The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis

International Journal for Parasitology 36 (2006) 1521–1534 www.elsevier.com/locate/ijpara

The phylogeny of Myxosporea (Myxozoa) based on small subunit ribosomal RNA gene analysis q Ivan Fiala

*

Institute of Parasitology and Faculty of Biological Sciences, Academy of Sciences of the Czech Republic, University of South Bohemia, Branisˇovska´ 31, 370 05 Cˇeske´ Bude˘jovice, Czech Republic Received 31 May 2006; received in revised form 27 June 2006; accepted 30 June 2006

Abstract The phylogeny of the Myxosporea was studied using the small-subunit ribosomal RNA gene sequences. Maximum parsimony and Bayesian inference were used to determine myxosporean phylogenetic relationships. The analysis included 120 myxosporean sequences retrieved from GenBank and 21 newly obtained sequences of myxosporeans representing nine genera. Members of the genera Palliatus and Auerbachia were sequenced for the first time. The phylogenetic analysis supported a split of myxosporeans into two main lineages separating most of freshwater species from marine ones as described by previous authors. In addition to the two main lineages, a third lineage consisting of three species was found (Sphaerospora truttae, Sphaerospora elegans and Leptotheca ranae) and additional exceptions to the marine/freshwater myxosporean split were recognised (Sphaeromyxa hellandi, Sphaeromyxa longa and Myxidium coryphaenoideum). All three myxosporean lineages were characterised by specific lengths of SSU rDNA sequences. The lineage of marine myxosporeans split into five well-defined clades. They consisted of species with a similar site of infection and spore morphology and were referred as the Parvicapsula clade, the Enteromyxum clade, the Ceratomyxa clade, the marine Myxidium clade and the Kudoa clade, respectively. The inner topology of the freshwater clade was more complex but the trend to branch according to site of infection was observed in this clade as well. Due to the number of sequences available, a histozoic (Myxobolus clade) predominated. Interestingly, five morphologically different species infecting urinary bladder clustered within the histozoic (Myxobolus) clade. The phylogenetic trees derived from this study differ in a number of respects from the current taxonomy of the myxosporeans, which suggests that several currently utilised characters may be homoplasious or that reliance on a single gene tree may not adequately reflect the phylogeny of the group.  2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Myxosporea; SSU rDNA; Phylogeny; Taxonomy; Bayesian analysis

1. Introduction Molecular data using SSU rRNA gene sequences of Myxosporea has indicated that the classification based mainly on the structure and shape of the myxospore is not consistent with phylogenetic relationships (Smothers

q

Nucleotide sequence data reported in this paper are available in GenBank, EMBL and DDBJ databases under the accession numbers: DQ377688–DQ377712. * Corresponding author. Tel.: +420 387775425; fax: +420 385310388. E-mail address: fi[email protected]

et al., 1994). This view was supported by Andree et al. (1999), who demonstrated that the genus Myxobolus is not monophyletic. Paraphyly of this genus was later confirmed by Kent et al. (2001). They also stressed the paraphyly of the genus Myxidium and polyphyly of the genera Henneguya and Sphaerospora. Phylogenetic analysis revealed the separation of freshwater and marine myxosporeans into two major branches (Kent et al., 2000, 2001). Ceratomyxa shasta, as well as some Myxobolus and Henneguya species, were the exception to this trend. Later, the phylogenetic positions of Chloromyxum leydigi (Fiala and Dykova´, 2004), Parvicapsula minibicornis (Jones et al., 2004) and Sphaeromyxa zaharoni (Diamant

0020-7519/$30.00  2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.06.016

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et al., 2004) were also found not to follow the marine/freshwater separation. Inner topologies within the marine and freshwater branches suggest that myxosporeans cluster in accordance with various characteristics. Salim and Desser (2000) analysed seven Myxobolus species grouped by spore morphology. In contrast, analysis of 10 different Myxobolus species (Andree et al., 1999) revealed relationships according to the site of infection. Similarly, Eszterbauer (2004) described two gill-infecting groups within Myxobolus spp. A relationship based on the site of infection was reported also by Holzer et al. (2004). In their study, five urinary bladderinfecting species assigned to different genera clustered together. Whipps et al. (2004) found the site of infection to be an important criterion in relationships of some Kudoa species. The earlier conclusion of Hervio et al. (1997), that five species of the genus Kudoa clustered by geographic location, was a reflection of small sample size (Blaylock et al., 2004). The aim of this study was to elucidate the relationships of a large set of myxosporeans based on SSU rDNA sequence data. Myxosporeans from both freshwater and marine environments were sequenced to test previously described branching of Myxosporea into freshwater and marine clades. Gall bladder infecting species were preferentially sequenced in order to enlarge the set of coelozoic species in the phylogenetic tree and to evaluate the relevance of site of infection in myxosporean evolution based on SSU rDNA. 2. Materials and methods 2.1. Collection of myxospores Fresh spores of Myxosporea were collected from their hosts in several regions of the world during 2002–2004 (Table 1). Two expeditions were arranged by the Fisheries Research Services Marine Laboratory in Aberdeen, Scotland (research vessel FRV Scotia). On the first expedition, myxosporeans were collected from marine fish from shallow waters off the coast of the British Isles. Deep-water fish from the seas to the west of Scotland were examined for myxosporeans on the second expedition. Myxosporeans were also collected from Caribbean fish caught in the brackish waters of Chetumal Bay and in the Caribbean Sea off the coast of Mexico. In addition, the myxosporeans from freshwater fish were collected from two geographic localities: the Trˇebonˇ pond area, Czech Republic, and lakes near Wuhan, Hubei Province, China. The spores of Myxidium chelonarum were found in the gall bladder of the turtle Kachuga smithi, which was imported from South Asia to Czech Republic. Samples were checked carefully by microscopic observation to ensure that they contained spores of only one species. Spores were cleaned from bile and host tissue with distilled water and used for morphological and molecular characterisation. The images of spores were captured using

