# Evolutionary relationships of sibling tapeworm species (Cestoda) parasitizing teleost fishes

O. Verneau, F. Renaud, F. Catzeflis

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O. Verneau, F. Renaud, F. Catzeflis. Evolutionary relationships of sibling tapeworm species (Cestoda) parasitizing teleost fishes. Molecular Biology and Evolution, 1997, 14 (6), pp.630-636. 10.1093/oxfordjournals.molbev.a025802. hal-04754447

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# Evolutionary Relationships of Sibling Tapeworm Species (Cestoda) Parasitizing Teleost Fishes

Olivier Verneau,* François Renaud,* and François Catzeflis†

*Laboratoire de Parasitologie Comparée and †Institut des Sciences de l'Evolution, Université de Montpellier II, France

DNA/DNA hybridization and sequencing of rDNA (partial 18S rDNA and ITS1) were used to investigate phylogenetic relationships among seven host-specific *Bothriocephalus* parasites (Cestoda, Pseudophyllidae). The small nucleotide divergence between six of the seven bothriocephalids suggests that isolation and differentiation of *Bothriocephalus* lineages in the different host species probably occurred recently and over a short time span. Comparison of the molecular phylogeny of the parasite species to the phylogeny of their hosts (teleostean fishes) revealed little congruence between the branching patterns of hosts and parasites, suggesting that bothriocephalids have not cospeciated with their hosts.

## Introduction

Tapeworms of the genus *Bothriocephalus* (Cestoda, Pseudophyllidae) have been described as endoparasites in a large number of teleost fishes belonging to several major lineages of Pleuronectiformes, Scorpaeniformes, Gadiformes, and Anguilliformes (Protasova 1977). The first described species, *Bothriocephalus scorpii* (Mueller 1776), originally reported from the bull-rout (*Myxozocephalus scorpius*) was not considered to be host-specific (Jones 1975). Indeed, this parasite was also found in other host species, such as the turbot (*Psetta maxima*), the brill (*Scophthalmus rhombus*), and the five-bearded rockling (*Ciliata mustela*). However, studies carried out on the population genetic structure of this parasite have shown strict reproductive isolation between the parasite populations of each host species (Renaud, Gabrion, and Pasteur 1983, 1986; Renaud and Gabrion 1984). These analyses have revealed that *B. scorpii* constituted a complex of sibling biological species comprising a number of similar morphological species with host preference. Thus, whether *B. scorpii* is only parasitizing the bull-rout, *B. gregarius* is found in the turbot, *B. barbatus* is found in the brill, and *B. funiculus* is found in the five-bearded rockling. In addition, it is known that two other pleuronectiform fishes, the scaldfish (*Arnoglossus laterna*) and the spotted flounder (*Citharus linguatula*), and one anguilliform, the eel (*Anguilla anguilla*), harbor three other *Bothriocephalus* species, *B. clavibothrium* (Ariola 1899), *B. andresi* (Porta 1911), and *B. claviceps* (Goeze 1872), respectively.

Thus, the high degree of biological specificity of the different parasite species among hosts belonging to so distantly related orders of teleost fishes (Lauder and Liem 1983) raises the question concerning the compared host-parasite phylogenesis in this heterospecific assemblage. We might ask if concomitant speciations (cospeciation) have occurred, leading to an evolutionary picture where the parasites' phylogeny mirrors the hosts'

phylogeny (Fahrenholz 1913). Several analyses of coevolutionary processes have been conducted (Brooks and Glen 1982; Mitter and Brooks 1983; Bandoni and Brooks 1987; Deets 1987; Klassen and Beverley-Burton 1987; O'Connor 1988; Brooks and McLennan 1991), all of which involve the appraisal of branching similarities between host and parasite phylogenies and between their taxonomic boundaries or geographic distributions. However, relatively few studies have compared genetic divergence and speciation events in hosts and their parasites, permitting a discussion of the relative timing of speciation within and between host and parasite lineages (Baverstock, Adams, and Beveridge 1985; Hafner and Nadler 1988, 1990; Rannala 1992; Hafner et al. 1994). In the literature, the clearest evidence for cospeciation derives from isozyme electrophoretic data and from sequences of the mitochondrial cytochrome oxidase I gene in a pocket gopher and chewing louse assemblage (Hafner and Nadler 1988; Hafner et al. 1994). These data, evidenced synchronous speciation events between sets of coevolving taxa.

