Phylogenetic Analysis of Complete Small Subunit Ribosomal RNA Coding Region of Myxidium lieberkuehni: Evidence that Myxozoa are Metazoa and Related to the Bilateria

Arch. Protistenkd. 147 (1996): 1-9 © by Gustav Fischer Verlag Jena

ARCHIV

FUR

PROTISTEN KUNDE

Phylogenetic Analysis of Complete Small Subunit Ribosomal RNA Coding Region of Myxidium Iieberkuehni: Evidence that Myxozoa are Metazoa and Related to the Bilateria MARTIN SCHLEGEL1) , JIRI LOM2 ) , ALEXANDRA STECHMANN1), DETLEF BERNHARD1 ) , DETLEF LEIPE3) , IVA DVKOVA2) & MITCHELL L. SOGIN4 ) 1) Unlversltat Leipzig, Zoologisches Institut, Leipzig, Germany; 2) Institute of Parasitology, Academy of Sciences of the Czech Republic, Geske Budejovice, Czech Republic; 3) National Center for Biotechnology Information, National Library of Medicine, Bethesda, U.S.A.; 4) The Marine Biological Laboratory at Woods Hole, Woods Hole, Massachusetts, U.S.A. v

Summary: The phylogenetic position of Myxozoa relative to other eukaryotes has been controversial. During their complexlife cyclesthey show both protistand metazoan characters. In contrast to their general classification as protists, phylogenetic comparisons of the complete 16Slike rRNAsequence of the myxosporean Myxidium Iieberkuehniwith other, unicellular and metazoan sequences showthat Myxozoa share a common evolutionary historywith Metazoa and are most closely related to the Bilateria. Key Words: Myxozoa phylogeny; Nematocyst evolution; Myxidium Iieberkuehni; Small subunit rRNA.

Introduction All members of the phylum Myxozoa are parasitic. These organisms have complex yet not completely understood life cycles that include multinuclear trophic stages and multicellular spores. Two classes have been recognized (LOM 1990). The Myxosporea contain approximately 1200 species which are generally coelozoic or histozoic parasites of bony fish. Since Myxosporea cause severe tissue destruction that may lead to death, these organisms pose a serious problem in fish husbandry. The class Actinosporea includes about 40 species that are responsible for benign infections in oligochaetes and sipunculids. There is growing evidence, however, that myxosporeans and actinosporeans do not constitute different classes. They may represent alternative forms of a single, complex myxozoan life cycle (WOLF & MARKIV 1984; KENT

et al. 1994). Nevertheless, a final proofthat such an alternation of myxosporean and actinosporean phase in one life cycle applies to all members of the phylum has yet to be presented. Infective myxosporean spores contain both generative cells (sporoplasm or infective germ) and products of somatic cells. Valvogenic cells with desmosome-like connections (DESPORTES-LIVAGE & NICOLAS 1990; DESSER et al. 1983; LOM 1990; SmA-BOBADILLA & ALvAREZ-PELLITERO 1993) transform into spore shell valves, and capsulogenic cells produce polar capsules with coiled extrusible filaments. These filaments anchor the spores at the intestine of the invertebrate host. After it has undergone a simple sexual process, autogamy, the sporoplasm initiates an actinosporean-

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M. SCHLEGEL et al.

