55 rRNA sequences`of 12 species of flatworms: implications for the

11ttlrrbittIttgiu.l05: 37 43. I995.
I-.R.(i. Cutrunr (ed.), Biology ol Turbellaria and sorne Related
i,;1995 Klutter Acadenic Publislters. Printed in Belgium.
Flatvtorms.
55 rRNA sequences'of 12 species of flatworms: implications
31
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
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
1
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 phylogeny of the Platyhelminthes is a promising
field for application of molecular methods because,
while some problems have already found reasonable
solutions and may serve as a 'control' (see E,hlers,
1985; Rieger et al., 1990; Rohde, 1990), many others
remain unsolved. By now, portions of l8S rRNA have
been sequence.l fiom various platyhelminthes (Baverstock e/ aI.,1990; Ruitort et a\.,1992 a,b). Yet, additional sequence data are still necessary, in particular,
from other molecules. With the 12 new sequences
presented here, a total of 14 sequences of 55 rRNA
may now contribute to this goal of elucidating platyhelminth phylogeny.
The shortness of the 5S rRNA limits its phylogenetic usage (Hendnks et a\.,1986; Hori & Osava, 1987).
Therefore, we focused our attention not on inferring
phylogenetic trees, but on the statistical estimation of
the existing phyiogenetic hypothesis, i. e. , on their compatibility with 5S rRNA sequence data. This aspect of
the problem is important irrespective of the length of
the anaiyzed sequences. Indeed, a tree computed from
molecular data is a statistical estimate, and one must
know whether there are statistically significant reasons
to prefer it (at least, under the assumptions of the used
tree constructing method) to some other topology, e.g.
one supported by morphological evidence.
Material and methods
The species studied, their systematic positions and collection sites are listed in Table l. The total RNA was
isolated by the hot phenol extraction procedure with
4M guanidinium isothiocyanate; 55 rRNA was purified by electrophoresis on 87o polyacrylamide gel and
sequenced by the Peattie's chemical method after 3'end labelling with (5'-32P)pCp as described earlier
(see Troitsky et al., l99l for the protocol used).
The sequences used fbr rooting the platyhelminth
tree were taken from the 'Berlin RNA databank'
(Specht et al., 1990) with the exception of 55 rRNA
of Echinorhynchus gadi which was sequenced by
Ttble l. Systematic positions and sites of collection
of the
platyhelminth species used in this study
3.3 package (Felsenstein, 1990). Maxirnum
likelihood (ML) method was always implementecl
with
empirical estimation of base fiequencils and
a transi_
tion/transversion ratio 2. Distances were
=
calculat_
ed using the 2-parameter Kimura,s model,
also with
a transition/transversion ratio 2. Lower probabili_
I]-IYLIP
'Turbellaria'
Archoophora
Acoela
=
ConvoIura crnvoluta (Abildgard, I g06)
ty tbr fixation of transversions is suggested
by a very
high level of conservation of the seilndary
strucrure
of rRNAs, which is always affectecl by transversions,
Polycladida
Noktp lttna humuli.r (stimpson, I g57)
'k
P
(2)
larutc era reticuldta (Stimpson, I g-5-5)
while the influence of transitions is moderated
by noncanonical pairing (GU). The value of 2 (rather
than,
e.9.. l.-5 or 3 t was empirically chosen based
Neoophora
Tricladida
Fam. Dugesiidae
on n series
of preliminary experiments with various rRNA data
sets. Computation of the ML trees was
repeatecl three
times with difTerent species input order (one
starting
*Duge.sia (Dugesia) joponlca
(lchikava et
Kawakatsu, I964)
D u ges
ia
(
G
irardh )
ti g rinu
(Girard,
Fam. Planariidae
Planuria ton)a O. F. Muller,
1773
(4)
Fam. Dendrocoelidae
Dendnrnel.uim lacreum ((). F. Muller, 1773)
Typhloplanoida
Bolhromesostoma e.ssenl (Braun,
Kalyptorhynchia
I gg-5)
(s)
N.hu:
cD, j
D. ti
D.7a
P.EO
14.
c. gz
P. at
D. sz
K. s:
F.h<
the
Dj
topologies, we ornitecl bootstrapping; rnorelver,
with
ML method, branch lengths which are not signif_
icantly positive themselves indicate the places
where
changes in the tree are most probable (Felsenstein,
r/h
DT
D7
PI
990).
