shows a relatively low
degree of sequence similarity, which results in long branch
lines 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 of
Vairimorpha necatriy, which is only 1244 nt in size. The SSU-
rRNA of Ciardiamuris is the next shortest, with 1432 nt; all
these rRNAs are much smaller, for instance, than 16S rRNA
Ie.nOpus laevlB
Zea maya
I__I chlamydoinonaa reinharcftii
I SaCCharOmYCCS ce.revislae
Cracllariopsls op.
Plasmodium malarlae
LlictyOBtelium discoideum
rrypan050ma brucel
- crith1da fascicuiata
- Euglena grac.iLia
Valrlmorpba neca trix
Hexamlta op.
Glardia ardeae
Giardia duodezza1.j
Giardla murio
II
Halobacteriimt vol canil
- Suit olobua soltataricus
Thezmotcga maritima
Hacberlch.ia coil
Figure 4. Phylogenetic dendrogram constructed for SSU-rRNA. The tree was constructed using the neighbor-joining method. The
horizontal component of separation represents the evolutionary distance between organisms, measured in units of the average number
of 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; Halobacterium
K00421; Sulfolobus solfataricus X03235; Therinotoga rnaritima M21774; Escheric/zia coli V00348; Giardia ardeag Z17210; Hexamita sp.
Z17224.

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 is
tempting to attribute prokaryotic features to the Hexamita
and 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 presence
of these signatures is independent of the G + C content, as
46 of the 49 are shared among the three Ciardia. Other typi-
cal eukaryotic features are that the eukaryote-specific loop E
8-1 around position 120, though small, is present in all
diplomonads. Also, variable region 2 is not split into two
stems as in all eubacteria, and the more problematic V4
region is unlike the typical eubacterial stem. However, it is
also unlike the V4 region of many eukaryotes, irrespective of
which folding model is used (20, 24). Region V6 has the typi-
cal stem structure found in archaebacterial and eukaryotic
SSU-rRNA (22). However, a number of “difficult” regions
are present in all the diplomonad rRNAs, namely, V2 and
V4, for which no phylogenetically conserved structure can be
presented. Note that the V4 region of G. duodenalis differs
from all other diplomonad rRNA structures (Fig. 3). The se-
quence that makes up region V7 in all other SSU-rRNA
structures, with the exception of Vairimorpha, is the most
truncated in the diplomonads (20). Other individual nt or nt
pairs show a strong resemblance with the domain-specific
signatures of Eucarya (Table 1). Finally, the bulge at position
810 (ACG; Fig. 2) is a typical eukaryotic feature (R. R.
Gutell, unpublished results).
For phylogenetic tree construction, a number of options
are possible. The entire sequence can be used or a selection
can be made disregarding “difficult” regions. Second, several
tree construction procedures are available, given an estimate
of the sequence distance matrix. To get a simple overall pic-
ture, the entire SSU-rRNA sequences of all taxa were used
for alignment and the resulting distance matrix was analyzed
only by the neighbor-joining method (Fig. 4).
The resulting dendrogram shows that, of the
diplomonads, all those representing the genus Giardia form
a distinct group. As observed by others (10), the Ciardia spe-
cies form the first branching in the eukaryotic line when the
neighbor-joining procedure is used. After this come Hexamita
and Vairimorpha, in that order. However, no definitive state-
ment on grouping can or should be made here, due to the
lack of additional diplomonad species and microsporidian
representatives.
A more selective procedure that restricts the number of
nucleotide positions might give different results, as might
other methods of phylogenetic tree analysis. These variations
are not reported in this study in view of the overall robust-
ness of the tree procedure selected (25-27). However, the
positioning of the Ciardia species as presented here agrees
with other information: all Giardia and the single Hexainita
appear to have the smallest SSU-rRNAs known, with only
Vairimorpha as an exception. All have a smaller than usual,
but free, 5.8S rRNA (9; unpublished data). The positioning
of Hexamita with respect to Ciardiais supported by ultrastruc-
tural characteristics, which place Giardia the furthest away
from a hypothetical common diplomonad ancestor (28).
Analysis of the sequence signatures shows that Hexamita
differs from Giardiain 11 of the 46 common Ciardiapositions.
The Hexamita signatures include three positions that are
unique. Vairimorpha,which also has a unique position in the
tree, shows a large difference in signatures with both Ciardia
and Hexamita. It differs in 20 of the 46 positions with Ciardia
and in 19 of the same 46 positions with Hexamita (a total of
22 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 observed
previously (9), the rDNA operon of C. muris differs from the
other two Ciardia rDNAs in a number of ways: the distance
between 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 three
Ciardia, 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 when
compared with those of the other two Ciardia; it is rounder
in shape, and has a different median body morphology and
a 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 different
mammals and birds). However, C. ardeae is also, as far as is
known, restricted to a few avian hosts (wading birds, e.g.,
great blue heron, egret, and green heron). Although the
SSU-rRNA genes of G. duodenalis and C. ardeae show a
greater degree of similarity when compared with each other
than when either one is compared with C. muris, it is clear
from these data that C. ardeae is sufficiently different in its
ribosomal genes to be recognized as a distinct species. Mor-
phological differences between C. duodenalis and C. ard.eae are
subtle, 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 Vairimorpha
and Giardia and of the free-living diplomonad Hexamita in the
phylogenetic trees and their overall eukaryotic, but also
unique 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 kingdom
within the domain of Eucarya, as suggested by Woese et al.
(11). Cavalier-Smith (30) has suggested the name Archezoa
for the kingdom that includes phyla with organisms that lack
mitochondria and peroxisomes. The size and organization of
the rRNA genes substantiates this division. However, the
new data presented here have complicated this view; now
two new Ciardiaspecies and another diplomonad are added.
The tree shows that the three species of Ciardiaconsistently
group together, leaving both Hexamita and Vairimorpha out-
side this group, which is in agreement with the differences in
signature sequences. A superkingdom of Archezoa, which
can then be divided into individual kingdoms, appears to be
a more appropriate classification. One of these kingdoms
could comprise the three species of the genus Ciardia. To es-
tablish several kingdoms as indicated and to solve the
problems of species identification within the genus Ciardia,
more information is needed from a greater number of
Diplomonadida, 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 cooperative
agreement CR-816637-01-0 to the University of Minnesota (S.L.E.)
and Cleveland State University (E.L.J.), and National Institutes of
Health grant GM 48207 (R.R.G.). We acknowledge the technical
assistance of Mrs. J. M. Robles-Resto, and we are grateful to Dr.
M. Nei for providingcomputer programs for the neighbor-joining
algorithm. We would also like to thank the W. M. Keck foundation
for its generous support of RNA science on the Boulder campus.
R.R.G. is an associate in the Evolutionary Biology Program of the
Canadian InstituteforAdvanced Research.

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Received for publicationSeptember30, 1992.
Acce/fled forpublicationNovember3, 1992.