A common origin of complex life cycles in parasitic flatworms: evidence from the complete mitochondrial genome of Microcotyle sebastis (Monogenea: Platyhelminthes)
© Park et al; licensee BioMed Central Ltd. 2007
Received: 28 July 2006
Accepted: 02 February 2007
Published: 02 February 2007
The parasitic Platyhelminthes (Neodermata) contains three parasitic groups of flatworms, each having a unique morphology, and life style: Monogenea (primarily ectoparasitic), Trematoda (endoparasitic flukes), and Cestoda (endoparasitic tapeworms). The evolutionary origin of complex life cyles (multiple obligate hosts, as found in Trematoda and Cestoda) and of endo-/ecto-parasitism in these groups is still under debate and these questions can be resolved, only if the phylogenetic position of the Monogenea within the Neodermata clade is correctly estimated.
To test the interrelationships of the major parasitic flatworm groups, we estimated the phylogeny of the Neodermata using complete available mitochondrial genome sequences and a newly characterized sequence of a polyopisthocotylean monogenean Microcotyle sebastis. Comparisons of inferred amino acid sequences and gene arrangement patterns with other published flatworm mtDNAs indicate Monogenea are sister group to a clade of Trematoda+Cestoda.
Results confirm that vertebrates were the first host for stem group neodermatans and that the addition of a second, invertebrate, host was a single event occurring in the Trematoda+Cestoda lineage. In other words, the move from direct life cycles with one host to complex life cycles with multiple hosts was a single evolutionary event. In association with the evolution of life cycle patterns, our result supports the hypothesis that the most recent common ancestor of the Neodermata giving rise to the Monogenea adopted vertebrate ectoparasitism as its initial life cycle pattern and that the intermediate hosts of the Trematoda (molluscs) and Cestoda (crustaceans) were subsequently added into the endoparasitic life cycles of the Trematoda+Cestoda clade after the common ancestor of these branched off from the monogenean lineage. Complex life cycles, involving one or more intermediate hosts, arose through the addition of intermediate hosts and not the addition of a vertebrate definitive host. Additional evidence is required from monopisthocotylean monogeneans in order to confirm the monophyly of the group.
The evolutionary origin of parasitism throughout the tree of life remains a central issue in evolutionary biology and has attracted intense theoretical and empirical laboratory-based attention. In this respect the Platyhelminthes ('flatworms') have received great attention from evolutionary biologists as a model system for investigating the adaptive radiation associated with the evolution of parasitism (e.g. see ). The phylum is represented by an assemblage of superficially simple metazoan animal groups and has long been considered to provide a key to understanding the evolutionary origin and diversification of bilaterally symmetrical metazoan groups . It includes about 100,000 extant species of both free-living and parasitic forms . The conventional view of the phylum 'Platyhelminthes' is that it contains three major clades Acoelomorpha (Acoela+Nemertodermatida), Catenulida, and Rhabditophora , but recent phylogenetic studies based on morphological  and molecular evidence [6–10] have suggested non-monophyly (mostly polyphyly) of the Platyhelminthes. This is inconsistent with long-held prevailing concept of "the phylum Platyhelminthes" as defined in most zoological textbooks. Subsequent studies have separated the Acoelomorpha from the remaining catenulid and rhabditophoran Platyhelminthes, with the acoelomorphs occupying a pivotal basal position among the Bilateria [11, 12] and the Platyhelminthes (sensu stricto) as relatively derived members of the Lophotrochozoa . Nevertheless, it is generally accepted that the Platyhelminthes contains four major groups, each having a unique anatomy, body size, and life style: the 'Turbellaria' (a paraphyletic assemblage of at least seven distinct lineages of mostly free-living forms), Monogenea (primarily ectoparasitic), Trematoda (endoparasitic flukes), and Cestoda (endoparasitic tapeworms). Among these, the latter three groups (called 'Neodermata'; ) are represented by diverse obligate parasitic flatworms of invertebrates and vertebrates that cause diseases in a variety of host animal groups, including domestic animals and humans.
The Monogenea are composed of mostly ectoparasitic species which live on external organs (e.g., gill, skin, etc.) of a broad range of aquatic vertebrate host, especially fishes, with some exceptional species occurring in internal organs of the host . As illustrated in Fig. 1, the phylogenetic position of the Monogenea, and its monophyly, remain outstanding issues, because the evolutionary origin of ecto- and endo-parasitism in major parasitic platyhelminth groups can be elucidated only if its phylogenetic position within the Neodermata clade is fully resolved . If Monogenea are resolved as paraphyletic, occupying the earliest branching lineages, then ectoparasitism as the earliest life habit of neodermatans may be inferred with confidence.