MC
Be
Gg
Pa
Ds
Dalyellioida
graffi Mitin,
I 97
0
udo g ralJilla are nic
o la
(Meixner.
G raJJtl La
P.s e
(6)
1938)
Monogenea
Di.scocoryle sagitattt (Leuckarr,
lg42)
0)
(g)
Cestoda
Khavia sinensis Hsu,
1935
(9)
Fasciola heparica Linne,
Results and discussion
Twelve 5S rRNA sequences of the species
studied are
presented in Fig. I together with the
sequences fiom
two species studied earlier (Oham a et al'., l9g6;
Hori
et al.,1988).
Trematoda
l75g
collection sites,
as well as the hosts for the parasitic species.
(.1) Dalnezelenetskaya Inlet, Barentz
Sea; (i) giotogicat
tion '^Vostok', Sea of Japan; (3) laboratory stock;
sta_
(4") a pond
near St-Petersburg; (5) a pool in Borok (yaroslavl
Disirict,
Russia); (6) from the marine gashopod, Neptunea
despec_
ta, Biological station ,Karresh., White Sea; (7) Bioiogical
station 'Kartesh', White Sea; (g) from the fi,sh Coreg'onus
albulct, Ladoga lake; (9) from the fish Cyprinu.r
,orpii fish_
farm in Estonia; (10) frorn cows from a slaughte.
house.
us and will be published elsewhere. All the platy_
helminth sequences wholly fitted to the alignment
universally used for 55 rRNA (see Specht er
a/.,
1990). 3'-terminal uracil residues were
analysis because variation in number
n;t used in the
of 3/_terminal
uracils (2 or 3) was observed in practically all stud_
ied species. Phylogenetic analysis was done using
Ks
Fh
F
i,q.
trtr
P.
long.
Approach kt the phl,logenetic. anall,sis
The first question we had to answer was whether
the
tree(s) constructed from 5S rRNA data reproduced
the
relations between the main groups of turbellarians
rep-
resented in our material (Fig. 2A,) which
had been
convincingly established from morphological
evidence
(Ehlers, I 985; Rieger e t al. , 1990;y"t,
,." Rohde, I 990
and present volume for diff'erent views _
these views
are not discussed below because no tree
topology suit_
able for sraristic estimation has been puUtistreOi.
In view of the fact that changes in ihe set
of species
are the most important test for the consistency
of the
tree topology (see, e.g. Wainwright et
at., 1993), we
constructed the trees for several sets of
species. Each
of them included all the l4 platyhelminih sequences
and diff'ered from the others in the single
our-o.orp
(rooting) sequence. This explicitly showea
the efTect
of each outgroup on the tree topology ancl provided
l
seque
(10)
+ Species -sequencetl by
other authors. For the species
sequenced by us, the numbers in parentheses indicate
the
cr
B. es
Cc
Dr
r
Mac rorhynchus crot:ea (O. Fabricius, 1826)
P.re;
with the root sequence and two random ones)
for each
species set with glabal rearrangements.