an Olympus BX51 microscope equipped with Nomarski differential interference contrast. 2.2. DNA isolation, cloning and sequencing DNA was extracted from fresh spores using the DNeasy tissue kit (Qiagen, Germany) according to the manufacturer’s protocol. The SSU rRNA gene was PCR amplified with a set of universal eukaryotic primers (ERIB1, 5 0 -ACCTGGTTGATCCTGCCAG-3 0 and ERIB10, 5 0 -CTTCCGCAGGTTCACCTACGG-3 0 ) (Barta et al., 1997) to obtain almost complete sequence. Negative results of some PCRs inspired the development of new myxosporean specific SSU rDNA primers MyxospecF (5 0 -TTCTGCCCTATCAACTWGTTG-3 0 ) and MyxospecR (5 0 -GGTTTCNCDGRGGGMCCAAC-3 0 ), based on a comparison of all available myxosporean sequences. PCR was carried out in a 25-ll reaction volume using 10 pmol of each primer, 250 lM of each – deoxyribonucleotide triphosphate, 2.5 ll 10 · PCR Buffer (Top-Bio, Czech Republic) and 1 U of Taq-Purple polymerase (Top-Bio, Czech Republic). The reactions were run on a Tpersonal cycler (Biometra). Amplification consisted of 30 cycles of 95 C for 1 min, 48 C for 1 min and 72 C for 2 min, followed by 10 min incubation at 72 C. One millilitre of the initial PCR with ERIB primers was used as a template for nested PCR with the MyxospecF and MyxospecR primers. The PCR conditions were the same as for the first PCR, with an annealing temperature 52 C instead of 48 C. The PCR products were isolated from the gel and cloned into pCR 2.1-TOPO vectors from the TOPO-TA Cloning Kit (Invitrogen). Single clones of PCR product were used for sequencing except for samples of Sphaeromyxa hellandi and Zschokkella nova (two additional clones for each sample) in order to determine the rate of intragenomic heterogeneity. Both strands of the clone were sequenced on a CEQ 2000 automatic sequencer (Beckman-Coulter), using a CEQ DTCS Dye Kit (Beckman Coulter) according to the manufacturer’s protocol. 2.3. Phylogenetic analysis Three datasets were prepared for analysis. The first consisted of newly obtained sequences and those retrieved from GenBank. This dataset contained 139 ingroup taxa and included sequences of Sphaerospora truttae, Sphaerospora elegans and Leptotheca ranae. The second (with mostly marine species) and the third (with mostly freshwater species) datasets were composed of 48 and 88 ingroup taxa, respectively. These two datasets were prepared in order to gain more information on the alignment of closely related taxa. Since myxosporeans are sister taxa to the malacosporeans (Canning et al., 2000), Malacosporea have been used as outgroup. However, test analyses with bilaterians Caenorhabditis elegans, Xenopus laevis and Polypodium hydriforme were performed.

I. Fiala / International Journal for Parasitology 36 (2006) 1521–1534 1523 Table 1 A summary of myxosporeans sequenced in this study Myxosporean species

Host

Site of infection

Locality

Length of sequence

Accession No.

Auerbachia pulchra Ceratomyxa sp. 1 Ceratomyxa sp. 2 Chloromyxum leydigi Henneguya sp. Myxidium bergense Myxidium chelonarum Myxidium coryphaenoideum Myxidium cuneiforme Myxidium gadi 1 Myxidium gadi 2 Myxidium incurvatum Palliatus indecorus Sphaeromyxa hellandi 1 Sphaeromyxa hellandi 2 Sphaeromyxa hellandi 3 Sphaeromyxa longa Sphaerospora sp. Zschokkella nova 1 Zschokkella nova 2 Zschokkella parasiluri Zschokkella sp. 1 Zschokkella sp. 2 Zschokkella sp. 3 Zschokkella sp. 4

Coryphaenoides rupestris Scyliorhinus canicula Notacanthus bonapartei Centroscymnus coelolepis Trachinotus goodei Helicolenus dactylopterus Kachuga smithi Coryphaenoides rupestris Cyprinus carpio haematopterus Pollachius virens Melanogrammus aeglefinus Callionymus lyra Alepocephalus grandis Mellanogrammus aeglefinus Helicolenus dactylopterus Helicolenus dactylopterus Trisopterus minutus Mugil curema Carassius auratus gibelio Ctenopharyngodon idella Pseudobagrus fulvidraco Eugerres plumieri Haemulon sciurus Diodon holocanthus Spectrunculus grandis

Gall bladder Gall bladder Gall bladder Gall bladder Peritoneum Gall bladder Gall bladder Gall bladder Gall bladder Gall bladder Gall bladder Gall bladder Gall bladder Gall bladder Gall bladder Gall bladder Gall bladder Wall of gall bladder Gall bladder Gall bladder Gall bladder Gall bladder Gall bladder Urinary bladder Gall bladder

North Atlantic North Atlantic North Atlantic North Atlantic Caribbean Sea North Atlantic South Asia North Atlantic China North Sea North Sea North Sea North Atlantic North Sea North Sea North Atlantic North Sea Caribbean Sea China Czech Republic China Caribbean Sea Caribbean Sea Caribbean Sea North Atlantic

1745 800a 1741 1866 2113 1752 2142 1992 2077 1753 775a 1735 1734 1976 1976 1976 905a 772a 2067 2070 2069 943a 945a 1954 805a

DQ377703 DQ377698 DQ377699 DQ377710 DQ377706 DQ377702 DQ377694 DQ377697 DQ377709 DQ377711 DQ377707 DQ377708 DQ377712 DQ377693 DQ377692 DQ377701 DQ377691 DQ377695 DQ377690 DQ377688 DQ377689 DQ377696 DQ377704 DQ377705 DQ377700

a

Indicates the partial sequences.

Preliminary phylogenetic trees constructed from the matrices were aligned using different gap-opening penalty, gap-extension penalty and DNA transition weights in order to check the influence of alignment parameters on the resulting tree topology. Final matrices were aligned using the Clustal_X program (Thompson et al., 1997) with arbitrarily chosen parameters (8.0 for gap opening penalty and 5.0 for gap extension penalty). The DNA transition weight was 0.5. Manual adjustments were done by eye to correct the alignment (mainly the long sequences of S. truttae, S. elegans and L. ranae) using the BioEdit sequence alignment editor (Hall, 1999). Ambiguous positions were selected by eye and then removed from the dataset. Bayesian inference (BI) analyses were conducted using MrBayes v. 3.0 (Ronquist and Huelsenbeck, 2003). The development of Bayesian inference using Markov Chain Monte Carlo techniques enables the evaluation of large datasets and the incorporation of parameter-rich evolutionary models (Huelsenbeck et al., 2001; Olson et al., 2003). Huelsenbeck et al. (2002) stressed that results of Bayesian analysis of phylogeny are contingent on the chosen model being correct. The hierarchical likelihood ratio test used to select the best model of evolution was chosen for this study. Models of nucleotide substitution were evaluated for the data using MrModeltest v. 2.2 (Nylander et al., 2004). For all three datasets, the most parameter rich model (general time reversible with estimates of invariant sites and gamma distributed among site rate variation) was found to fit the data best. Thus, BI was performed with

parameters (rates = invgamma, NST = 6, ncat = 4) corresponding to the model estimated (GTR + I + C). No a priori assumptions about the topology of the trees were made and all searches were provided with a uniform prior. Posterior probability distributions were generated using Markov Chain Monte Carlo (MCMC) methods. The MCMC processes were set so that four chains were run simultaneously for 1,000,000 (for the first dataset) and 500,000 (for the second and third datasets) generations, with trees being sampled every 100 for a total of 10,000 and 5000 trees, respectively. Burn-in, when a process reached a stationary state, was determined when visual inspection indicated that the log-likelihood values achieved an asymptote over a large number of generations. The length of burn-in period was 85,000 generations for the first dataset and 35,000 generations for the second and third datasets. The Maximum parsimony (MP) analysis was conducted with PAUP*, Version 4.0b10 (Swofford, D.L., 2001. PAUP*: Phylogenetic Analysis Using Parsimony (*and other methods), Version 4.0b8. Sinauer Associates, Sunderland, Massachusetts), using an heuristic search with tree bisection–reconnection (TBR) branch swapping, random addition of taxa (10 replications) and the ACCTRANoption. Gaps were treated as missing data. The matrices were analysed using the 1:1, 1:2 and 1:3 Ts/Tv ratios. Clade support was assessed with bootstrapping of 1000 replicates. In addition to MP and BI, the distance method was performed using an heuristic search with minimum evolution