The aim of this paper is to estimate the evolutionary relationships of seven species of teleostean parasite (*Bothriocephalus*) by using two complementary approaches. First, we will employ the nonrepeated DNA/DNA hybridization methodology which allows an estimation of the overall nucleotide divergence between the entire genomes of compared species (Werman, Springer, and Britten 1990). This is the first time that this approach has been applied to parasitic helminths to directly ascertain levels of interspecific genomic divergence. Second, we will use sequence data from nuclear rDNA (the first internal transcribed spacer, or ITS1, and partial 18S rDNA) to investigate the evolutionary relationships within bothriocephalids. Finally, we compare the phylogeny of these parasites with the evolutionary relationships of their teleostean hosts to elucidate biological and ecological factors that may have governed the evolution of this host-parasite association.

## Materials and Methods

### Specimens Examined

Pleuronectiform fishes were trawled in the Mediterranean Sea, along French coasts (Sete) for turbots, brills, and scaldfishes, and along Spanish coasts (Valen-

Key words: evolution, helminth, teleostean, DNA/DNA hybridization, 18S rDNA, ITS1.

Address for correspondence and reprints: Olivier Verneau, Laboratoire de Parasitologie Comparée, UMR 5555, CC 105, Université de Montpellier II, Pl. E. Bataillon, 34095 Montpellier Cedex 5, France. E-mail: verneau@crit.univ-montp2.fr.

Mol. Biol. Evol. 14(6):630–636. 1997
© 1997 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
cia) for spotted flounders; scorpaeniforms (bull-rout) and gadiforms (five-bearded rockling) were obtained from the British Channel (Roscoff), and anguilliforms were caught by electrofishing in a river in southern France (Montpellier). After capture, fishes were anesthetized in the laboratory with 3-aminobenzoic acid ethyl ester (Sigma A-5040). Digestive tracts were dissected to collect tapeworms, which were rinsed several times in 0.9% NaCl.

## Methods

DNA was extracted from 5-20 individuals of each parasite species, following the procedures of Sambrook, Fritsch, and Maniatis (1989) and Verneau, Renaud, and Catzeflis (1991). Purified DNA was sheared by sonication with a Branson 250 Sonifier calibrated to yield an average fragment size of 500 bp or 2,000 bp, respectively. The size of sonicated fragments was confirmed by agarose gel electrophoresis, where DNA was stained with ethidium bromide and compared with the pBR 322-Taq I marker. Two thousand base pairs of DNA was heat denatured (100°C) and incubated at 55°C until all repeated DNA sequences were reassociated (Verneau, Renaud, and Catzeflis 1991). Fractionation of single-copy versus repeated DNA sequences was performed on hydroxyapatite columns (Britten, Graham, and Neufeld 1974). Twenty-five nanograms of the resulting nonrepeated DNA was radiolabeled with 5-¹²⁵Iodo-2'-deoxycytidine 5'-triphosphate (Amersham, code IM.5103) by using random primer extension (Feinberg and Vogelstein 1983, 1984). Nonincorporated nucleotides were removed from radiolabeled DNA on G50 Sephadex columns (Sambrook, Fritsch, and Maniatis 1989). Duration of the enzymatic reaction was calibrated to yield radiolabeled DNA with a size of 300-500 bp, which was confirmed with sizing gels following standard procedures (Hall et al. 1980; Hunt, Hall, and Britten 1981; Caccone, Amato, and Powell 1987). Nonrepeated radiolabeled DNA, used as "tracer" in the DNA/DNA experiments, was hybridized to a thousand-fold excess of nonlabeled 500-bp DNA (driver) in homologous hybridization (i.e., DNA tracer and driver from the same species), and heterologous hybridization (i.e., DNA tracer and driver from different species). The mixtures were denatured at 100°C and then incubated at 60°C until completion of complementary strand reassociation at equivalent Cot of 16,000 (Cot = the product of DNA concentration × time of reassociation). The hybrids were passed through hydroxyapatite columns preheated in a water bath at 55°C, and single-stranded DNA was eluted with 12-15 ml of 0.12 M phosphate buffer (Kohne and Britten 1971; Werman, Springer, and Britten 1990). Temperature of the water bath was then increased by steps of 2.5°C until it reached 95°C, and the radioactivity contained in all collected samples was counted. The percentage of single-stranded DNA was estimated for each elution temperature. For each available tracer (size around 300-500 bp), hybrids were formed with two to five replicates. After reassociation, all thermal elution experiments were performed on the same day in order to minimize experimental error resulting from variation

among experimental runs of the thermal chromatography (Dickerman 1991). The parameter Tm, which represents the median temperature at which 50% of hybrid DNA has been dissociated, was deduced from the melting curves of DNA hybrids and used for the analysis of evolutionary relationships.