like developmental cycle that includes meiosis, formation and fusion of gametes, and, eventually, production of multicellular spores that can infect fish. After initial invasion, the infective cells proliferate and migrate to target organs where multinuclear trophic plasmodia develop. The plasmodia may divide by plasmotomy or budding and ultimately produce spores which can reinitiate a new cycle of infection. In summary, there are clearcut metazoan features present in the Myxozoa, including the separation into generative and somatic cells, the differentiation of the somatic cells, and the occurrence of desmosome-like structures in the envelope or valve cells. Indeed, Myxozoa have already been classified as Metazoa by PATIERSON (1994). However, germ cells are not differentiated in sperms and eggs, and the development does not include a morula-blastula stage. Thus, other metazoan characteristics are lacking and the question remains open, whether the Myxozoa are an independent multicellular evolutionary line, or whether they have reduced these metazoan traits. Following much earlier but rather vague assumptions about metazoan affinities of myxozoans (see LOM 1990), WEILL (1938) proposed a possible phylogenetic link with the Cnidaria based upon similarities of polar capsules and their extrusible filaments with nematocysts of cnidarians. Furthermore, myxosporean pansporoblast formation and larval endoparasitic forms of the Cnidaria such as the fish-infecting Polypodium hydriforme display striking parallels (WEILL 1938; RAIKOVA 1980). Electron microscopy revealed structural and morphogenetical similarities of polar capsules and nematocysts (LOM & DEPuYTORAC 1965) difficult to explain by convergent evolution. Extrusible filaments are also found in protists, such as the dinoflagellate Polykrikos (WESTFALL et al. 1983). However, they are distinctly different both in their final structure and morphogenesis. In order to provide information on the unresolved question, whether Myxozoa are protists or Metazoa, and to further elucidate their yet even basically unresolved phylogenetic relationships, we have started to investigate both myxozoan and hydrozoan rRNA sequences. The comparison of the 16S-like rRNA sequence of Myxidium lieberkuehni with those of different Metazoa, including the cnidarian sequences available to date, and representatives of the alveolates suggests that Myxozoa are metazoans. In contrast to the ultrastructural evidence, however, they are not linked with the cnidarians, but are more closely related to the bilaterians.

Material and Methods Myxidium lieberkuehni trophic stages were collected and pooled from pike (Esox lucius) urinary bladder, fixed in

80% ethanol and stored at 4 °C until further processing. Prior to DNA isolation, fixed specimen were washed in 1 mM TrisIEDTA, pH 8.0, 3x20 min. Cells were lysed in 10 mM Tris, 1 mM EDTA, pH 8.0, 1% SDS for 20 min at 65°C. DNA was isolated using standard procedures (SAMBROOK et al. 1989). 10 to 100 ng of bulk DNA were used for amplification of the 16S-like RNA coding region using eukaryote specific primers complementary to the 5' and 3' end of the gene (ELWOOD et al. 1985) and standard PCR amplification techniques (MEDLIN et al. 1988). As a control, DNA from the host Esox lucius was isolated from muscle tissue and amplified. Amplified products were checked for appropriate size on 1% agarose gels and subsequently cloned into the (RF) of the single stranded vector of both M13 mpl8 and Ml3 mp19 (MESSING 1983). Single stranded templates were prepared for dideoxynucleotide chain termination protocols (SANGER & COULSEN 1975). Synthetic oligonucleotide primers complementary to regions that are well conserved in eukaryotic 16S-like rDNA (ELWOOD et al. 1985) were used to initiate DNA sequencing reactions on both DNA strands. The sequences (EMBL accession numbers X76638 and X76639) were initially aligned with other rRNAs according to primary structure conservation. The sequences were further refined by juxtaposing positions that define conserved secondary structures. Sequences from metazoan and protist representatives for phylogenetic comparisons were retrieved from GenEMBL data bank. Phylogenetic relationships were inferred using distance matrix, maximum parsimony and maximum likelihood methods [provided by the PHYLIP software package, version 3.5c of 1. FELSENSTEIN (FELSENSTEIN 1993)]. Bootstrap methods (FELSENSTEIN 1985) provided rough estimates of support for topological elements in neighbor-joining and maximum parsimony analyses. For all methods we used randomized orders of taxon addition to avoid potential bias from ordered input of taxons. For neighbor-joining analysis (SAITOU & NEI 1987) that did not assume a molecular clock, we converted pairwise similarities between sequences to evolutionary distances using a correction for multiple unseen mutation events (KIMURA 1980). The numbers of extra steps necessary to obtain alternative tree topologies were estimated using the "user-tree-option" in parsimony analyses of the PHYLIP package.