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(ME) objective settings (using PAUP* program) and log determinant distances (LogDet). This method was used to examine whether base compositional nonstationarity was a factor influencing topology. Myxosporeans (Figs. 4 and 5) were supplemented by the following data (if available): site of infection, systematic classification of fish host (data from www.fishbase.org), geographic origin and host environment. 3. Results 3.1. Sequence analysis The SSU rRNA gene sequences of 21 myxosporean species were obtained (Table 1) in the present study. Table 2 summarises morphological characteristics of eight

unnamed species. Selected microphotographs of myxosporeans (examples of species representing the genera sequenced in present study) are shown in Fig. 1. Almost complete sequences were obtained from 15 myxosporean species. The length of almost complete sequences varied from 1734 to 2142 nucleotides (nt) (see Table 1). Shorter sequences of SSU rDNA (about 1800 nt) were found in species that clustered in the marine clade in the phylogenetic analysis. Most species of the freshwater clade have SSU rDNA sequences longer than 2000 nt. When the PCR with universal eukaryotic primers failed, nested PCR successfully amplified partial sequences (800–950 nt) from six myxosporean species. Myxidium gadi, Z. nova and S. hellandi were found in different host species and/or different geographic localities and their sequences were compared. Three sequences of

Fig. 1. Spores of myxosporeans. (A) Myxidium bergense (apical view) from Helicolenus dactylopterus. (B) Myxidium bergense (frontal view). (C) Myxidium coryphaenoideum from Coryphaenoides rupestris. (D) Zschokkella sp. 1 from Eugerres plumieri. (E) Auerbachia pulchra from Coryphaenoides rupestris. (F) Zschokkella parasiluri from Pseudobagrus fulvidraco. (G) Sphaerospora sp. from Mugil curema. (H) Sphaeromyxa hellandi (frontal view) from Mellanogrammus aeglefinus. (I) Sphaeromyxa hellandi (apical view). (J) Chloromyxum leydigi from Centroscymnus coelolepis. (K) Henneguya sp. from Trachinotus goodei. (L) Palliatus indecorus from Alepocephalus grandis. (M) Ceratomyxa sp. 1 from Scyliorhinus canicula. Scale bar = 5 lm (A–K) and 10 lm (L and M).

I. Fiala / International Journal for Parasitology 36 (2006) 1521–1534 1525 Table 2 Spore characteristics of unnamed myxosporean species (20 spores measured) Myxosporean species

LS

WS

Ceratomyxa sp. 1 Ceratomyxa sp. 2 Henneguya sp. Sphaerospora sp. Zschokkella sp. 1 Zschokkella sp. 2 Zschokkella sp. 3 Zschokkella sp. 4

11.5 (10–12) 10.3 (10–12) 11.4 (11–12) 6.9 (6–8) 11.5 (11–12) 13.0 (12–14) 13.1 (12–14) 13.0 (12–14)

102.0 (92–111) 14.8 (12–16) 7.6 (7–8) 8.6 (8–9) 13.4 (13–14) 8.7 (8–10) 8.2 (8–9) 8.7 (8–9)

LCP

11.2 (10–13)

LPC

WPC

NC

3.5 3.0 5.0 4.0 6.0 4.5 3.5 3.0

3.5 3.0 2.5 2.5 4.5 4.0 3.5 3.0

4 2–3 4 5–6 4 5 6 5

LS, length of spore; LCP, length of caudal projection; WS, width of spore; LPC, length of polar capsules; WPC, width of polar capsules; all measurements in lm; NC, number of coils of polar filament.

S. hellandi from two different hosts and localities differed at 3–10 nt sites (99.51–99.85% similarity). Two sequences of M. gadi differed at only one nt position (700 bp compared). The comparison between two representatives of the genus Zschokkella, both morphologically identified as Z. nova, showed a difference in 49 nt (97.56% similarity). Newly obtained SSU rDNA sequence of C. leydigi from Centroscymnus coelolepis from the North Atlantic was compared with sequence of C. leydigi from Torpedo marmorata from the Mediterranean Sea. These sequences revealed a difference of 47 nt (97.49% similarity). Intragenomic variability was ascertained in S. hellandi (from Hellicolenus dactylopterus from North Sea) and Z. nova (from Ctenopharyngodon idella from Czech Republic). Comparison between three cloned PCR products showed 99.60– 99.80% similarity in S. hellandi and 99.05–99.65% similarity in Z. nova. 3.2. Phylogenetic analysis Based on SSU rDNA sequence data, myxosporeans split into three lineages (two major and one minor clade). The two major clades divided myxosporeans into a lineage with mostly freshwater species (freshwater clade) and one with mostly marine species (marine clade). A minor clade, containing three taxa, S. truttae, S. elegans and L. ranae, was distinguished by the extraordinary long nucleotide inserts in their SSU rDNA sequence. Sphaerospora truttae, S. elegans and L. ranae clustered as a basal branch to all myxosporean species and this basal position was confirmed by distance analysis with LogDet distances. All preliminary phylogenetic trees constructed from the matrices aligned under different parameters revealed almost the same topology, congruent with the topology shown in Fig. 3. The marine clade split into five distinct lineages (Kudoa clade, marine Myxidium clade, Ceratomyxa clade, Enteromyxum clade and Parvicapsula clade). Two species diverged (Zschokkella sp. 3 and C. shasta). Although the BI tree (Fig. 2) and the MP tree (Fig. 3) were almost fully resolved, low nodal support made relationships within the marine lineages unclear. Moreover the topology of the BI and MP trees differed within marine clade. The Parvicapsula + Enteromyxum clades were closely related to the marine Myxidium