Being main_
ly interested in the statistical eirimates ot.given
tree
Neorhabdocoela
C. coJ
a
seve
,.si
seqr
the
ed
catt
cep
lcttr
SUS
bra
SPC
nel
(w
bu
i
tl'
39
Maximum
rented with
rd a transi-
:
calculatalso with
probabitiby a very
structure
sversions.
d by non:her than,
n a series
NA
data
ted three
: starting
for each
rg mainven tree
'er, with
t signifs where
enstein.
ied are
s fiom
j; Hori
er the
ed the
)
p.reticuTata* (Pr)
N.humuTis (Nh)
cD.japonica (Dj)
(Dt)
D.tigrina
D.Tacteum (DL)
(Pt)
P.torva
(Mc)
M.crocea
(Be)
B.esseni
(cS)
G.graffi
P.arenicoTa (Pa)
(Ds)
o.sagittata
(Ks)
K.sinensis
(Fh)
F.hepatica
C.
CONVOTUtA
( CC
60
10
70
80
:cies
f the
,we
lach
nces
'oup
1-ect
:da
700
770
/'
severe test for its consistency, especially, in the root
re-9ion.
To provide maximal homogeneity of the set of
suit-
90
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.
sequences in the analysis (see, e.g., Felsenstein, 1990),
1990
,iews
50
720
CC UAAGCAACAWIGGGCCUGCUUAGUACUUGCAUGGGUGACCGCWGGGAACACCGGGUGUUGUAGECWru
Pr
.OUG.C.....CA..
..C......U..U......C.CUAC....CUc.c.A. . .YA. . .U. . .
Nh
. .C. . . . . .U. .U. . . . . .A. .UA. . . ._
Dj
.c.c..A... ..C. .
. .. .A...C.. . .e..uc. ... . .u..cA....G.. .C.A..._
Dt ....u...uAAc...UGC.
....AU.
.....A
..u..ccA...CCA.U.A...D7
cuAcAAu. ..uAc.
u..u.
..
. .uA.cc.. ....u..cuA. . . .c....A..._
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.._
Fh
...G.c.....c...
....C.
cC......U.
ccccc.._
iiiiu
ii
iuiuiiiiii
ii uiiii
iiiiiiuii
iii
ii
rep-
lence
40
30
.AUAo..U......C.1AC....A..
..c.-..
..UA..Uc.....Uc.YAc....A.y.
.u.c...cu...,.u.e....ce......c.A.
u.....u.c...u..
.U.A.u.c..
Uc....cC..
.A.......U....U.
.U.....GU.....AUCU?..c.A.Uc..U.A.
.U....U.A.
.U.A.c..U.....USCCG..c.A.U..AU.A
..U....U.A-..C..
.U.Ac.
...C.CCS....A.U.
.tl .A. -.c.....UC..Ac..cCA..
..U..
..c.-..
.Ac.e..c.....Uc.
c.A..
..U..
.AG.c..C.....uc.c....cCA..
...Ac.
...u.c....
N.cAc.
...U.c..A....GU.
yc.Cc.
...U.c....c.A.U.
..U..
u iiii
ui u uiu i iiiiii
iiii i i i ii
been
s
20
GCCUACGACCAUACCAUGUUGAAUACACCGGUUCUCGUCCCAUCACCCAA-Gt]
we used for rooting all the sequences known from
the anirnals generally considered more closely relat-
ed to the Platyhelminthes, that is Brachionus plicdrills (Rotatoria), Echinorhl,nchus gadi (Acanthocephala), Emplectonema gracile, and Lineus genicu/arrzs (Nemertini).
In addition, the majority
consen-
sus sequences fbr fbur more distant lower invertebrate groups were composed and used: cPorifera (3
species), cCoelenterata (6), cNematoda (3), and cAnnelida (3). The rooting sequences were rather diverse
(with distances between them of 0.071-0.348 K.,") ,
but did not seem to markedly increase the heterogeneity of the analyzed data sets
at least, the distances
-
between the outgroup and platyhelminth sequences
(0.090-0.597 K,,.) varied within the same range as the
distances between the platyhelminth sequences themselves (0.093 -A.625
K...
).