Results

Amplification of 16S-1ike rDNA The amplification of Myxidium lieberkuehni bulk DNA yielded a 2100 base pair product. This compares with a fragment length of 1.8 kb for rRNA sequences amplified from DNA of Esox lucius. The 2100 base pair products were cloned into the single stranded phage M13. A single mp 18 and two mp 19 clones were sequenced but the mp 18 product differed from the mp 19 sequences at 54 sites. The extent of possible microheterogeneity in rRNA gene copies of Myxidium was not further investi-

3

SmallSubunit rRNA Phylogenyof Myxidium lieberku ehni

80

90

100

110

120

130

r-G~T~T~ TAA ~T~G~T~ ( AGACTGCGAAAGGCTCAGTAAATCAGCTATAATCTATTT - - - GAT GTTAACTCCCAGTG A • .. . . . . . •.• .• . . . . . . . •.••••... . . ..•. • •.• • • . . . . . .. --- ••.

-- . C.. -140

A. T

T .C

150

T .. G.G

1 60

A

C.C .. TA.CCGG

170

180

. . . . . . •. •.•.. .

( CGG. T . CT . ATA..

190

200

f.1~~~~~~~~~~~~~~~~~~~~~~~~ . ~~~~~~~~~~~~~~~~~~~~~~~~~~~~

U

T.C .. A. . TA.. G

--- - -

IA

CAA .GC. A. . CG . -- . GT

210 220 230 240 250 260 270 TTATTGGAA-ATACCAACTTTGGGCGAGTGGGTTTA--CCTGCTCGTC- -TAGATAGGTGAATCTGGATA · G AA . T - -C .

·

A

- - - - - - - - - - - - - - - - -- . - - . G. TT

C.G

GACG .T .T .T.T

C

A

.

280 290 300 310 320 330 A TGGCTGATCGTATGGCC---TTG1PCTGACGACGTTTCAATTGAATTTCTGCCCTATCAACTTGTTGG

· --A .. A. T . -- .. CT. T .AGT.. CG~

TCTA .CCGG . AA . GG. G

T

A.A

.

350 360 370 380 390 400 TAAGGTAGTGGCTTACCAAGGTTGTAACGGGTAACGGGGAATCAGGGTTCGATTCCGGAGAGGGAGCCTG

· . GTT . . T

AC

T

T

A

A

C

T

420 430 440 450 460 470 AGAAATGGCTACCACATCCAAGGGAGGCAGCAGGCGCGCAAATTACCCAATCCACAATGTGGGAGGTGGT

. . .. . C

G

A

A. . C

490 500 510 520 GACGAGAAATATTAGGT TTTTCCCTCAGGGAATGCGATAC

T

C. GTTG . -- . . AT

A.A . .

530 540 AAATGAGCACAATTTAGAACATTTGT

:::~ :•:::::~: •~:l~ :~~: ::~ :~~: :~~: ::~: ~ : :~: .•:::::.;::~~ :~: .,::~~ :

560 570 580 590 600 610 CGAGTAACAACTGAAGGGCAAGTCTGGTGCCAGCAGCCGCG-TAATTCCAGCTTCAGTAGTGTATATTAA •• • • • • • • • . . . • • • • • • • • • . . .• •• • • • . • • . . . . • • • • G .••.. • • .. ••• .. . . ... ••• •• •. • •

·

G

GGAG

G

CTCC

C. CGT

620 630 640 650 660 670 680 TGCTGCTGCGGTTAAAACGCTCGTAGTTGAA ACGAAGCACGGGACGATACTTTAACGTATGCTACATC . . ... . G . . . . . . . . • . ....• .. . • • • . .•. ... ....• T . . . . . . . . . . • • .. . .... . .. • • • ....

. AT

A

G.. ---------- ------ ------------- - -

GG.T

690 700 710 720 730 740 750 AAACCGTGTTGCAGTATTGCGAANNAGACCACGTCTGTCTAGCGATGGTGCAGCATGGCGTGGCGTATGT .. . . . . . . C. . T . . ~ GC T .G . . C T .C .

. -- - . . . . CC .. .. . - - ---- - - ---------------- .C .. A. . ------- - - - -- -- .T

-----

Fig.1. l6S-like rDNA sequences of Myxidiumlieberkuehni (first line: Myxidium forward strand , mpl8 sequence; second line: Myxidium reverse strand, mpl9 sequence), aligned with l6S-like rDNA of Caenorhabditis elegans (third line) . Differences in the sequences are shown. The boxes indicate the locations of variable regions as identified from the alignment of all 25 l6S-like rRNA sequences used for phylogenetic analysis as shown in Figs. 2 and 3.

