Infection, Genetics and Evolution

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1 Infection, Genetics and Evolution 10 (2010) Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: Molecular diversity of Trichobilharzia franki in two intermediate hosts (Radix auricularia and Radix peregra): A complex of species D. Jouet a, *, K. Skírnisson b, L. Kolářová c, H. Ferté a a JE 2533 USC AFSSA «VECPAR» UFR de Pharmacie, Université de Reims Champagne Ardenne, 51 rue Cognacq-Jay, Reims, France b Institute for Experimental Pathology, University of Iceland, Keldur, Reykjavík, Iceland c Institute of Immunology and Microbiology of the First Faculty of Medicine, Charles University in Prague and General Teaching Hospital, Studničkova 7, Prague 2, Czech Republic ARTICLE INFO ABSTRACT Article history: Received 23 March 2010 Received in revised form 19 May 2010 Accepted 4 August 2010 Available online 11 August 2010 Keywords: Trichobilharzia franki Radix auricularia Radix peregra Molecular analyses Cercariae Snails Recently, the systematic use of the molecular approach as a complement to the other approaches (morphology, biology, life cycle) has brought help for the identification of species considered as different in the past to be regrouped and synonymised, and distinctions to be drawn between species similar at the morphological level. Among these species, we tried to clarify the situation of Trichobilharzia franki Müller and Kimmig, 1994, species that today include more than 50 haplotypes notably coming from larval stages isolated from intermediate hosts belonging to gastropods of the Radix genus. Cercariae were isolated in France and Iceland from various molluscs, before being analyzed, with their hosts, by molecular analysis of various fields such as the D2 and ITS of the ribosomal DNA and the COX1 of mitochondrial DNA. We thus show the presence of two clades depending on the specificity of their intermediate host in which they were isolated (Radix auricularia or Radix peregra), thus allowing separation of the species T. franki that had been described in the past as a probable new species. ß 2010 Elsevier B.V. All rights reserved. 1. Introduction Among schistosomatids, representatives of the genus Trichobilharzia, with more than 40 described species, are probably the most frequent in the field. The parasite life cycle comprises various birds (mainly waterfowl) and water snails as definitive and intermediate hosts, respectively. Due to a complicated life cycle and the difficulty of isolation and morphological identification of the adult worms, the taxonomy within this genus is difficult. In the past, characterization of the species was based mainly on morphology of the larval (cercariae) and adult stages. Unfortunately, in many cases cercariae and adults were described separately with regard to their further development in definitive or intermediate hosts, respectively, meaning that the morphology of all developmental stadia of such species remained unknown. Valuable data were obtained by studies on schistosomatids developing in experimentally infected hosts, but numerous problems such as unavailability of compatible hosts or isolation of adult flukes from the birds (Kolářová et al., 2010) limited the wide use of these methods. The use of recent molecular tools such as the 28S and ITS of the ribosomal DNA and the COX1 of the * Corresponding author. Tel.: ; fax: address: damienjouet@hotmail.com (D. Jouet). mitochondrial DNA (Littlewood and Johnston, 1995; Mollaret et al., 1997; Snyder and Loker, 2000; Picard and Jousson, 2001; Lockyer et al., 2003; Olson et al., 2003; Snyder, 2004; Brant et al., 2006; Littlewood et al., 2006; Webster et al., 