Phylogenetic trees
Figure
2
shows that six
of the eight used rooting
sequences inferred maximum likelihood (ML) trees
(Figs 28,C) which correspond well to recent views on
the phylogeny of the Turbellaria (Fig. 2A) and differ
only in the position of the parasitic flatworms. The
seventh tree was very similar, though Echinorhynchus
'branched offtogether with polyclads' instead ofroot_
ing the platyhelminth tree (Fig. 28, arrowhead). The
tree rooted with Lineus was very different (Fig. 2D),
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
LnL
cCoelenterata
logy
LnL
Echinorhynchu.t
LnL
LnL
ML
- 1038.459
-1044.60
- 1053.06
- 1041.01
C
-1045.945
1.06
D
-1047.508
1.49
B
-1047.020
1.37
AI
A2
A3
-1059.0M
1.66
-1064.366
-106'7.434
1.94
2.13*
- 1051 .56 0.96
-1052.96 1.1'r
- 1053.20 1.49
-1065.21
- 1068.82
1.61
-1071.52
- 1057.90 .68
-i059.54 1.91
-t059.82 1.99*
-1060.18
1.00
-t061.75
1.23
-1061.71
1.49
-1073.40
- 1076.89
1.64
t.76
- 1071.49
1.75
2.09*
2.09*
-1074.84
-
2.t3*
1077.85
2.37*
t
080.
14
1
*
2.0'l*
Rooting sequence
topo-
cAnnelida
cPorifera
cNematoda
logy
LnZ
LnL
Lineus
LnL
LnL
ML
-
-
-1078.22
-
- 1083.03 0.62
- 1085.00 1.03
- I 084.80 0.93
-r058.37
- 1060.05
- I 059.99
- tog7 .17
-ll02.6s
- 1071.06
C
D
B
1026.779
- 1030.887
0.53
-1054.75
-1056.61
0.63
-1032.716
0.73
-1032.17',l
0.89
- 1056.66
0.95
- 1068.74 1.41
- 1073.18 1.59
- 1077.08 2.05*
A1
-1042.349
1.21
A2
- 1046.519
- 1050.101
1.39
A3
1049.8-s
1.82
1.02
is statistically
(distances to the other rooting sequences
0.101_0.244
those to the platyhelminth species O.l4Z_0.475
see the previous paragraph). This confirms
that
our series of outgroup sequences was an effective test
for the consistency of the dominant tree topology. yet,
two details of this topology are probably erroneus:
Dugesia is def,nitely a monophyletic hx;n, and the
Cestoda is generally considered more closely related
to the Monogenea, than to the Trematoda. This ques_
tions the reliability of other branching patterns and
outlines the importance of statistical estimation of the
tree topologies (see below).
On the position of the Neodermata
Parasitic flatworms could be grouped together in the
same way in all ML trees, though two branches with
zero length are to be taken in account here (Fig.
2). Maximum parsimony (Mp) and Fitch trees near_
ly always support monophyly of the Neodermata. The
*1074.87
*1078.69
- I 105.89 1.89*
r between gir"n o;(r";E;;;;
Ln L, logarithm likelihood, , - student's /-ratio for the
difference in Ln
ML tree for corresponding data set. * denotes that difference
K,,",
K,,";
1.42
1.70
1047 .73
0.74
0.87
0.85
1.24
1.42
1.70
ol
l:t
cl
,ignin.oriif>].c6 for2<0.05).
phylogenetic relationships of the Neodermata
remain
controversial. The Neodermata is most often
consid_
ered either the sister group for the Dalyellioida,
rhabdocoels- with doliiform pharynx (see, e.g.,
Ehlers, l9g5;
Brooks, 1989), or as an earlier branch of rhabdocoels
which retained the less specialized rosulate pharynx
(see Joffe et al.,l98l.,Joffe & Chubrik,
lggg; Koriko_
va & Joffe, 1988 for review and evidence from
the
morphology of the pharynx and nervous system).