clade in BI, in contrast to the close relationship of the Parvicapsula + Enteromyxum clade with the Ceratomyxa clade in the MP analysis. In both BI and MP trees, the Parvicapsula clade was closely related to the Enteromyxum clade. The position of the Kudoa clade also differed in the BI and MP analyses. The Kudoa clade was the sister taxon to the Parvicapsula + Enteromyxum + Myxidium clade in the BI analysis. The Ceratomyxa clade was the most basal clade within the marine clade. By contrast, MP analysis placed the Kudoa clade as a sister taxon to the Parvicapsula + Enteromyxum + Ceratomyxa clade. The Myxidium clade was the basal marine clade in the MP analysis. The unstable branching pattern of these clades was reflected by a polytomy in the analysis of the marine species only (Fig. 4). Two species of Sphaerospora branched within the Kudoa clade while Unicapsula sp. (AY302725) branched as its basal taxon. Besides the Myxidium spp., the marine Myxidium clade included representatives of the genera Ellipsomyxa, Zschokkella and Auerbachia. Zschokkella sp. 3 did not cluster with the marine Myxidium clade and branched separately (MP analyses) or clustered with Parvicapsula spp. (BI analysis). Similarly, C. shasta did not cluster with the Ceratomyxa clade. This clade included four Ceratomyxa species and Palliatus indecorus. The monophyletic clades Enteromyxum and Parvicapsula clades contained three and four species, respectively. The topology of the freshwater clade was almost identical in both BI and MP analyses (Figs. 2 and 3) and did not differ substantially from results of analyses of freshwater species only (Fig. 5). Chloromyxum leydigi was basal followed by the freshwater gall bladder (GB) clade. Within this clade, C. truttae and Chloromyxum cyprini were the basal species. The freshwater GB clade included Chloromyxum trijugum, Myxidium coryphaenoideum clustering with the Sphaeromyxa clade and the freshwater Myxidium clade. The latter clade included species of the genera Myxidium, Zschokkella and Sphaerospora sp. (AY735411). Three species of different genera (Myxidium lieberkuehni, Sphaerospora oncorhynchi and Chloromyxum legeri) represented a sister clade (named the Myxidium lieberkuehni clade) to the Myxobolus clade. The conspicuous group of five species infecting the urinary bladder (UB clade) clustered inside the Myxobolus clade.

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Fig. 2. The phylogenetic tree of the Myxosporea based on Bayesian inference (GTR + I + C) of the SSU rDNA data. Buddenbrockia plumatellae and Tetracapsuloides bryosalmonae are as outgroup species. The numbers at the nodes show the clade posterior probability, which signifies the proportion of the sampled trees containing that branch. Newly sequenced species are marked with asterisk. GenBank Accession Nos. are shown in Figs. 4 and 5. Thick branches indicate the three main lineages within myxosporeans.

I. Fiala / International Journal for Parasitology 36 (2006) 1521–1534 1527 Myxobolus siddalli Myxobolus algonquinensis Myxobolus martini Myxobolus smithi clade Myxobolus parviformis 53/60/66 Myxobolus impressus Myxobolus macrocapsularis Henneguya cutanea Myxobolus bramae -/53/- 70/81/83 Myxobolus pavlovskii Myxobolus dispar 72/67/55 Myxobolus basilamellaris Thelohanellus wuhanensis 82/72/64 92/89/85 Thelohanellus hovorkai 67/78/79 Myxobolus rotundatus Myxobolus rotundus 77/75/69 Sphaerospora renicola Sphaerospora molnari 71/77/75 Myxobolus obesus 80/80/82 Myxobolus intimus 87/90/90 Myxobolus hungaricus 92/88/84 100/100/100 Myxobolus pseudokoi 98/98/98 Myxobolus pendula 94/92/88 Myxobolus pellicides 88/88/90 Myxobolus longisporus 67/83/83 88/91/83 Myxobolus musculi 99/99/98 Myxobolus cyprini -/59/65 Myxobolus pseudodispar Myxobolus bartai Myxobolus bibullatus Myxobolus elegans Myxobolus bononiense Myxobolus xiaoi 83/75/70 Myxobolus cultus Myxobolus lentisuturalis 53/-/Myxobolus kubanicum Myxobolus portucalensis Henneguya nuesslini 100/100/100 Henneguya salminicola 100/100/100 Henneguya zschokkei Myxobolus constrained spp. Myxobolus cerebralis Myxobolus squamalis 92/95/96 Myxidium giardi 98/99/99 UB Chloromyxum sp. 89/92/88 Zschokkella sp. clade Hoferellus gilsoni 72/67/59 Myxobilatus gasterostei 100/97/97 Myxobolus ichkeulensis 51/58/64 Myxobolus spinacurvatura 92/93/93 Myxobolus bizerti 98/95/94 Myxobolus episquamalis 100/100/100 Myxobolus exiguus Myxobolus muelleri 96/94/89 Myxobolus procerus 100/100/100 Henneguya lesteri Henneguya ictaluri Henneguya exilis 100/100/100 Henneguya weishanensis Henneguya doori 85/86/87 Henneguya sp. Henneguya sp. * Myxobolus osburni Myxobolus acanthogobii 100/100/100 Sphaerospora oncorhynchi Myxidium lieberkuehni 79/93/93 Myxidium lieberkuehni clade Chloromyxum legeri 82/86/90 Sphaerospora sp. 52/-/Zschokkella parasiluri * freshwater Myxidium cuneiforme * 100/100/100 Myxidium truttae Zschokkella nova 1 * 83/81/72 clade Zschokkella nova 2 * 94/97/94 Zschokkella sp. 1 * 82/83/73 Zschokkella sp. 2 * 65/69/62 Myxidium sp. Myxidium chelonarum * Chloromyxum trijugum 64/63/64 Sphaeromyxa hellandi 1* 100/100/100 Sphaeromyxa hellandi 3 * 94/95/95 100/97/95 Sphaeromyxa hellandi 2* clade Sphaeromyxa longa * Sphaeromyxa zaharoni 97/95/85 Myxidium coryphaenoideum * 70/61/Chloromyxum cyprini Chloromyxum truttae Chloromyxum leydigi 100/100/100 Chloromyxum leydigi * -/-/50 Kudoa neurophila 96/99/97 Kudoa yasunagai 55/-/Kudoa thalassomi 71/77/78 Kudoa scomberomori clade Kudoa permulticapsula 67/71/71 95/98/98 Kudoa grammatorcyni Kudoa thyrsites Kudoa minithyrsites Kudoa lateolabracis Kudoa quadricornis 80/80/83 Kudoa clupeidae -/-/51 Kudoa funduli Kudoa rosenbuschi Kudoa sp. CMW 89/89/88 Kudo aminiauriculata Kudoa dianae Kudoa paniformis Kudoa ovivora Sphaerospora dicentrarchi 79/75/70 Sphaerospora sp. * 92/93/90 Kudoa iwatai Kudoa shiomitsui 99/97/95 97/99/99 Kudoa hypoepicardialis 98/99/98 Kudoa amamiensis -/65/81 Kudoa crumena Unicapsula sp. 77/82/84 Parvicapsula asymmetrica 100/100/99 Parvicapsula unicornis 98/100/100 Parvicapsula pseudobranchicola clade 98/97/94 Parvicapsula minibicornis 100/100/100 Ceratomyxa shasta 100/100/100 Enteromyxum fugu Enteromyxum Enteromyxum scophthalmi 100/100/100 clade Enteromyxum leei Ceratomyxa labracis Ceratomyxa sparusaurati Ceratomyxa sp. 2 * Palliatus indecorus * 74/68/67 clade Ceratomyxa sp. 1 * Myxidium gadi 1* 67/70/66 67/60/Myxidium gadi 2 * marine 100/100/100 Myxidium bergense * Myxidium incurvatum * 86/88/85 Ellipsomyxa gobii 64/55/81/72/54 Zschokkella mugilis clade Auerbachia pulchra * Zschokkella sp. 4 * Zschokkella sp. 3 * 97/100/100 Sphaerospora truttae 100/100/100 Sphaerospora elegans Leptotheca ranae Buddenbrockia plumatellae Tetracapsuloides bryosalmonae 80/78/74 62/66/58 98/99/96