4

M.

SCHLEGEL

et al.

760 770 780 790 800 810 820 TAATTAACAGTCGTTCCGGGCTACTAGATTGTGAGAATGGTGAATGGTCTTCACTGACTGCTCATCGTGT •..• A . . . . . . . . . . . . . . . . . • • • • . • . . . . • • . • • • • . • • . . . . . . . . • T .. C ..•••....•••...

---.C ... T .. -.G .. GT.A .. T... AT... CTG .TT .. AG.TTG ... TCG.C.. ------ ... A. -' .. -

830 840 850 860 870 880 890 CTCGCAGAGT(TGCCTTGAATAAACCAGAGTGCTCAAAGCAGG ATGTTGCT GAAT-GTTAATAGCATG .CA

- .. 'T .A

T

TA.. A.. -GC

A.C.C .. --

.

900 910 920 930. 940 950 960 GAACGAACAAATGTGATTTCGTGTACTGACAAGTAATGCACAATGATTTACATTGTTGTGCACTAACTGG ... ------------------------------ ..... A.AC.G .. C.. CGG .. C.. T.-----------

970 980 990 1000 1010 1020 1030 CAGTACATAGCACCAACCACCAAGGCGGA GTTGGTTTCCGTTTTGGGGTGATGATTAAAAGGGGCGGTT CTA.AAC .. ATT.A

----------~------------------

G

G

A.AAAC

1040 1050 1060 1070 1080 1090 1100 -GGGGGCATTGGTAT TGGCCGCGAGAGG AAATTCTTGGACCGGCCAAGGACTAACAAATGCGAAGGC C

C

ATTA

G

TAGTGA

GCC

CA

A..

1110 1120 1130 1140 1150 1160 1170 ATCTGCCAAGACCGTTCCCGTTGATCAAGAGCGATAGTGAGAGGTTCGAAGACGATCAGATACCGTCCTA · .T

AT.. CTT.A .. A

1180

·

1190

G.. C

A

A

C

G

T.

C

.

1240

1200

G

T

..C

---------T .GG.. GG.TTTT.. C. TGCCGA .. AG.TA

1320 1330 1340 1350 1360 1370 1380 GAAGGGCACCACCAGGAGTGGAGCCTGCGGCTTAATTTGACTCAACACGGGAAAACTCACCCGGTCCGGA . . . . . . . . . • . . A •.. C •.....• T . . . . . • . . . . . . . . . . . . . . . . . . . . • . • . • . . . . • . . . . . . . . .

1390 1400 1410 1420 1430 1440 1450 CATCGATAGGATCGACAGACTGATAGATCTTTCGTGATACGGTGTATGGTGGTGCATGGCCGTTCTTAGT · .C .AT

CT

T

A.. C

TC

TT

GT

.

1460 1470 1480 1490 1500 1510 1520 TCGTGAAGTGATTTGTCAGGTTTATTCCGGTAACGAACGAGACCATATTCTCCATTTACGGAGTCGATGC .G

G

Fig. 1. (Continued)

T

A

G

-----

- -----------------

5

SmallSubunitrRNAPhylogeny of Myxidiumlieberkuehni

1530 1540 1550 1560 1570 1580 1590 AGCATCAGCCTGTCAGGGGCAACTCTGCCAGGCGAAGTGCGACGATTAAATTGGTGATTCCCCGGTACCT · c T A TG.. TAG.A T.

------

c .. A.. TT

------------------------------.T.AAT.G

GTT.-

1600 1610 1620 1630 1640 1650 1660 GTATCGAGGGATTGCTTTTTTTCCATTGACTAAGCATTGTGTAGCCTTAAAAACTGCATAGTGCGCTATG · T .CT.A T.C c c .

.... ----------------------------~-----------------1670

1680

1690

1700

1710

T.T .. ----------

1720

1730

GAGAGACAACCGGGT~TATCCAAAGCCGGAGGACGTAT(GCAATAACAGGTCTGTGATGCCCTTAGATGT

· .. G.. T .. G. -

:;.T. ----

C.C .. GA.TG .. G

.