2007) finally allowed definitive and precise taxonomical identification of the parasites. On the basis of morphological and molecular characteristics, including intermediate-host specificity, three Trichobilharzia species maturing predominantly in waterfowl (for a review see Horák et al., 2002) are currently recognized in Europe: Trichobilharzia szidati Neuhaus, 1952 developing in Lymnaea stagnalis, Trichobilharzia franki Müller and Kimmig, 1994 developing in Radix auricularia and Trichobilharzia regenti Horák Kolářová and Dvořák, 1998 developing in Radix peregra. Four species that had been discovered some time ago were recently characterized in North America Trichobilharzia physellae (Talbot, 1936) McMullen and Beaver, 1945 developing in Physa parkeri, Trichobilharzia querquedulae McLeod, 1937 developing in Physa acuta (experimentally), Trichobilharzia stagnicolae (Talbot, 1936) McMullen and Beaver, 1945 developing in Stagnicola emarginata and Trichobilharzia brantae Farr and Blankemeyer, 1956 developing in Gyraulus parvus. For the latter, phylogenetic analysis of DNA suggests that this species probably does not belong in the genus Trichobilharzia (Brant and Loker, 2009). T. franki has been reported in many European countries: Czech Republic (Kolářová et al., 1997; Rudolfová et al., 2005), Germany /$ see front matter ß 2010 Elsevier B.V. All rights reserved. doi: /j.meegid

2 D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) (Brant and Loker, 2009), Switzerland (Picard and Jousson, 2001), France (Ferté et al., 2005; Jouet et al., 2008, 2009), Iceland (Skírnisson and Kolářová, 2008; Skírnisson et al., 2009; Aldhoun et al., 2009a), Russia (Semyenova et al., 2005), Belarus (Chrisanfova et al., 2009), Poland (Rudolfová et al., 2005) and Finland (Aldhoun et al., 2009b). At present, many haplotypes of cercariae and/or adults originating from naturally infected snails and aquatic birds, respectively, are available in GenBank. Except for one record of T. franki isolated from L. stagnalis by Rudolfová et al. (2005), the larval stages were found only in snails of genus Radix: mainly R. auricularia but also R. peregra were then identified as the intermediate hosts. Since T. franki has been found in a variety of geographical areas in a large number of hosts and considering the increasing number of sequences available in the databases (in particular the recent description of new American species), we studied the possible variations of the parasite within the species and revised the complex of parasites with regard to species-type described by Müller and Kimmig (1994). We attempted to clarify the position of the haplotypes of parasites found in various Radix snails, using molecular analysis. 2. Materials and methods This study was carried out for an international project (EGIDE) in the aim of studying the common parasites between France and Iceland. Snails of the genus Radix were collected between 2000 and 2009 in France and during summer and autumn 2009 in Iceland. Cercarial emergence was stimulated according to Kolářová et al. (2010). Ocellate furcocercariae were preserved in 95% ethanol and frozen ( 20 8C) until the DNA analysis. Positive snail hosts were frozen directly at 20 8C in individual sterile bags for storage. Only the samples from which cercariae and their corresponding snails were sequenced were used for molecular analyses. The sequenced samples are listed in Table 1. Photographs and measurements of cercariae were made on fresh material or preserved in formalin, with a digital camera (Leica DC 300) attached to a microscope (Leica DMLB or Olympus BX50) equipped for differential interference contrast or Nomarski system. After removing