With a few exceptions, animal mitochondrial genomes are circular in form, ranging from 13~16 kb in size. Each genome contains 37 genes: 13 protein-coding genes, two ribosomal RNA genes and 22 tRNA genes . Comparative analysis of the mitochondrial genome information (e.g., gene arrangement, nucleotide and amino acid sequences) has become a popular molecular tool for resolving the deeper node of phylogenetic interrelationship in a variety of metazoan groups [24–27]. To date, the complete mitochondrial genome sequence has been determined for 13 flatworm species (six from cestodes and seven from digenean trematodes), but the taxon sampling is highly biased toward endoparasitic flatworms i.e. cestodes and trematodes that are of medical or economic importance (for details see ). No complete mitochondrial genome information from monogenean species has been available as yet and this lack of information has hindered better understanding of the impending phylogenetic issue as well as mitochondrial genome evolution among major lineages of the parasitic Platyhelminthes. For these reasons, comparisons of a monogenean mitochondrial genome with other flatworms are expected to be very useful for resolution of phylogenetic relationships in conjunction with gene rearrangement among the mitochondrial genomes of three neodermatan groups (Cestoda, Trematoda, and Monogenea). In the present study, we revisited the phylogenetic issue of the Monogenea within the parasitic Platyhelminthes based on the comparisons of the complete mitochondrial genome sequences of a polyopisthocotylid monogenean,Microcotyle sebastis Goto, 1894 with published data from other flatworm species.
Results and Discussion
General Features of M. sebastis mtDNA Genome
Nucleotide composition and AT- and GC-skewnesses of M. sebastis mtDNA sequences for potein-coding, rRNA, tRNA genes and non-coding regions.
Ribosomal RNA gene sequence
Transfer RNA gene sequence
Highly repetitive region (HRR)
Unassigned region (UAR)
The majority of protein-coding genes (ten of 12 genes; cox2, cox3, cob, atp6, nad1, nad2, nad3, nad4L, nad5 and nad6) appear to use ATG as the start codon, while the other genes are predicted to start with ATT (cox1) and ATA (nad4), respectively. Seven of the 12 genes terminate with TAA (cox3, nad4, atp6, nad2, nad3, cox1 and nad6) and the other three use TAG (cob, nad4L and cox2) as the termination codon. Although incomplete termination is common in metazoan mtDNAs [23, 24], it is relatively rare in the flatworms studied thus far. Only two genes (nad5 and nad1) are inferred to end with incomplete codon TA and T, respectively, each of these is immediately adjacent to the downstream tRNA genes trnE and trnN.
Twenty-two nucleotide sequence segments (ranging in size from 59 nt [trnS1] to 69 nt [trnL1]) were predicted to fold into a cloverleaf secondary structure (see Additional File 2). The putative secondary structures common in 22 tRNA genes include an amino-acyl stem of 7 nucleotide pairs (ntp), a DHU-stem of 3-4 ntp with a 4-9 nt loop, an anticodon stem of 5 ntp with a loop of 7 nt, and a TΨC stem of 3-6 ntp with a loop of 3-6 nt. Some exceptions to these common features are trnS1 (AGN) and trnS2 (UCN) in which each of DHU arms is missing and replaced with an unpaired loop (10-14 nt) as found in all other flatworm species . Anticodon sequences of 22 tRNAs were identical to each of their corresponding tRNA genes found in other flatworm species with an exception that the trnR has a TCG anticodon sequence, rather than ACG as those found in other platyhelminth groups.