1740 1750 1760 1770 1780 1790 1800 TCGGGGCCGCACGCGCGCTACAATGGTAACAACAGAAACAGTCTGGTT---CpAAAGAATCAGGTAATCA . . . . . . . . . . . . . . . . . . . • • . • . . • . • . • . . . . . . T .........•• --- •.....•••..••••••••

C

T

T

C

GGAGT

CGG TT.T.CC.ATGC

GTATC

A.C

1810 1820 1830 1840 1850 1860 1870 --TAAATTGTTACCGTAATGGGGACTGTGCTTTGTAATTATCGCACACGAAAGAGGAATTCCTAGTAAGT

GT .G .. A. TC. T .. A.GTCC .... TA.G. TA...

0

•

0

••••

T .. C. TTA .. C.

0

•

0

0

••

Go .

0

0

••••••

1880 1890 1900 1910 1920 1930 GCTCGGAATCAACGAGTGCTGATTGCGTCCC-GCCCTTTGTACACACCGCCCGTCGCTACTACCGAGTGA

· TGA. TC

0

G. TCAC . T

1950

A

1960

T

0

1970

ATGGTGTCATGATGCCT~~TGACCGGACGTCG ••••••••

0"

0

•••

0

••••

0

•••••••

0.0.--

••••••••••••••

2020

2030

.

1980 1990 2000 TAGACGGTGCAAGCCGTCGAACGATGCTGGAATCAA GoA.. A ToT ••••

0

. CT.AT .. GA .. A.AG.G .G ... ----T .... CT.C .. G. T. TA.C .A. T 2010

TCCGG .. C

2040

2050

-TT T 0

2060

••••••

G.A .. C.. T 2070

~TGGCGCAAT---TTCGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCAGAAG

••

~r~:~~::::~:~~:: :~~:~~~~:::::::::::::::::::~~:~::::::::::::::: 2080 GATCAAGCTT

..... --TCG

Fig. 1. (Continued)

gated but most differences resided within hypervariable regions not included in phylogenetic reconstructions. The large size of the Myxidium 16S-like rONA (relative to most other eukaryotic rRNAs) is accomodated by extra nucleotides within hypervariable regions rather than by the presence of intron sequences (Fig. 1).

Phylogenetic analysis Phylogenetic comparisons of the two Myxidium sequences were investigated in analyses that included

alveolate protists, plants, fungi, and both diploblast and triploblast metazoans. Fig. 2 shows a tree constructed with the neighbor-joining method (SAITOU & NEI 1987). Bootstrap values indicated in the figure are based upon 100 replicates. The myxozoans consistently group with the bilaterian metazoans (bootstrap value 100%), and weak support (bootstrap values 60%) suggest a possible relationship with nematodes. In maximum parsimony trees (Fig. 3) the grouping of Myxidium with the bilaterians is also strongly supported (99% bootstrap value) but there is no evidence of a sister group relationship

6

M. SCHLEGEL et al. Xenopuslaevis

73

] Chordata

Herdmania momus

100

Arthropoda

Artemia salina 37'1r- 100

46 ~

-

Mollusca

Limicolaria kambeul

_

Schistosomahaematobium

100 ,.-----------------------

Platyhelminthes

Caenorllabditis elegans ]

Strongyloides stercoralis 46

6

100

L..-

~

~

:Jl

s

Nematoda

Myxidium lieberkuehni (mp1 8) Myxidium lieberkuehni (m p1 9)

67

lJl

11r--- Trichoplax adhaerens Tripedaliacystophora Anemoniasulcata Mnemiopsis leidyi

] Myxozoa

Placozoa ] Cnldarla Ctenophora Porifera

100

Diaphanoecagrandis Acanthocoepsis unguiculata

95

Candida albicans

100

Saccharomyces cerevisiae Oryza sativa

Fig. 2. Dendrogram inferred from 25 l6S-like rDNA sequences using the neighbor-joining method (SAITOU & NEI 1987). Evolutionary distance between the nodes is representedby the horizontalcomponent of their separation. Numbers indicate bootstrap values of a consensus tree with identical tree topology (i.e. how many times the respective node occurred in 100 replicates).