-/-/51

-/-/53

Myxobolus

56/51/52

Sphaeromyxa

freshwater GB clade

Myxidium

Kudoa

90/94/94

freshwater clade

marine clade

Parvicapsula

100/100/100 -/52/53

98/97/96

Ceratomyxa

Myxidium

Fig. 3. Strict consensus tree of the 2690 maximum parsimonious trees (9245 steps) of Myxosporea resulted from maximum parsimony analysis of the SSU rDNA data. Buddenbrockia plumatellae and Tetracapsuloides bryosalmonae are used as outgroup species. Numbers at the nodes represent the bootstrap values (1000 replicates, Ts/Tv ratio 1:1, 1:2 and 1:3) gaining more than 50% support.

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0.80 0.76

1.00

1.00 1.00 1.00 1.00 1.00 0.85

1.00

1.00 1.00 0.92

1.00

Kudoa neurophila Kudoa yasunagai Kudoa thalassomi Kudoa grammatorcyni Kudoa scomberomori Kudoa permulticapsula Kudoa thyrsites Kudoa minithyrsites Kudoa lateolabracis Kudoa quadricornis Kudoa ovivora Kudoa clupeidae Kudoa funduli Kudoa rosenbuschi Kudoa sp. CMW Kudoa miniauriculata Kudoa dianae Kudoa paniformis Kudoa shiomitsui Kudoa hypoepicardialis Kudoa amamiensis Kudoa crumena Sphaerospora dicentrarchi Sphaerospora sp. Kudoa iwatai Unicapsula sp Enteromyxum fugu Enteromyxum scophthalmi Enteromyxum leei Myxidium gadi 1 Myxidium gadi 2 Myxidium bergense Myxidium incurvatum Ellipsomyxa gobii Zschokkella mugilis Auerbachia pulchra Zschokkella sp. 4 Ceratomyxa labracis Ceratomyxa sparusaurati Ceratomyxa sp. 2 Palliatus indecorus Ceratomyxa sp. 1 Parvicapsula pseudobranchicola Parvicapsula asymmetrica Parvicapsula unicornis Parvicapsula minibicornis Ceratomyxa shasta Zschokkella sp. 3 Buddenbrockia plumatellae Tetracapsuloides bryosalmonae

*

1.00 0.85 1.00

0.94 0.94 0.82 0.94 0.96

1.00 0.98

0.69

1.00 1.00 1.00

1.00

1.00 0.58 1.00 1.00

* *

*

* * *

* * *

*

B

M

B Mu

M M

Mu Mu AY078429 Mu AF031412 Mu

M M M

AY172511 AY302741 AY302738 AY302739 AY302737

AY152749 Mu AY382606 Mu AY078428 Mu AY152750 E AY197771 Mu AY312279 Mu AY623795 Mu AY302723 Mu AF034639 Mu AF414692 Mu AF034640 Mu AY302724 Pc AY302722 AY152748 AF378347 AY278564 DQ377695 AY514038 AY302725

M M M M M M M M M M M M M

H

M

Mu Mu Ct Gbw

M M

Mu

M M

AY520573

Mu I

AF411335 AF411334

I I

DQ377711 Gb DQ377707 Gb DQ377702 Gb DQ377708 Gb AY505126 Gb AF411336 Gb DQ377703 Gb DQ377700 Gb AF411472 Gb AF411471 Gb DQ377699 Gb DQ377712 Gb DQ377698 Gb AY308481 G AY584191 K AY584190 K AF201375 K AF001579 I DQ377705 Ub AY074915 U70623

M M

M M M

Perc Pleu

Australia Japan

Perc Perc

Australia

Perc Perc

Australia, Great Barrier Reef Japan

Perc Perc Clup Cyprd

Australia

Scor Tetr Gadi Tetr

USA, California Mexico, Pacific ocean Canada, British Columbia

Australia Perc Australia Perc Australia Salm Canada, British Columbia

Caribbean Sea USA, Virginia Canada, Nova Scotia Gadi South-west Atlantic Pleu USA, Gulf of Mexico

Perc Perc Perc Perc Perc Perc Perc Tetr Pleu Perc

USA, Gulf of Mexico USA, Gulf of Mexico Australia, Great Barrier Reef USA, North Carolina Spain Caribbean Sea Israel, Red Sea, Gulf of Eilat Australia Japan, Kumamoto Spain Greece

M M M M

Gadi North Sea, off Scotland Gadi North Sea, off Scotland Scor North Atlantic

M M M

Perc

M M M M M M M M M M F M

Perc

North Atlantic Denmark, Niva Bay

Perc Spain Gadi North Atlantic Ophi North Atlantic Perc Spain Perc Nota Osme Carc Salm

Spain North Atlantic North Atlantic North Atlantic Norway, Lyngen, Troms

Scor Norway Gadi Norway Salm Canada, British Columbia Salm North America, Pacific Northwest Tetr Caribbean Sea

Fig. 4. The phylogenetic tree of mostly marine myxosporeans based on Bayesian inference (BI). Buddenbrockia plumatellae and Tetracapsuloides bryosalmonae are used as outgroup species. The BI posterior probability is indicated at the nodes. Newly sequenced species are marked with an asterisk. The GenBank Accession No. follows the species name. If available, the GenBank Accession No. is followed by the information about the site of infection, host environment, taxonomic classification of the fish host and the geographic locality. Abbreviations: B, brain; Ct, connective tissue; E, eye; G, gills; Gb, gall bladder; Gbw, gall bladder wall; H, heart; I, intestine; K, kidney; Mu, muscle; Pc, pericardial cavity; Pe, peritoneum; Ub, urinary bladder; F, freshwater; M, marine; Clup, Clupeiformes; Cyprd, Cyprinodontiformes; Gadi, Gadiformes; Nota, Notacanthiformes; Ophi, Ophidiiformes; Osme, Osmeriformes; Perc, Perciformes; Pleu, Pleuronectiformes; Salm, Salmoniformes; Scor, Scorpaeniformes; Tetr, Tetraodontiformes.