The complete matrix of delta-Tm values was subjected to a bootstrapping procedure to assess its robustness (Krajewski and Dickerman 1990). Resampling of distance data was performed 1,000 times, and the resulting asymmetric matrices were adjusted by using the procedure of Sarich and Cronin (1976). An unrooted network was constructed by the least-squares approach of Fitch and Margoliash (1967) from each pseudoreplicate matrix. The percentage of occurrence of each node observed in the resulting networks was calculated by the program Consense in the PHYLIP package (Felsenstein 1990). Because the bootstrapping procedure does not generate normally distributed data (the variance is slightly biased; Weir 1990), a random sample of 100 replicate trees was used to calculate the modal value and 95% confidence interval for each branch length. The resulting network was unrooted.

Because the parasites studied are specific to distinctly related hosts belonging to four major fish lineages, we expected to find high levels of molecular changes between bothriocephalid species if cospeciation had occurred. Therefore, partial sequences of the 18S rDNA, a slow-evolving nuclear gene, were obtained. Preliminary results obtained from the 18S rDNA sequences showed few substitutions between the species studied. So we decided to sequence one intron (ITS) which evolves more rapidly than 18S rDNA.

Partial 18S rDNA (about 650 bases) and the entire ITS1 region (about 570 bases) were amplified from genomic DNA by using the polymerase chain reaction (PCR). This was done for the seven species of bothriocephalids studied and another parasite *Triaenophorus nodulosus* hosted by *Esox lucius*, which belongs to the pseudophyllidean family *Ancyrocephalidae*. Oligonucleotide primers used for the PCR reactions were designed from regions of 18S rDNA and 5.8S rDNA that are highly conserved in a wide range of organisms: *Caenorhabditis elegans* (EMBL accession number X03680), *Opisthorchis viverrini* (X55357), *Drosophila melanogaster* (X70692 and X15707), *Alligator mississippiensis* (M59383 and M36342), and *Mus musculus* (X56974). The first primer (L7) hybridized in the 18S rDNA (5'-TGATTTGTCTGGTTTATTCCGAT-3') and is homologous to the sequence of the nematode *C. elegans* at positions 1143-1165 (Ellis, Sulston, and Coulson 1986). The second primer (H7) hybridized in the 5.8S rDNA (5'-GCTGCGTTCTTCATCGATACTCG-3') and is the complementary sequence of the 5.8S rDNA gene of *C. elegans* at positions 2132-2154 (Ellis, Sulston, and Coulson 1986). PCR products were immediately cloned in the pGEM-T vector (Promega) and transfected in competent bacteria (JM101 strain of *Escherichia coli*). Recombinant clones were identified by color screening on indicator plates and were sequenced directly by the method of Sanger, Nicklen, and Coulson
<table><caption>**Table 1**
Delta-Tm and Tamura-Nei Distances Between Bothriocephalid Parasites</caption><thead><tr><th></th><th>B. gregarius</th><th>B. barbatus</th><th>B. clavibothrium</th><th>B. andresi</th><th>B. funiculus</th><th>B. scorpii</th><th>B. claviceps</th><th>T. nodulosus</th></tr></thead><tbody><tr><td>B. gregarius</td><td>0.0</td><td>2.05 ± 0.23<br>n = 8</td><td>2.90 ± 0.14<br>n = 9</td><td>2.91 ± 0.08<br>n = 7</td><td>2.89 ± 0.35<br>n = 6</td><td>3.50 ± 0.51<br>n = 8</td><td>—</td><td>—</td></tr><tr><td>B. barbatus</td><td>0.0097</td><td>0.0</td><td>3.15 ± 0.36<br>n = 7</td><td>3.19 ± 0.28<br>n = 8</td><td>3.29 ± 0.11<br>n = 6</td><td>3.62 ± 0.33<br>n = 7</td><td>—</td><td>—</td></tr><tr><td>B. clavibothrium</td><td>0.0167</td><td>0.0123</td><td>0.0</td><td>0.31 ± 0.15<br>n = 9</td><td>3.09 ± 0.52<br>n = 7</td><td>3.12 ± 0.54<br>n = 8</td><td>—</td><td>—</td></tr><tr><td>B. andresi</td><td>0.0203</td><td>0.0158</td><td>0.0052</td><td>0.0</td><td>3.40 ± 0.21<br>n = 8</td><td>3.56 ± 0.15<br>n = 7</td><td>—</td><td>—</td></tr><tr><td>B. funiculus</td><td>0.0132</td><td>0.0880</td><td>0.0141</td><td>0.0176</td><td>0.0</td><td>3.12 ± 0.05<br>n = 8</td><td>—</td><td>—</td></tr><tr><td>B. scorpii</td><td>0.0105</td><td>0.0079</td><td>0.0096</td><td>0.0132</td><td>0.0097</td><td>0.0</td><td>—</td><td>—</td></tr><tr><td>B. claviceps</td><td>0.1592</td><td>0.1549</td><td>0.1583</td><td>0.1614</td><td>0.1561</td><td>0.1562</td><td>0.0</td><td>—</td></tr><tr><td>T. nodulosus</td><td>0.2167</td><td>0.2153</td><td>0.2122</td><td>0.2130</td><td>0.2182</td><td>0.2110</td><td>0.2489</td><td>0.0</td></tr></tbody></table>