all ethanol from samples, DNA was extracted using the Qiamp DNA Mini Kit (Qiagen, Germany) following manufacturer s instructions. During the first step (tissue lysis), cercariae or a small part of the foot of each positive snail were crushed using a piston pellet (Treff, Switzerland). The DNA was eluted in 50 ml of the buffer provided. Polymerase Chain Reaction was performed in a 50 ml volume using 5 ml of DNA, and 50 pmol of each of the primers. The PCR mix contained (final concentrations) 10 mm Tris HCl (ph 8.3), 1.5 mm MgCl 2, 50 mm KCl, 0.01% Triton X-100, 200 mm dntp each base, and 1.25 units of Taq polymerase (Eppendorf, Germany). Sequencing of the D2 domain of the 28S subunit, internal transcribed spacers (ITS-2 and ITS-1) of ribosomal DNA and COX1 domain of the mitochondrial DNA were used for the identification of avian schistosomes obtained from naturally infected snails. PCR was performed under conditions that had been published previously (Jouet et al., 2009). Ribosomal DNA: ITS-2 and ITS-1 of furcocercariae were amplified using primers ITS3Trem (5 0 -GCG TCG ATG AAG AGT GCA GC-3 0 ), ITS4Trem (5 0 -TCC TCC GCT TAT TGA TAT GC-3 0 ), ITS2Trem (5 0 -GCT GCA CTC TTC ATC GAC GC-3 0 ) and ITS5Trem (5 0 - GGA AGT AAA AGT CGT AAC AAG G-3 0 )(Dvořák et al., 2002). The D2 domain was amplified using the primers C2 0 B(5 0 -GAA AAG TAC TTT GRA RAG AGA-3 0 ) and D2 (5 0 -TCC GTG TTT CAA GAC GGG-3 0 ) according to Mollaret et al. (1997). Mitochondrial DNA: The domain COX1 was amplified using the primers SchistoCox1-5 0 (5 0 -TCT TTR GAT CAT AAG CG-3 0 ) and SchistoCox1-3 0 (5 0 -TAA TGC ATM GGA AAA AAA CA-3 0 )(Webster et al., 2007). ITS-2 was also used for snail identification: Amplification of the positive snails was made by using the specific primers of Radix spp. DIX1 (5 0 -CGC GCT CTG GWC CKT CGC GGC-3 0 ) and DIX2 (5 0 -ATY TYG TYC GAT TTG AGG TTG-3 0 )(Jouet et al., 2008). PCR products were directly sequenced in both directions with the primers used for DNA amplification (QIAGEN, Germany). The sequences are deposited in GenBank under the accession numbers HM HM and HQ HQ Sequences were aligned using the ClustalW routine included in the MEGA version 3.1 software (Kumar et al., 2004) and checked by eye. The D2 domain (554 bp) and ITS region (1637 bp) of the ribosomal DNA and the COX1 domain (822 bp) of the mitochondrial DNA for the parasite and ITS-2 (440 bp) of the rdna for the snails were used for tree construction and rooted with the outgroup taxon (Bilharziella polonica (D2), T. szidati (ITS and COX1) and L. stagnalis (ITS-2)). Phylogenetic analyses were performed using haplotypes obtained in this study (Table 1) and sequences available in GenBank: T. regenti, T. szidati, T. physellae, T. querquedulae, Trichobilharzia sp., T. stagnicolae, T. brantae, Allobilharzia visceralis, Dendritobilharzia pulverulenta, Gigantobilharzia huronensis, B. polonica for ocellate furcocercariae; R. auricularia, Radix balthica (=peregra, ovata), Radix ampla, Radix lagotis, Radix labiata and Radix sp. for snails (Tables 2 and 3). Phylogenetic reconstruction using Neighbour-Joining (NJ) with the Kimura-2 parameter and uniform rates among sites was performed using the MEGA version 3.1 software (Kumar et al., 2004). Maximum Likelihood (ML) analysis was performed in Phyml online (Guindon et al., 2005). The model and the parameters were chosen using the hierarchical likelihood ratio test implemented in Modeltest 3.7 (Posada and Crandall, 2001). Maximum Parsimony (MP) analysis was performed in MEGA version 3.1 software (Kumar et al., 2004) using the Maxi-mini Branch and Bound method. For all NJ, ML and MP analyses, gaps were treated as missing data and internal node support was assessed by bootstrapping over 500 replicates. 