Two
other hypotheses which could be tested with
our data
would consider the Neodermata the sister group
either
for the Neorhabdocoela + Seriata
Ruitori
er
1c.f.
al.,
1992b), or for the Seriata (note again that the hypothe_
ses suggested by Rohde, (1990 and present
volume;
cannot be discussed here).
Phylip allows a sratistical test of whether the difference between logarithm likelihoods or lengths
of
two given trees is significant. We estimated 6 topolo_
gies representing these four hypotheses (Fig.
3) in
each
of our 8 species sets. Statistical estimates
lstudent,s
t_
ri.
s(
rl
(
-5
N
(
o
S
!
(,
ll
I
I
I
(
41
Neodermata
Acoela
Polycladida
Tricladida
C
B
C.convoluta
Neorhabdocoela
D
C.convoluta
P.reticulata
P.reticulata
N.humulis
N.humulis
G.grafJi
G.graffi
P.are4icola
P.are4icola
.essem
.essent
.crocea
.crocea
F.hepatica
C.convoluta
Linaeus
G.grafft
P.areqicola
.essent
P.reticulata
N.humulis
M.crocea
D.sagittata
K.sinensis
K.sinensis
K.sinensis
F.hepatica
D.ianonica
D.iaoonica
, D.tierina
PJorva
, D.tisina
-
Plorva
-
D.lacteum
D.lacteum
D.lacteum
Fig 2 A Phylogenetic tree representing recent views on the evolution of the 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).
:main
rnsid-
rbdo1985;
coels
rynx
tikor the
Iwo
data
ther
al.,
.he-
ne)
rif-
of
loch
t-
ratio) fbr the trees based on the 'dalyellioid' hypothesis
were 1.15-2 fold higher than fbr three other hypothe_
ses, and its variant (Fig. 3,A3) was significantly worse
than ML tree in 6 of 8 data sets (Table 2). Thus, the
'dalyellioid' hypothesis fits poorly with the available
5S rRNA data compared with other tested assumptions.
None of the 3 other hypotheses may be preferred.
Compatabilitt, of recent hypotheses on the phl,logeny
of the Turbellaria with rRNA molecular data
Statistical estimates described
in the previous
para-
graph also demonstrated that the tree topologies repre_
senting widely accepted recent views on the phylogeny
of the Turbellaria, with Neodermata not branching with
the Dalyellioida (Figs 2A,3F,, b,c,d), fit well with 55
rRNA data (Table 2).
The maximum parsimony (MP) method returned
-1 0 shortest trees, depending of the species set. Estimates of various hypotheses about the relationships
of Neodermata were the same as obtained from ML
l
method. The trees corresponding to recent views on
the platyhelminth phylogeny, provided that rhe Neo_
dermata were not derived together with the Dalyel_
lioida, were less than 5Vo longer than Mp trees, and
the difference was far from being statistically signifi_
cant.
In addition, we unired the data published by Ruitort
et al., 1992a and Baverstock et al., 1991, corrected
alignement and, thus, obtained two data sets includ_
ing 16 and 13 platyhelminrh species with 443 and 536
well aligned positions correspondingly. For the sake of
comparison, they were rooted as in Baverstock er al.
(1991). Studied both by ML and Mp methods,
these
data also demonstrated no statistically significant dis_
agreement with phylogenetic concepts corresponding
to the tree shown on Fig. 2A.
A'
+L
A1
A2
rE
Lg
J-
Typhloplanoida
Kalyptorhynchia
Dalyellioida
Neodermata
{
C
l'yphloplanoida
Kalyptorhynchia
Dalyellioida
Neodermata
--tTricladida
{
Tricladida
Neorhabdocoela
Neodermata
Neodermata
Neorhabdocoela
C.cortvoluta
P.reticulata
B
1
Neodermata
Neorhabdocoela
Tricladida
Neodermata
B.esseni
M.crocea
N.humulis
G.graffi
outgroup
G.graffi
P.arenicokt
B.esseni
M.crocea
r
F.hepatica
K.sinensis
D.sagittata
Kalyptorhynchia
Typhloplanoida
Dalyellioida
Djaponica
D.tigrina
P.torva
D.lacteunt
P.arenicola
B.esseni
M.crocea
G.graffi
P.arenicola
M.crocea
B.esseni
Q.sra.ffi
P.aretticolrt
3' A'D' Trees, representing four hypotheses about the relationships ofthe Neodermata with the Turbellaria,
the .dalyellioid, 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, left) was grafted to the
base turbellarian tree (bottom, center) at the positions
marked 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.