NOTE.—Upper right matrix: average delta-Tm values (in °C) with standard deviation estimated from DNA/DNA experiments. *n* = number of replicate experiments for each pair of hybrids. Dashes (—) indicate nondetermined values. Lower left matrix: Tamura-Nei distances estimated from sequence analysis.

(1978). For sequencing, we used the Reverse and Forward plasmid primers and two other primers designed for 18S rDNA and ITS1 regions. Sequences of these oligonucleotides were as follows: 18S-1 (5'-ATTGAGCAATAACAGGTCTGT-3') and ITS1-1 (5'-AAACAATCATGATGGGGCGTAG-3').

Sequences from ITS1 and partial 18S rDNA were aligned using the MUST package (Philippe 1993). When alignments were ambiguous, in particular for regions with insertions or deletions, all sites including a gap (insertion, deletion) were removed from the phylogenetic analyses which were conducted using combined sequences obtained from ITS1 and the partial 18S rDNA. The parsimony analysis was done using the PAUP program version 3.1.1., with the branch-and-bound option (Swofford 1993). All informative sites were retained and equally weighted. For the phenetic analysis, the Tamura-Nei distance (Tamura and Nei 1993) was computed between all pairs of sequences. This distance was chosen because the transition/transversion ratio was slightly biased and the four nucleotides

deviate substantially from equality. Trees were then constructed using the neighbor-joining method (Saitou and Nei 1987). Robustness of resulting trees was assessed using a bootstrapping procedure on all sites (1,000 replications), and trees were rooted using *T. nodulosus* as the outgroup. Indeed, preliminary studies based on parsimony and distance analyses on partial 18SrDNA sequences including all bothriocephalids, *T. nodulosus* and another species of helminth belonging to the order Proteocephalidea (*Proteocephalus anguillae*) supported bothriocephalid parasites as a monophyletic group (data not shown). Sequences have been deposited in EMBL Data Bank under accession numbers Y09670-Y09685.

## Results

### DNA/DNA Hybridization

Table 1 depicts divergence estimates (average delta-Tm values with standard deviation) between each pair of parasite taxa. It was not possible to measure delta-Tm values between *B. claviceps* and any other species because the level of genomic divergence between this taxon and the others exceeds the resolving power of the methodology. The tree (unrooted) is represented in figure 1. A single branching pattern was observed between the parasite species where all resulting nodes are supported at the 100% level.

### Nucleotide Sequences

The alignments of 18SrDNA and ITS1 sequences for the seven bothriocephalid species and *T. nodulosus* have been deposited in EMBL under accession numbers DS28072 and DS28073. The length of the 18SrDNA region was 647 bp for *B. claviceps* and 644 bp for all other bothriocephalids and *T. nodulosus*. When alignments were obtained and compared to the sequence of *C. elegans*, bothriocephalids and *T. nodulosus* revealed an insertion of about 150 bp within the 18S rDNA. The first internal transcribed spacer was sequenced completely for all parasites studied. Length of this intron

<figure><img src="image_3.png" /><figcaption>**FIG. 1.**—Evolutionary relationships among six species of *Bothriocephalus* constructed by phenetic analysis (FITCH) of the delta-Tm parameter from DNA/DNA hybridization. Numbers correspond to frequencies of occurrence of the different nodes in 1,000 bootstrap replicates. The letter F indicates parasites of flatfishes (Pleuronectiformes).</figcaption></figure>
a higher rate of nonrepeated DNA change, such as mice and rats, delta-Tm values of ca. 3.0°C correspond to a divergence time of ca. 2–3 Myr (Catzeflis et al. 1993).