3. Results 3.1. Morphological comparison of cercariae Measurements of the cercariae isolated from R. auricularia and R. peregra show variations between them and relative to the cercariae of T. franki, T. querquedulae and T. physellae, particularly in the overall length of the cercariae (Table 4). However, cercariae of avian schistosomes are contractile, which may explain such variability. It is thus impossible to determine to which species the parasites belong on the basis of morphological criteria alone Molecular analyses of cercariae The molecular analysis of the D2, ITS and COX1 domains are congruent (Figs. 1 3): they show that the cercariae isolated from Radix belong to Trichobilharzia. Our analyses clearly show that the haplotypes T. franki auricularia and T. franki peregra belong to two distinct clades, separated by the haplotypes corresponding to the recently described American species: T. querquedulae and T. physellae. For the D2 domain, each clade ( auricularia and peregra ) is composed of 100% homologous haplotypes. In spite of the conserved character of this domain (specific level of the D2), three variations are present between these two haplotypes. In comparison, T. franki auricularia and T. physellae are also separated by three variations, two variations between T. franki peregra and T. physellae, and also between T. franki peregra and

3 1220 D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) Table 1 Isolates of bird schistosomes used for molecular analysis. Host: snail Geographical data Taxa Species Taxa Life cycle stage Locality GPS coordinates RAN79 Radix pergera a EAN79 Cercariae Annecy Lake, France N/ E RAN80 Radix pergera a EAN80 Cercariae Radix pergera ICR1 Cercariae Raudavatn, Iceland N/ O Radix pergera ICR21 Cercariae Helgavogur-Myvatn, Iceland N/ O Radix pergera ICR22 Cercariae Radix pergera ICR23 Cercariae Radix pergera ICR24 Cercariae Radix pergera ICR25 Cercariae IS1F Radix pergera a F1IS Cercariae Botnsvatn, Iceland N/ O IS2F Radix pergera a F2IS Cercariae IS3F Radix pergera a F3IS Cercariae Raudavatn, Iceland N/ O IS4F Radix pergera a F4IS Cercariae IS5F Radix pergera a F5ISA Cercariae IS5F Radix pergera a F5ISB Cercariae IS6F Radix pergera a F6IS Cercariae RAN77 Radix auricularia a EAN77 Cercariae Annecy Lake, France N/ E RSFO1 Radix auricularia a FORS1 Cercariae Der-Chantecoq Lake, France N/ E AY Radix auricularia a FORS3 Cercariae RSFO4 Radix auricularia a FORS4 Cercariae RSBE1 Radix auricularia a BERS1 Cercariae Beauvais, France N/ E RSBE2 Radix auricularia a BERS2 Cercariae RSBE13 Radix auricularia a BERS13 Cercariae RSBE67 Radix auricularia a BERS67 Cercariae Radix auricularia STRS2 Cercariae Strasbourg, France N/ E Radix auricularia STRS4 Cercariae Radix auricularia STRS5 Cercariae Radix auricularia STRS6 Cercariae a Molecular identification. Table 2 GenBank sequences of bird schistosomes used for molecular analysis. Species Host Locality GenBank accession numbers D2 ITS2 ITS1 COX1 Trichobilharzia franki peregra Radix peregra France EU Radix peregra France EU Radix peregra France EU Radix peregra France EU Trichobilharzia franki auricularia Radix auricularia France AY Radix auricularia France AY Trichobilharzia physellae Aythya affinis USA FJ FJ Bucephala alveola USA FJ FJ FJ FJ Physa gyrina USA FJ FJ FJ Aythya affinis USA FJ FJ FJ Aythya affinis USA FJ FJ FJ Aythya valisineria USA FJ FJ Aythya collaris USA FJ FJ FJ Mergus merganser USA FJ FJ FJ Physa gyrina USA FJ FJ FJ Mergus merganser USA FJ FJ FJ Aythya affinis USA FJ FJ FJ Clangula hyemalis USA FJ Trichobilharzia querquedulae Anas discors USA FJ FJ FJ FJ Anas discors USA FJ FJ FJ FJ Anas cyanoptera USA FJ FJ FJ FJ Anas clypeata USA FJ FJ FJ Anas clypeata USA FJ FJ Anas clypeata USA FJ FJ FJ Anas discors USA FJ FJ FJ Anas clypeata USA FJ FJ FJ Anas clypeata USA FJ FJ FJ Anas cyanoptera USA FJ FJ FJ Anas discors USA FJ FJ FJ Anas clypeata USA FJ FJ FJ Anas cyanoptera USA FJ FJ FJ Anas clypeata USA FJ FJ FJ Anas cyanoptera USA FJ Anas discors USA FJ Trichobilharzia sp. Radix peregra France EU Radix peregra France EU413964