Fig
The rates of nucleotide base replacement and phylogenetic trees
The relative rate of change may be approximated from
the lengths of the branches in the trees (ML, Mp, or
Fitch-Margoliash) or directly from a disrance matrix.
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
completely agree with the estimates obtained from lgS
rRNA by Ruitort et al. (1992b). A high rate of evo_
lution may cause inappropriate early divergence of a
group in the tree or incorrect clustering of the .quick
clocks' together (see, e.g., Lake, 1991). Therefore, the
uncertainty of the position of the Neodermata and/or a
relatively too early position of the planarian branch in
various trees constructed from 55 and l8S rRNA data
(see Hendriks et al., 1986 Hori & Osava, 1987; Baver_
stock e/ al., 199\; Ruitort et al., l992a,b) may result,
correspondingly, from too low and too high rates of
base replacement. The influence of the heterogeneity
in the rates of base replacement is important foi it can
drastically affect the topology of phylogenetic trees
independently of the sequence lengths.
Acknowledgments
We are very grateful to Dr O. N. yunchis (Research
Institute for Lake and River Fishery, St_petersburg)
and to Dr T. A. Timofeeva (Zoological Institute, St_
Petersburg) who collected for us two parasitic species.
Dr E. N. Grusov from the same Institute let us use his
computer, though it notably hindered his own work. We
are also very grateful to our colleagues fiom Moscow
State University: to Dr V K. Bobrova for the help in
a\l)cl lll
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il
\lll()11()
r
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/nchia
toida
I
a
)ela
43
c\perimental work, to Miss Katya Drusina
for perf.ect
rind willing technical assistance, and
to p.ot. e S
..\ntonov for constant attention to this
work and critical
re
tions between Tremat
Perersburg) 22:
Lake, A. J., 1999. Origin
o.f the eukaryotic nucleus
determined by
rare-invariant analyses
RNo *.r".," l" iilo*rnrrn,
K. Bremer & H. Jornval
:f,.lbo.s:m:l
1eds.r" The
;; if.. Err"ui".
Science publishers. Amsterdam: g7_
l0l.
^.
Ohama,
T., T. Kumazaki. H Hori,
S..Osawa & M. Takai,
19g3. Fresh
water plararians ard
aI
in r he is,* *
::11
Rieger. R M . S. ryrer.
p s smiii,
"r.
prary_
helminthes: Turbellaria_
f, e. W. ff*.iror*A i."r. ,"J,un
ptaryhelminrhes
f"Orl,
and Rhvnchoc,oer"
iiie" ri. "rlir,ron ,.0.,
""i.
anaromy ot rnvertebrates.
witey-Liss,
york:
References
h";;;;;
& I. Bev_
lryp"ri,"r"^iJ. the para_
\iric praryhelminrhs tested by pa-rrior
,.qr.niinglll ai'.ruorornor
ItNA. Int. J. parasirol. 2t:329_339.
R.,
1999. The phylogeny
i'"'Jlljllli r:i:?t 1.?,
an
a
gen
of the
erat
"
""
",.v'p.,
"i
ium, Berkley, California
Hendriks, L., E. Huysmanr,
re86. primary srrucrure
pr..
3.3. University ofCalifornia
Herbar_
Berghe & R. De Wachter,
3' Vg den
jh: ,ib"r";;i RiA; J , i **"pods and applicability of"f55 ls
RNA ro rhe ;;;;;;;;;*
lution. J. Mol, Evol. 2,1: 103-109.