## Comparison of Host and Parasite Phylogenies

Comparison of the branching patterns among hosts and their associated parasites can provide information on cospeciation and host-switching events that have occurred during the evolutionary history of the association. Few molecular studies have been conducted to discern relationships between orders of teleost fishes. Le, Perasso, and Billard (1989) and Le, Lecointre, and Perasso (1993) showed, from 28S rRNA sequence analysis, that pleuronectiforms (in our survey, four species are examined: *P. maxima*, *S. rhombus*, *A. laterna*, and *C. linguatula*) are more closely related to scorpaeniforms (for example *M. scorpius*) than they are to gadiforms (for example, *C. mustela*) and, finally, to anguilliforms (for example, *A. anguilla*) which are the most divergent. However, relationships between the major orders examined in this study are moderately supported with low bootstrap values (Le, Lecointre, and Perasso 1993). Concerning relationships within pleuronectiform fishes, Verneau et al. (1994) showed from DNA/DNA hybridization and electrophoretic analyses that the two scophthalmids (i.e., *P. maxima* and *S. rhombus*) are more closely related to each other than either is to a citharid (i.e., *C. linguatula*) or a bothid (i.e., *A. laterna*). Moreover, the two latter families (Citharidae and Bothidae) are as divergent from Scophthalmidae as they are from each other (Verneau et al. 1994). So, it seems that speciation events leading to the lineages of *C. linguatula* and *A. laterna* occurred before the split between *P. maxima* and *S. rhombus*. In the light of a tree based on the combination of these sets of data (Le, Perasso, and Billard 1989; Le, Lecointre, and Perasso 1993; Verneau et al. 1994), we can discuss the evolution of this host-parasite association when the combined host tree (fig. 3B) is compared to the parasite phylogeny (fig. 3A).

Besides the early separation of *B. claviceps*, all remaining bothriocephalid parasites are grouped in just two clades: (1) *B. gregarius*, *B. barbatus*, *B. funiculus*, and *B. scorpii*; and (2) *B. clavibothrium* and *B. andresi* (fig. 3A). Thus, flatfish parasites (i.e., tapeworms of Pleuronectiformes labeled F on figs. 1, 2, and 3A) do not appear to be monophyletic. Consequently, the discrepancies observed in the branching patterns between the two biological assemblages (i.e., hosts and parasites) suggest that pleuronectiform fishes and their associated parasites have not cospeciated. The branch lengths of the parasites trees (figs. 1 and 3A) suggest that the speciation between *B. andresi* and *B. clavibothrium* is more recent than the one between *B. gregarius* and *B. barbatus*, whereas the branching pattern of the fishes indicates that *P. maxima* and *S. rhombus* are more related to each other than either is to *C. linguatula* and *A. laterna* (fig. 3B). As bothriocephalids are subdivided into two clades on the phylogenetic tree of figure 3A, the unrooted network (fig. 1) shows that *B. andresi* and *B. clavibothrium* are more closely related to each other than is *B. gregarius* to *B. barbatus*. This contrasting

pattern in evolutionary relationships between hosts and parasites suggests that a host-switching event has occurred during the evolutionary history of *C. linguatula* and *A. laterna* and their parasites.

Two different—but nonexclusive—evolutionary processes can be proposed for explaining the results of this study: (1) sequential colonizations of different host species followed by speciation of parasites, as proposed elsewhere in helminth faunas by Hoberg (1986, 1992) and Hoberg and Adams (1992); or (2) a rapid radiation of a ubiquitous ancestral parasite species, leading to isolation and differentiation of *Bothriocephalus* lineages in the different host species. As judged from the small amounts of genomic divergence measured between most pairs of tapeworms, these processes have occurred rather recently and over a short time span.

## Acknowledgments

We thank Julio Herrero and Santiago Mas Coma for their help in collecting bothriocephalid samples from palayas in Valencia (Spain), Louis Euzet for his help in collecting bothriocephalid samples from parpeilles in Sete (France), and Roland Marin for his help in electrofishing eels in the south of France. We also thank Rodney Honeycutt, Thierry De Meeûs, Guillaume Lecointre, and an anonymous referee for their helpful comments and discussion. Laboratory facilities were provided by Service Commun de Systématique de l'Université de Montpellier II, and Olivier Verneau was financially supported by the Ministère de la Recherche et de la Technologie.

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RODNEY L. HONEYCUTT, reviewing editor

Accepted February 18, 1997