] Fungi

] Plantae

Volvox carteri ,........--- Paramecium tetraurelia L......._ _

] Choanoflagellata

Oxytricha granulifera

] Ciliophora

...--- Theileria annulata Plasmodium berghei

L..-

Prorocentrum micans

] Aplcomplexa Dinoflagellata

0,05

with any particular bilaterian group. Maximum likelihood estimates of the same sequences also show Myxidium to be the sister group to all bilaterians (not shown). All methods reveal a phylogenetic origin of the Myxozoa within the triploblast Metazoa. There is no evidence for a closer relationship to the diploblast cnidarians nor to the protistan alveolates. At present, we cannot decide with our data whether Myxozoa are the sister group to all bilaterians or whether they branch off later with the nematodes.

Test of alternative hypotheses Using maximum parsimony analyses we estimated alternative tree topologies which consider a putative phylogenetic relationship of Myxidium with 1) alveolates, 2) cnidarians, 3) nematodes, and 4) arthropods. The topology of all other taxa was kept identical to that of the most parsimonious tree which had a length of 2409 steps (the same topology as shown in Fig. 3). Under these conditions, 47-59 extra steps were

required, if Myxidium was placed as the sister group of the "alveolates" (47 for a sister group relationship to the ciliates, 55 extra steps for the dinoflagellates, and 59 for the apicomplexans), 28 additional steps were necessary to place Myxidium with the cnidarians, 23 extra steps to group arthropods and Myxozoa, and 8 additional steps to group Myxidium with nematods. In summary, the phylogenetic analysis of Myxidium 16S-like rDNA indicates, that myxozoans have their phylogenetic origin within the metazoans and are closely related with the bilaterians. They are neither related to the cnidarians, nor to the alveolates.

Discussion Sequence variation within Myxidium lieberkuehni Sequence similarities between 16S-like rRNAs of eukaryote congeneric species range from 98.6% to 100% identity (SOGIN et al. 1986; Huss & SOGIN 1990;

Small Subunit rRNA Phylogeny ofMyxidium lieberkuehni Xenopus laevis Herdmania momus

61 100

99

100

100

Schistosoma haematobium

] Platyhelminthes

S

Arlemia salina

] Arthropoda

:;

CaenorfJabditis elegans

Tripedalia cystophora

100

] Placozoa ] Clenophora

Scypha ciliata

] Porifera

O;aphanoeca grandis Acemnocoepsis unguiculata

100

Oryza sativa Volvox carle ri Paramecium tetraurelia

Fig. 3. Consensus tree of maximum parsimony replicates. Numbers indicate bootstrap values.

SCHLEGEL et al. 1991). Thus, it is striking that an even higher sequence difference (2%) was found between forward and reverse strand of Myxidium lieberkuehni rRNA, which cannot be explained by PCR amplification artifacts (MEDLIN et al. 1988). The DNA used for PCR amplification was isolated from trophozoites that were pooled from different host individuals. However, all samples were clearly determined as members of M. lieberkuehni by light microscopy. Consequently, populations of rONA molecules with considerable sequence differences must exist within M. lieberkuehni. Two explanations are possible: M. lieberkuehni consists of (at least two) morphologically indistinguishable, but genetically different cryptic species. Since sexual reproduction is thought to be mostly autogamous (LOM 1990), it may also be possible, that Myxidium consists of several, genetically independent lineages, which have accumulated numerous point mutations in the 16S-like rRNA-genes. Similar levels of microheterogeneity in rRNA genes have been observed in dinoflagellates (SCHOLIN et al. 1993). Site variation of 11% occurs in different rRNA gene copies in plasmodium species, e.g. P. vivax (GUNDERSON et al. 1987). It is not evident why these genes do not appear to be undergoing concerted evolution as proposed by DOVER (DOVER et al. 1982). Comprehensive analyses of more Myxidium lieberkuehni 16S-like

99

] c nidaria

Trichoplax adhaerens

Saccharomyces cerev isiae

~

Myxozoa

Mnemiopsis leidyi Candida albicans

m

] Nematoda

Myxidium lieberkuehni (mp18) ]

Anemonia sulcata

44 2

c:l

] Mollusca

Myxid ium lieberkuehni(mp19)

4S 3

] Chordata

Limicola ria kambeul

Strongyloides stercoralis

54,4

7

] Fungi ] Choanoflagellata ] Plantae ] Ciliophora

Oxytricha granulifera Prorocentrum micsns Plasm odium berghe i Theileria.annulata

] Dinotlagellata ] Apicomplexa

rRNA sequences will be required to explain this phenomenon.