"r"
Hori,
H., A. N1uro, S. Osawa. M. Takai,_K._y
Lue & M, Kawakatsu,
I988.
Evoturion of turbeilorra
r. a"ar..J?ir"is'ltororno,
Zool./progress ;;; i;, i
ia_, u,
Hori. H. & S. Osawa. 19g7.
Origins anj.""",*,i"
deduced fr.ont -5S rRNA ser
"r"".*ii,rrr.,
ro'e, B r, G s,a;;;;;; &;lTlh#$":'?i,:::ii"l[.'r11i,
mongenei i ih phylogeneticheskie
sviasi , ,r.n.ii*iy"ri.
Lpt".
structure
theses
rnding
rUous
ashed
NA
)nx
seq uences. Forrschrirte.
in the ntonor
:l;;,ru,;,:,:i#:llin:,T,#:,,'::[iiii",:;::Jl,,;;
Jofli, B. I. & G. K. Chubrik,,l9gg.
Srroenie glotki trenratorl
i
phylogenericheskie sviasi
mezhrju fr..otoJu l"ir.i"ii*1".
ffr"
stlucture of the pharynx in
trematocles ona pfyog"nlrl.
,.fo_
f
;ult.
jof
:ity
ran
-es
:h
)
o,i,.,*Tff .rJilillffi i:".ll,rj:,Jr;
a;:;)].;:.;e0.
-
Cercomeria (platy_
r,ii
Ehlcrs. U ,^l 995 Das phylogenetishe
System der plathelminthes.
G.
Fisher, Sturrgaft _ New york,3l7
pj,
Feisensrein, J., I990. pHyLIp
R
re88 on the nervous svsrem
or
rans. Fortschritte Zool./progress
Zool. 36:
Igl-'1g4.
l]rooks, D.
and rurbellarial Parasitologia
(st-
*"1["Jiri;,i;,1,#;,1,""'.
rrrrrks to the manuscript.
l]rverstock, P R., R. Fielke, A M. Johnson,
R. A. Bray
cridge, 1991. Conflicting ohylogenetic
,a
2974;:.a
I.t;;.."r,.
New
t,' he c tvhe m n thes
"i"',I,.T.;
w h
.PI
i 3'r1,l,ll :"::J,'^ :
Rui:::: Mj K"
i"Ji;l?i.l,iflTll
I
i
a'ffi
i t
il,lf
1992a. Enzyme
s
peci ar
;,g,,iJ.
eleclroohoresis. lgS rRNA
sequences. and lev_
els of phylogeneric resoturion
among ..u.rrii|".1"..o,
r."rn
water planariaas (plarvhe
m -"-"-'"
i n thes, rrrLlduruil'
rf,Ii"",r,.
raludlco
J.Zool.tO:
I
l425.14:j9
r.:"i;;;,
Ruiron. M., K. G. Field. F
A Rxff &
rRNA \equences ,ra pttllogenY
on,li
syrr.
E.;i ;l';;' "ru
Specht, T., J. Wolters
rRNA and 55 rRNA
2230
J
a_
Bagufla' l992b l8s
of Platvhelminthes. Biochem.
&VI
lgg0 Compilation of 55
n.r" )^-u:1T*':.
iequences. Nucl. Acids
Res. lg:221-5_
Troitsky, A. V, yu E.
Melekhr
;:,x x, ".*,x[:ffi #: i,i:,]:ilr;i x,,[;"liifi
*o .;o;;J.;;;;,J,:":: ,;:l:;:;i,:!:lri:i:i.,
,,
il
warnwrighr,
^,
p.
-
G. Hinklc. M..L. Sogin
a S-X. S,l.t.f. fnS:.
Monophyletic origins of
the Merazoa: an evotutionarl.lint<
with
Fungr. Science 260: 140_-.141