4. Discussion The length of SSU rRNA gene sequences is not fixed in eukaryotic organisms and can be unusually long (Milyutina et al., 2001). In myxosporeans, S. truttae was found to

possess a very long SSU rDNA sequence (Holzer et al., 2003). Since the almost complete sequence of S. truttae consists of 2541 nt, a similar length was expected from the sequences of S. elegans and L. ranae (deduced from the length of their partial sequences). Their sequences

c Fig. 5. The phylogenetic tree of predominantly freshwater myxosporeans based on Bayesian inference (BI). Buddenbrockia plumatellae and Tetracapsuloides bryosalmonae are used as outgroup species. The BI posterior probability corresponds with numbers at the nodes (). Newly sequenced species are marked with asterisk. The GenBank Accession No. follows the species name. If available, the GenBank Accession No. is followed by the information about the site of infection, host environment, taxonomic classification of fish host and the geographic locality. Abbreviations: B, brain; C, cartilage; Ct, connective tissue; E, eye; G, gills; Gb, gall bladder; Hd, head; I, intestine; K, kidney; Ms, mesenteries; Mu, muscle; L, liver; Pe, peritoneum; S, skin; Sc, scales; Ub, urinary bladder; Vo, various organs; F, freshwater; M, marine; Angu, Anguilliformes; Cypr, Cypriniformes; Cyprd, Cyprinodontiformes; Esoc, Esociformes; Gadi, Gadiformes; Gast, Gasterosteiformes; Perc, Perciformes; Percop, Percopsiformes; Salm, Salmoniformes; Scor, Scorpaeniformes; Silu, Siluriformes; Squa, Squaliformes; Torp, Torpediniformes.

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contained numerous nucleotide insertions that are not homologous with the sequences of other myxosporeans. The length of the SSU rDNA sequence for the two main clades of myxosporeans was noted by Fiala and Dykova´ (2004) and their observations were confirmed in this study. A majority of the marine species revealed shorter sequences of SSU rDNA than freshwater ones. The intermediate length of the SSU rDNA sequence of marine C. leydigi (1866 nucleotides) corresponds with the phylogenetic position of C. leydigi as a basal species of the freshwater clade. The length of sequences of the other marine species (Sphaeromyxa spp. and M. coryphaenoideum), which cluster in the freshwater clade, is comparable with the length of freshwater species. It seems that the length of myxosporean sequences corresponds with the phylogenetic position of myxosporeans and is specific for representatives of all three clades in the myxosporean SSU rDNA tree. The question of the extent of difference in sequence data required to assign two organisms to different species taxa remains unclear (Kunz, 2002) given known intragenomic rDNA diversity (Buckler et al., 1997). Schlegel et al. (1996) found a relatively high difference (2.6%) among various samples of M. lieberkuehni. Similarly, two morphologically identical samples of myxosporeans identified as Zschokkella nova from distant geographic localities revealed considerable differences in the sequence of SSU rDNA. The same difference in SSU rDNA sequences appeared when sequences of C. leydigi (AY604199) from T. marmorata and newly sequenced C. leydigi from C. coelolepis were compared. By contrast, a 1.4–1.6% difference occurs in two morphologically different species, Kudoa thyrsites and Kudoa minithyrsites (Whipps et al., 2003), and a remarkably small (0.1%) difference occurs between Myxobolus pellicides and Myxobolus pendula (Kent et al., 2001). The spores of the three samples of S. hellandi were morphologically identical and the SSU rDNA sequences of these samples revealed high similarity. Comparison between the three PCR clones of SSU rDNA sequence of S. hellandi suggested that the difference among three samples of S. hellandi corresponded with intragenomic variability and that these three samples could be identified as a single species. However, it is difficult to decide, whether the sequence differences within Z. nova samples reflect intraspecific variability or can be attributed to the existence of cryptic species. Intragenomic SSU rDNA variability found in cloned PCR products of Z. nova was much lower than the sequence variability between the samples from different geographic location. These allopatric specimens of Z. nova could be the two closely related species as indicated in Lom and Dykova´ (1992). SSU rDNA is widely used for revealing phylogenetic relationships among taxa (Avise, 2004). Single gene phylogenies may not correspond with the true phylogeny, particularly, in the case of mitochondrial genes with frequent horizontal transfer of the genetic information. This should not be the case of the presented study. The main source of possible phylogenetic distortion is likely to be connected to

weak support of some branches (particularly in the marine clade) and to artefacts due to the aberrant behavior of some sequences (e.g. possible long-branch attraction of S. truttae as discussed below). Nevertheless, SSU rDNA is the only molecular marker available for the broad range of Myxosporea at present. Consequently, SSU rDNA sequences are the only source of information for reconstruction of evolutionary history of myxosporeans. Observed discrepancies between SSU rRNA gene phylogeny and taxonomy based on morphology may indicate that the molecular phylogeny is not congruent with the true phylogeny or may indicate that the characters of spore morphology are homoplasious. Although partial LSU rDNA sequence data of a limited number of myxosporeans support the results of SSU rDNA analysis (Whipps et al., 2004), molecular data of other genes are needed to confirm the myxosporean rDNA phylogeny. Although, an increasing number of marine myxosporeans has been incorporated into the clade of mostly freshwater species (Bahri et al., 2003; Diamant et al., 2004; Fiala and Dykova´, 2004; Yokoyama et al., 2004; present study), the phylogenetic separation of myxosporeans into two major clades with freshwater and marine species (Palenzuela, O., Bartholomew, J.L., 1999. Phylogeny of the genus Ceratomyxa and other marine myxozoans based on small subunit ribosomal RNA gene sequences. 5th International ˇ eske´ BudeˇjoSymposium on Fish Parasites, 9–13 August, C vice, Czech Republic, Abstract of Papers, p. 106; Kent et al., 2001) is still valid. One of the ‘‘exceptions‘‘ to the marine/freshwater separation is the remarkable group of three marine Sphaeromyxa species clustering inside the freshwater GB clade. Sphaeromyxa species are closely related to Myxidum/ Zschokkella species, M. coryphaenoideum from deep-sea fish being the closest species. It seems that the common ancestor of the marine Sphaeromyxa spp. was the freshwater myxosporean with Myxidium-shaped spores. The position of myxosporeans from reef-associated or coastal fish among the clade of freshwater species seems logical since hosts of these species often enter estuaries and rivers. Bahri et al. (2003) described six marine Myxobolus spp. from mullets (Mugil cephalus and Liza ramada), commonly found in estuaries or rivers. Hence, it is questionable whether these Myxobolus spp. should be considered marine myxosporeans. The situation that fish hosts that live in both marine and freshwater environments occurs also in Sphaerospora sp. from Mugil curema and Sphaerospora dicentrarchi from Dicentrarchus labrax. These two Sphaerospora species clustered together in a close relation to marine Kudoa spp. The marine clade is composed of myxosporeans infecting marine fish but includes two exceptional species, C. shasta and P. minibicornis. Ceratomyxa shasta is a unique parasite infecting salmonids in a freshwater environment, that clusters with marine myxosporeans. To date, C. shasta represents an independent lineage with no firm relationship to any group of marine myxosporeans including the four