A total of 21 intergenic regions, varying from a single nucleotide to 472 nt long in size, were found in the M. sebastis mtDNA genome. Of these, two intergenic sequences, i.e., the highly repetitive region (HRR; 472 nt) and unassigned region (UAR; 348 nt) adjacent to each other are most prominent. The HRR located between trnK and UAR contains seven identical repeat units of a 53-nt sequences plus two additional repeat units, each abutting directly onto upstream and downstream of seven consecutive repeat units, respectively, with some sequence modifications: two substitutions (T→G and A→T substitutions at the second and fifth positions of 53-nt repeat unit; upstream unit) or with a truncated sequence of 5-nt from 3' end of the repeat unit (downstream unit). A 53-nt repeat unit is predicted to form a stem-loop secondary structure (not shown) with a 21 base paired-long stem and a loop of 9 nucleotides. Of 21 base pairs in the stem region, there are 12 A:T, five G:T, one G:C, three mismatch pairings (1 G:G and 2 A:G) and two unpaired A's, respectively. The highly structured A:T and G:T base pairings with avoiding C (only a single C is detected in each of the repeated unit) account for an extremely high level of GC skewness (0.85) in this repeat region. Although there are some mismatched base pairings and two unpaired A's in the stem region, the predicted putative secondary structure is considered to be analogous to those reported in cestode species (H. diminuta ; T. asiatica ). This stem-loop structure, although its function is still unclear, has often been assumed to be associated with replication origin. The second largest intergenic sequence, an unassigned region (UAR), is located between the HHR and nad6. This region contains a peculiar ORF (open reading frame)-like sequence segment of 174 nt comprising 58 codons including the starting codon (GTG for Val) and termination codon TAA. Although atp8 gene has never been found in flatworm mitochondrial genomes, the size of the unattributed ORF is more or less similar to the atp8 genes reported in other metazoans. In order to confirm its identity, we conducted the hydropathic profile comparison with other published atp8 genes (Geodia neptuni [Porifera]; Paratomella rubra [Acoelomorpha]; Trichinella spiralis [Nematoda]; Lumbricus terrestris [Annelida]; Limulus polyphemus [Arthropoda]; and Homo sapiens [Vertebrata]) using MacVector program (Accelrys Inc.). The predicted hydropathy profiles of the ORF candidate displayed very different patterns from the other sequences compared. Direct sequence comparisons of the ORF candidate using both nucleotide and amino acid sequence were also performed, but nearly no similarity was detected. Moreover, the AT content of the ORF candidate (78.3%) was much higher than the average (69.6%) of 12 protein-coding genes. Thus, taken together the evidence does not provide unambiguous support that this sequence is atp8 and therefore we designated it as an unassigned region (UAR).
Molecular Phylogeny of the Neodermata
The statistics of tree topology test using Shimodaira-Hasegawa  and Templeton  tests for comparisons of alternative hypothesis.
Difference in -lnL
Unconstrained best tree
Monophyly of Cestoda+Monogenea
Monophyly of Trematoda
Unconstrained MP tree
Monophyly of Cestoda+Monogenea
Monophyly of Trematoda
Evolution of Parasitism in the Neodermata
Evolutionary diversification and adaptive radiation of the parasites are strongly correlated with their life cycle patterns. Accordingly, the evolutionary history of major parasitic platyhelminth groups can be traced by examining the evolutionary changes of parasite-host associations . Tracking back to their historical transformations of different life history forms during the evolutionary process can convincingly be clarified only if it is interpreted within a robust phylogenetic framework. The interrelationships of the major neodermatan lineages is required to determine how parasitic platyhelminths evolved, and which life cycle pattern (ectoparasitism vs. endoparasitism) arose first within the Neodermata clade. If monogenean monophyly is true, as supported by the majority of previous works, it is expected that a single shifting event from ecto- to endo-parasitism or vice versa is equally possible to explain the evolutionary scenario of the parasitic patterns found in the neodermatan groups. An endoparasitic life style appears to be plesiomorphic when inferred from morphological [1, 34, 35, 38] and molecular [7, 43] phylogenies where Trematoda are sister group to Cestoda+Monogenea. In contrast, in gene trees where Cestoda and Trematoda are sister taxa [19, 20] (Fig. 1B), it can be postulated that ectoparasitism is the plesiomorphic condition. Indeed, it has often been considered among parasitologists that a parasitic group with a simple (direct) life cycle and higher specificity to its host groups is more primitive than the group with more complicated life cycles [15, 44]. Therefore, the simple, direct life cycle pattern displayed by monogenean groups has been assumed to be primitive and also correlated with high level of host specificity : Trematodes and cestodes display rather complicated life cycle, such as utilizing many invertebrates i.e., molluscs (trematodes) or arthropods (cestodes) as intermediate hosts prior to entering into the final life stage in various vertebrate hosts ('multiple host system'). In contrast, the majority of Monogenea are ectoparasitic on a broad range of aquatic vertebrates and have only one host with high host specificity during their life cycle, not involving intermediate hosts ('single-host system'). The position of Monogenea in the mitochondrial gene tree (Fig. 2) is compatible with the idea that a parasitic group maintaining simpler life cycle (being ectoparasitic) is more primitive than the group with more complicated life cycles (being endoparasitic), if we accept that a single host life cycle precedes a two or more host life cycle. Considering it in conjunction with the interpretation of life cycle patterns within the Neodermata, our mitochondrial gene tree corroborates the idea that ectoparasitism arose first in the neodermatan phylogeny (i.e. primitive condition), probably in an early lineage of monogenean groups, and that endoparasitism was secondarily acquired (i.e. derived condition) in cestodes and trematodes (comprising all extant obligate endoparasitic forms) after their common ancestry diverged from the Monogenea. This result contradicts the previous hypotheses that the common ancestor of the Neodermata acquired endoparasitism as the first mode of parasitism.