Phylogenetic relationships and evolution of extrusible filaments The position of Myxidium lieberkuehni in the tree based on 16S-like rRNA sequences provides evidence that Myxozoa are a metazoan lineage. This result is in perfect congruence with an analysis by SMOTHERS et al. (1994) that included five myxozoans. Despite their larger data set, it cannot be decided whether the Myxozoa are the sister group of all bilaterians, or if they are related to a particular bilaterian lineage. The analyses presented here as well as that of SMOTHERS et al. clearly show that myxozoans are not specifically related to Cnidaria as suggested by ultrastructural similarities (LOM 1990). The common ancestor of the metazoans most likely differentiated egg and sperm cells and developed a morula-blastula stage during embryonic development. Thus, their phylogenetic affinity with bilateral metazoans suggests that Myxozoa must have undergone considerable reductions during their evolutionary history which are remarkable even for parasites. On the other hand, highly differentiated structures such as the polar capsules with their extrusible filaments must have evolved independently in other metazoan

8

M. SCHLEGEL et al.

and protist lineages. Furthermore the puzzle of their astonishing similarity to nematocysts is still open to speculations. It has been suggested that interstitial cells, which give rise to the nematocysts in cnidarians may have evolved from an endobiont that already possessed extrusible filaments, such as the Myxozoa (SHOSTAK 1993). However, it is assumed that nematocysts or cnidocysts were already present in the cnidarian ancestor (KAESTNER 1984). According to the sequence comparisons myxozoans emerged after the cnidarians, and thus could not give rise to a cnidarian endobiont that eventually might have evolved into the nematocyst. Further phylogenetic analyses of myxozoans are needed to refine the phylogenetic position to, or within the bilaterians. For this purpose it is necessary to enlarge the ribosomal RNA data base. This will also be useful for the study of myxozoan systematics including the obscured relationships between and within the two families Myxosporea and Actinosporea (WOLF & MARKIV 1984). In addition, it is necessary to search for other molecular information, e.g. protein coding genes such as those coding for a-tubulin, p-tubulin or elongation factor 1a (BALDAUF & PALMER 1993). Mini collagens, which are discussed to be characteristic for metazoans, may also be good candidates for this purpose.

Acknowledgement: This work was supported by the Deutsche Forschungsgemeinschaft (Schl 22911-4), nih grant gm 23964 and the Unger G. VetlesenFounation.

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morphology of the site for extrusion. J. Cell Sci. 63: 245-261. WOLFF, K. & MARKIV, M. E. (1984): Biology contravenes taxonomy in the Myxozoa: New discoveries show alternation of invertebrate and vertebrate hosts. Science 225. 1449-1452. Accepted: January 5, 1996 Authors' addresses: MARTIN SCHLEGEL, ALEXANDRA STECHMANN and DETLEF BERNHARD, Universitat Leipzig, Zoologisches Institut, Spezielle Zoologie, TalstraBe 33, D - 04103 Leipzig, Germany; JIRf LOM and IVA DYKOVA, Institute of Parasitology, Academy of Sciences of the Czech Republic, Branisovska 31, 37005 Ceske Budejovice, Czech Republic; DETLEF LEIPE, National Center for Biotechnology Information, National Library of Medicine, NIH, Building 38A, 8600 Rockville Pike, Bethesda, MD 20984, U.S.A.; MITCHELL L. SOGIN, The Marine Biological Laboratory at Woods Hole, Water Street, Woods Hole, Massachusetts 02543, U.S.A. All correspondence should be directed to MARTIN SCHLEGEL.

Note added in proof: While this study was in press a deviating result was published by Siddall et al. (J. Parasitol. 81,961-967). In their data set three myxozoan sequences group with the 16 S-like rDNA of the narcomedusan Polypodium hydriforme.