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Ceratomyxa species sequenced to date. Both its phylogenetic position and interesting life cycle, which includes a freshwater polychaete as a host for the actinosporean phase (Bartholomew et al., 1997), indicate the marine origin of C. shasta. Polychaetes are presumably involved in marine myxosporean life cycles (Køie et al., 2004). The assumed origin of C. shasta is also supported by the hosts: salmonids are anadromous fish occurring in a marine environment and polychaetes are typically marine. Species of the genera Myxidium, Zschokkella and Sphaerospora occur in basal clades within both the major marine and freshwater clades. Phylogenetic analysis of SSU rDNA suggests that these myxosporeans evolved independently into species of similar spore type in both environments. By contrast, Myxobolus and Henneguya spp. from marine hosts occur in the freshwater clade and share a common ancestor. Phylogenetic analyses of SSU rRNA gene suggest that spore morphology is of minor importance in phylogenetic relationships of myxosporeans (Hervio et al., 1997; Andree et al., 1999; Eszterbauer, 2004; Holzer et al., 2004). This view is also supported by the present study, which encompasses numerous paraphyletic and polyphyletic taxa. It seems that change in spore shape may be frequent in myxosporean evolution, with the occurrence of similar spore morphology in different clades due to convergence. Nevertheless, species with the same spore morphology predominate in several clades. This applies particularly to the marine clade (Parvicapsula + Enteromyxum). The phylogenetic position of Sphaerospora species and the lack of correlation with spore morphology was reported by Kent et al. (2001) and Eszterbauer and Szekely (2004). The present phylogenetic analysis placed Sphaerospora spp. in five distant branches. Phylogenetic analysis of the recently-submitted myxosporean sequences of S. truttae, S. elegans and L. ranae (Holzer et al., 2004; Jirku˚ M., unpublished data) revealed that these species occurred near the malacosporean/myxosporean divergence. This position might be influenced by Long-branch attraction (LBA) (Siddall and Whiting, 1999). Long branches may be attracted to each other (often long ingroup branch to the outgroup sequence), particularly in the maximum parsimony analysis. The position of S. truttae, S. elegans and L. ranae was confirmed by BI and minimum evolution methods with LogDet distance not affected by LBA. Holzer et al. (2004) discussed the possible relationships of S. truttae and S. elegans within the clade of mostly marine species, but their observations were not confirmed in this study. The unexpected phylogenetic position of Sphaerospora dicentrarchi within Kudoa species (Diamant et al., 2005) was not supported with sufficient bootstrap values. The nodal support remained low even when the newly sequenced Sphaerospora sp. was added. These Sphaerospora species may be sister taxa to Kudoa spp. or they may be two-valved Kudoa species. Nevertheless, basal position of multivalvulid Unicapsula sp. supported the inclu-

sion of bivalvulid Sphaerospora spp. inside the multivalvulid cluster. Deducing relationships from SSU rDNA sequences in this study, the multivalvulid myxospores either arose several times in the evolution of myxosporeans, or Sphaerospora sp. and S. dicentrarchi evolved from an ancestor of species of the genera Kudoa and Unicapsula and lost their multivalvulid character. Relationships within Myxobolus have been analysed many times (Andree et al., 1999; Salim and Desser, 2000; Dykova´ et al., 2002; Molnar et al., 2002; Bahri et al., 2003; Eszterbauer, 2004). Representatives of the genus Myxobolus were intermixed with the morphologically distinct Sphaerospora spp., Thelohanellus spp. and several Henneguya spp. This paraphyly of the genus Myxobolus has been observed since the first molecular study was published (Smothers et al., 1994). The species of the polyphyletic genus Henneguya probably arose from Myxobolus ancestors several times in myxosporean evolution. Kent et al. (2001) stressed that caudal appendages do not represent a valid character for distinction of the genera Myxobolus and Henneguya. Based on SSU rDNA analysis, Auerbachia pulchra evolved from the Myxidium/Zschokkella type and apparently lost one polar capsule in its evolutionary history. Diamant et al. (2004) described a new species, Sphaeromyxa zaharoni, and based on results of their phylogenetic analysis, they questioned whether the presence of ribbon-like polar filaments was sufficient to warrant a separate suborder Sphaeromyxina. The present analysis supported the phylogenetic position of S. zaharoni and its close relationship to two other Sphaeromyxa species. They formed a monophyletic group with a close relationship to the freshwater Myxidium clade. Consequently, the analysis suggested that the assignment of Sphaeromyxa species to a separate suborder is not needed. Lom and Dykova´ (1992) have stated that the distinction between species of the genera Myxidium and Zschokkella is sometimes difficult to determine. Conspicuous similarity was found between the sequences of Z. nova and M. truttae. They have very similar spore morphology and the same site of infection, differing only in fish host. The close relationship of Z. nova and M. truttae is an example of how difficult it is to distinguish between the above-mentioned genera, often leading to arbitrary classification within one or the other genus. The importance of the site of infection as a factor in myxosporean evolution, as revealed in previous studies (Andree et al., 1999; Eszterbauer, 2004; Holzer et al., 2004), was supported by the results of SSU rDNA analysis obtained in this study. The gall bladder infecting clades are well defined in both freshwater and marine clades. The freshwater GB clade contained myxosporeans from different genera. However, the most numerous species of the genera Myxidium and Zschokkella are similar in spore shape and hence the close relationships were predictable. Nevertheless, Myxidium and Zschokkella species from this freshwater GB clade are phylogenetically distant, as

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inferred from SSU rDNA, from the other freshwater Myxidium/Zschokkella spp. – Zschokkella sp. (AJ581918), M. lieberkuehni and M. giardi (all from the excretory system) and were more distant from Myxidium/Zschokkella species clustering in the marine clade. Species of the genus Myxidium from marine fish (M. gadi, Myxidium bergense and Myxidium incurvatum) are morphologically similar to Enteromyxum spp., but they clustered with other gall-bladder infecting species of the genera Zschokkella, Ellipsomyxa and Auerbachia. Enteric myxosporeans of the genus Enteromyxum clustered together (Palenzuela et al., 2002; Yanagida et al., 2004) and they are monophyletic with no firm relationships to the other myxosporeans. BI analysis of SSU rDNA revealed the possible relationship of C. shasta (also infecting the intestine) with the Enteromyxum clade (Fig. 2), but a relatively low value of posterior probability did not support this clustering. The other marine gall bladder-infecting clade (Ceratomyxa clade) supported the clustering of myxosporeans by site of infection. Furthermore, Zschokkella sp. 3 from the urinary bladder clustered (BI analysis) with the excretory system-infecting Parvicapsula clade (the exception is Parvicapsula pseudobranchicola from the gills). Following the redescription of the multivalvulid species of the genera Pentacapsula, Haxacapsula and Septemcapsula (Whipps et al., 2004), the genus Kudoa appeared monophyletic. However, the presence of S. dicentrarchi within the Kudoa clade renders Kudoa paraphyletic (Diamant et al., 2005). Kudoa species are mostly histozoic while Sphaerospora species are mostly coelozoic. Sphaerospora dicentrarchi, together with newly sequenced Sphaerospora sp., represent a minority of the histozoic Sphaerospora species. It supports the hypothesis that these two Sphaerospora species evolved from marine histozoic myxosporeans loosing the multivalvulid type of spore. Species of the genus Myxobolus are mostly histozoic, infecting various tissues. Eszterbauer (2004) described two groups of Myxobolus species infecting gills. The present study supports only the group containing M. longisporus, M. pellicides, M. pendula, Myxobolus pseudokoi, Myxobolus intimus, Myxobolus hungaricus and Myxobolus obesus. The second gill-infecting group, sensu Eszterbauer (2004), is not uniform with respect to the site of infection due to the presence of species not infecting the gills (e.g., Myxobolus smithi, Myxobolus martini or Myxobolus rotundatus). To date about 50 species of Myxobolus have been sequenced, which represents less than one-tenth of the currently described species of the genus. Hence, the interpretation of relationships of Myxobolus spp. may change with the increasing number of available SSU rDNA sequences. Shulman (1966) suggested that histozoic species evolved from coelozoic myxosporeans. The overall results of the phylogenetic analysis based on SSU rDNA support this hypothesis. However, the group of species infecting the excretory system (Holzer et al., 2004) diverged within Myxobolus in this study, i.e., in the UB clade. Nevertheless, the most derived myxosporeans (Kudoa spp. and Myxobo-