Evolution of Host-Parasite Association in the Neodermata
The evolutionary origin of parasitism within the Neodermata was inferred for the first time from a phylogeny of the Neodermata, estimated with complete mitochondrial genomes from all parasite classes. Comparisons of inferred amino acid sequences and gene arrangement patterns of a polyopisthocotylid monogenean Microcotyle sebastis with other published flatworm mtDNAs indicate that Monogenea are sister group to the Trematoda and Cestoda within the Neodermata clade. From this finding, we suggest that ectoparasitism likely arose first in the neodermatan phylogeny (i.e. primitive condition), probably in an early lineage of monogenean groups, and that endoparasitism was acquired secondarily (i.e. derived condition) in cestodes and trematodes after their common ancestry diverged from the Monogenea. In association with the evolution of life cycle patterns, our result lends strong evidence that the most recent common ancestor of the Neodermata giving rise to the Monogenea adopted vertebrate ectoparasitism as its initial life cycle pattern and that the intermediate hosts of the Trematoda (molluscs) and Cestoda (crustaceans) were subsequently added into the endoparasitic life cycles of the Trematoda+Cestoda clade after the common ancestor of these branched off from the monogenean lineage. Complex life cycles, involving one or more intermediate hosts, arose through the addition of intermediate hosts and not the addition of a vertebrate definitive host.
Sampling and Molecular Techniques
Live specimens of M. sebastis were isolated from the gill of host fish Sebastes schlegeli from a fish farm at the Namhae County of Gyeongsangnam-do Province of South Korea (N 34°42'23", E 128°03'97"). The total genomic DNA was extracted from a single individual using a QIAamp tissue kit (Qiagen Co.). Two partial fragments of cob (~450 nt) and rrnL (~430 nt) were initially PCR-amplified and cycle-sequenced using two primer sets: The cob primers (Cytb-424F [5'-GGW TAY GTW YTW CCW TGR GGW CAR AT-3'] and Cytb-876R [5'-GCR TAW GCR AAW ARR AAR TAY CAY TCW GG-3']) were originally designed by von Nickisch-Rosenegk, Brown, and Boore  and the rrnL primers (PL16SF [5'-WYYGTGCDAAGGTAGCATAAT-3'] and PL16SR [5'-AWAGATAAGAACCRACCTGGCT-3']) were directly designed on the basis of conserved regions of mitochondrial 16S rDNA sequences of diverse platyhelminth species. The sequences obtained in these two regions were then used to design species-specific primer sets for long PCR reactions. Two pieces of overlapping long PCR products (~7.8 and ~7.4 kb each in size) covering the entire mitochondrial genome were amplified using the Expand Long Template PCR System (Roche Co.) with the following conditions: 1 cycle of initial denaturation (45s at 94°C), 35 cycles of denaturation-primer annealing-elongation (10s at 92°C, 30s at 63°C, and 8 min at 68°C), and 1 cycle of the final extension (12 min at 72°C). A negative control (no template) was also performed for every PCR run to determine any potential contamination of the PCR products. The amplified long PCR products were isolated on a 0.8% agarose gel containing crystal violet, excised in ambient light and extracted using the TOPO Gel Purification reagents supplied with the TOPO XL Cloning kit (Invitrogen Co.). After gel purification, each of two long PCR products was ligated using the cloning kit (TOPO XL Cloning kit) and then transformed into E. coli competent cells. Cyclic sequencing reactions for each of the long PCR products were performed in both directions with a Big Dye Terminator Cycle-Sequencing Kit (Applied Biosystems) using primer walking. A full strand of the entire mtDNA sequence was then assembled by double-checking the sequences of overlapping regions of the two long PCR fragments.
Gene Annotation and Phylogenetic Analyses
Twelve protein-coding genes and two ribosomal RNA genes of M. sebastis were identified by sequence comparison with those published in other flatworm species, with the aid of a web-based automatic annotation program for organellar genomes (DOGMA; ). We identified the putative secondary structures of 22 tRNA genes by using tRNAscan-SE program  or by recognizing potential secondary structures and anticodon sequences by visual inspection. The amino acid sequences for protein-coding genes of M. sebastis mtDNA were inferred using the flatworm mitochondrial genetic code (the genetic code table 9 in GenBank). Amino acid sequences, and gene starts and stops, were verified by alignment against homologous genes from other flatworms. In addition to the mtDNA of M. sebastis, the mitochondrial genome sequences for 13 neodermatan species and four lophotrochozoan species (used as outgroups) were retrieved from the GenBank for phylogenetic analyses: Echinococcus multilocularis [GenBank: NC_000928], E. granulosus [GenBank: NC_008075], Fasciola hepatica [GenBank: NC_002546], Hymenolepis diminuta [GenBank: NC_002767], Paragonimus westermani [GenBank: NC_002354], Schistosoma japonicum [GenBank: NC_002544], S. mansoni [GenBank: NC_002545], S. mekongi [GenBank: NC_002529], S. spindale [GenBank: NC_008067], S. haematobium [GenBank: NC_008074], Taenia asiatica [GenBank: NC_004826], T. crassiceps [GenBank: NC_002547], T. solium [GenBank: NC_004022], Loligo bleekeri [GenBank: NC_002507; Mollusca], Phoronis psammophila [GenBank: AY368231; Phoronida], Platynereis dumerilii [GenBank: NC_000931; Annelida], and Terebratulina retusa [GenBank: NC_000941; Brachiopoda]. The sequence information of the mitochondrial protein-coding genes for rhabditophoran turbellarian species Microstomum lineare is limited to smaller number of gene loci (full lengths of nad5, cox3, atp6 and partial lengths of cox1 and cob; ). For this reason, three independent amino acid sequence datasets were prepared for phylogenetic analyses: (1) the dataset containing all complete neodermatan sequences and the incomplete M. lineare sequences as ingroups and four complete lophotrochozoan sequences as outgroups, (2) the dataset containing the complete genome sequences only (excluding M. lineare sequences), and using four lophotrochozoan sequences as outgroups, and (3) the dataset comprising five gene loci only (nad5, cox3, atp6, cox1 and cob) obtained universally from all platyhelminth ingroup and lophotrochozoan outgroup taxa. A multiple alignment for each gene loci was performed using ClustalX  with the following options: gap opening penalty = 10, gap extension penalty = 1.0 with a "delay divergent sequence" setting of 30% of the BLOSUM similarity matrix. The result of multiple sequence alignment is not always unambiguous due to the length and sequence variation among the taxa, causing the poorly aligned profile. Therefore, a conserved block of concatenated alignment was selected using the Gblocks program  for each of protein-coding loci of all species examined and then subjected to subsequent phylogenetic analyses. This was compared with an alignment where ambiguously aligned positions had been identified by eye; the two alignments were almost identical and yielded identical phylogenetic estimates. Molecular phylogenetic analyses of flatworm mitochondrial genomes were conducted using several methods applied to the protein sequence data. Bayesian analysis was performed using MrBayes 3.1 . We set parameters to "ngammacat = 4", "rates = invgamma" for likelihood setting. Four Markov Chain Monte Carlo (MCMC) chains were run for 106 generations, sampled every 100 generations. Bayesian posterior probability values representing the percentage of samples recovering particular clades were estimated after initial 1,000 trees (the first 105 generations) were discarded. Maximum likelihood (ML) analysis was carried out using quartet puzzling method of the TREE-PUZZLE 5.2 program  under the mtREV24 matrix , as an evolution model for mitochondrial protein, with four categories of gamma-distributed rates estimated from the dataset. The analysis was run for 5×104 puzzling steps. For ML phylogenetic analyses, the mtREV substitution model of Adachi and Hasegawa was used as it is widely recognized to represent much better fit to the mtDNA-encoded protein sequence data than the Dayhoff and the JTT models [55, 56]. The maximum likelihood mapping method  was conducted to assess the amount of phylogenetic signal in the dataset. We also conducted maximum parsimony (MP) analysis in PAUP* 4.0b10 version  and nodal support in the resulting tree was estimated by nonparametric bootstrap analysis with 1,000 random replications using a heuristic search option. Statistical tests for the alternative phylogenetic hypotheses were performed using the likelihood-based Shimodaira-Hasegawa test  and parsimony-based Templeton test  implemented in TREE-PUZZLE 5.2 and PAUP* 4.0b10, respectively.
We thank Michael Cummings for his careful reading of the manuscript and Rod Bray for comments. Bo Young Jee (National Fisheries Research and Development Institute) and Jeong-Ho Kim provided materials for DNA works. We are indebted to three anonymous referees and the editor for their constructive comments. Materials used for morphological identification were deposited (PRB#06_00024_00031) in the Parasite Resource Bank of Korea National Resrarch Resource Center, Republic of Korea. This work was financially supported by the Korea Research Foundation Grant (KRF-2005-070-C00124). DTJL was funded by a Wellcome Trust Senior Research Fellowship (043965).
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