lus/Henneguya spp.), are generally histozoic species. The assignment of myxosporeans as being either histozoic or coelozoic is often difficult. For example, Sphaerospora renicola infects kidney tissue, but it is considered as a coelozoic species in renal tubules. Moreover, S. renicola clustered with the histozoic Sphaerospora molnari in the Myxobolus/Henneguya clade. The role of geography in myxosporean evolution was advanced by Hervio et al. (1997) from the analysis of SSU rDNA sequences of five Kudoa spp. Further analyses with additional Kudoa sequences did not support the relationships identified by geographic location. However, the analysis based on more SSU rDNA sequence data of Kudoa species suggested a close relation of the species from Australia (Fig. 4). The group of about 30 myxosporean species infecting cypriniform fish emerged within Myxobolus. The group was statistically well-supported, and contained fish-infecting species from various geographic locations. Except for Myxobolus species, this group included Henneguya, Thelohanellus and Sphaerospora spp. These myxosporeans infect mainly the gills and muscles but are found in other tissues as well. Host preference was the only common feature of myxosporeans in this group. Nevertheless, the reflection of host and geographic origin seemed to be less well expressed in the phylogenetic relationships of myxosporeans than the environment of host species or site of infection. The use of molecular data in taxonomy of myxosporeans together with morphological characteristics has been instrumental in establishing a new genus Enteromyxum (Palenzuela et al., 2002), enabling also the synonymy of the families Pentacapsulidae, Hexacapsulidae and Septemcapsulidae with Kudoidae (Whipps et al., 2004). Revision of the other myxosporean taxa, particularly genera, using the results presented here, will be necessary, but should be approached cautiously. Regardless of the increasing number of available SSU rDNA sequences, the proportion of sequenced species is low and many species are still unknown to science. The SSU rDNA sequences of more than half of the described myxosporean genera are lacking. Future taxonomic modification of genera must also be based on their type species. For instance, P. indecorus may be assigned to the genus Ceratomyxa, but without knowledge of the phylogenetic position of the type species C. arcuata, which may not be related to other Ceratomyxa spp. and P. indecorus, the possible transfer of P. indecorus to the genus Ceratomyxa would be premature. To date, only seven type species have been sequenced: Myxobolus muelleri, M. lieberkuehni, S. elegans, C. leydigi, Parvicapsula asymmetrica, Kudoa clupeidae and Enteromyxum scophthalmi. Present study supported findings that SSU rDNA phylogeny of Myxosporea does not correspond with the current taxonomy. Phylogenetic analysis based on SSU rRNA gene revealed many paraphyletic and polyphyletic genera. In the future, other type species of myxosporean genera should be sequenced. It would enable to propose

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new definition (diagnoses, delimitation) of individual genera based on the phylogenetic positions of their type species and subsequent taxonomic changes. Future research should be also focused on the analyses of other genes to confirm whether SSU rDNA phylogeny corresponds to organismal phylogeny. The combined analyses of appropriated genes could also increase the resolution of phylogenetic trees (Olson et al., 2003; Hypsˇa et al., 2005; Kim et al., 2006) and could help to clarify the phylogenetic relationships of myxosporeans. Acknowledgements The author thanks Iva Dykova´ and Va´clav Hypsˇa for their helpful suggestions and advice on this article. The author also thanks Roman Kuchta, David Gonza´lez-Solı´s and Andy P. Shinn for their kind assistance with the collection of parasites. David Modry´ is acknowledged for providing Myxidium chelonarum material. This article was supported by research project of the Institute of Parasitology, Academy of Sciences of the Czech Republic (Z60220518), Research Centre ‘‘Ichthyoparasitology’’ (LC522), Ministry of Education, Youth and Sports of the Czech Republic (6007665801) and Grant Agency of University of South Bohemia (58/2002/P-BF). References Andree, K.B., Szekely, C., Molnar, K., Gresoviac, S.J., Hedrick, R.P., 1999. Relationships among members of the genus Myxobolus (Myxozoa: Bilvalvulidae) based on small subunit ribosomal DNA sequences. J. Parasitol. 85, 68–74. Avise, J.C., 2004. Molecular Markers, Natural History, and Evolution. Sinauer Associates Inc. Publishers, Sunderland, Massachusetts. Bahri, S., Andree, K.B., Hedrick, R.P., 2003. Morphological and phylogenetic studies of marine Myxobolus. J. Eukaryot. Microbiol. 50, 463–470. Barta, J.R., Martin, D.S., Liberator, P.A., Dashkevicz, M., Anderson, J.W., Feighner, S.D., Elbrecht, A., Perkins-Barrow, A., Jenkins, M.C., Danforth, H.D., Ruff, M.D., Profous-Juchelka, H., 1997. Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences. J. Parasitol. 83, 262–271. Bartholomew, J.L., Whipple, M.J., Stevens, D.G., Fryer, J.L., 1997. The life cycle of Ceratomyxa shasta, a myxosporean parasite of salmonids, requires a freshwater polychaete as an alternate host. J. Parasitol. 83, 859–868. Blaylock, R.B., Bullard, S.A., Whipps, C.M., 2004. Kudoa hypoepicardialis n. sp. (Myxozoa: Kudoidae) and associated lesions from the heart of seven perciform fishes in the northern Gulf of Mexico. J. Parasitol. 90, 584–593. Buckler, E.S., Ippolito, A., Holtsford, T.P., 1997. The evolution of ribosomal DNA: divergent paralogues and phylogenetic implications. Genetics 145, 821–832. Canning, E.U., Curry, A., Feist, S.W., Longshaw, M., Okamura, B., 2000. A new class and order of myxozoans to accommodate parasites of bryozoans with ultrastructural observations on Tetracapsula bryosalmonae (PKX organism). J. Eukaryot. Microbiol. 47, 456–468. Diamant, A., Ucko, M., Paperna, I., Colorni, A., Lipshitz, A., 2005. Kudoa iwatai (Myxosporea: Multivalvulida) in wild and cultured fish in the Red Sea: redescription and molecular phylogeny. J. Parasitol. 91, 1175–1189.

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