4 D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) Table 2 (Continued ) Species Host Locality GenBank accession numbers D2 ITS2 ITS1 COX1 Trichobilharzia regenti Radix peregra Czech Republic AF AF Radix peregra Czech Republic AY AY Radix peregra France EU Anas clypeata Poland EF EF Aythya fuligula Poland EF EF Anas platyrhynchos Poland EF EF Anas platyrhynchos Poland EF EF Anas platyrhynchos Poland EF EF Aans clypeata Czech Republic EF EF Radix peregra France EU EU Radix peregra UK DQ Radix peregra UK NC Trichobilharzia szidati Lymnaea stagnalis Czech Republic AF AF AY Lymnaea stagnalis Czech Republic AY Lymnaea stagnalis Czech Republic AY AY Lymnaea stagnalis Germany AY AY Lymnaea stagnalis Netherlands AY AY Lymnaea stagnalis Czech Republic AY AY Lymnaea stagnalis Poland AY AY Lymnaea stagnalis Czech Republic AY AY Anas platyrhynchos Poland EF EF Anas platyrhynchos Czech Republic EF EF Lymnaea stagnalis Finland FJ FJ Lymnaea stagnalis Finland FJ FJ Lymnaea stagnalis France AY AY Lymnaea stagnalis France AY AY Lymnaea stagnalis USA FJ FJ FJ FJ Stagnicola elrodi USA FJ Trichobilharzia stagnicolae Stagnicola sp. USA FJ Mergus merganser USA FJ Stagnicola emarginata USA FJ Allobilharzia visceralis Cygnus columbianus USA EF Cygnus columbianus USA EF Trichobilharzia brantae Gyraulus parvus USA FJ Chen caerules USA FJ Dendritobilharzia pulverulenta Gallus gallus USA AY Chicken USA AF Gigantobilharzia huronensis Agelaius phoeniceus USA AY Agelaius phoeniceus USA AF Bilharziella polonica Anas platyrhynchos Ukraine AY Planorbis planorbis Bohemia AF T. querquedulae, two variations between T. querquedulae and T. physellae and a single variation between T. franki auricularia and T. querquedulae. For the total ITS and the COX1, only sequences corresponding to the species T. szidati, T. regenti, T. querquedulae, T. physellae and haplotypes T. franki auricularia and T. franki peregra were compared. Taking into account the considerable variability of these fields, the sequences were compared using the pairwise distance between each group, whose results appear in Table 5. These results show very small distances for these four groups in comparison with distances separating them to the haplotypes of T. regenti and T. szidati. The results obtained for these two domains confirm those obtained for the D2, with the separation of the haplotypes T. franki auricularia and T. franki peregra into two distinct clades Prevalence and molecular identification of snails Among snails of the Radix genus isolated, the prevalence of ocellate furcocercariae pertaining to the species T. franki varied Table 3 GenBank sequences of snails used for molecular analysis. Determination Accession number Origin Radix auricularia AJ Czech Republic, Austria, UK AJ Czech Republic AJ319630, AJ319631, AJ319632, AY France Radix peregra (=R. ovata; =R. balthica) AJ Iceland AJ319634, AJ319635, EU EU France Radix labiata AJ Turkey AJ Germany Radix lagotis AJ Czech Republic AJ Austria Radix ampla AJ Austria Radix sp. AJ Turkey Lymnaea stagnalis AJ Germany AJ319615, AJ France AJ Italy, France, Germany

5 1222 D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) Table 4 Dimensions and comparison of cercariae of Trichobilharzia franki, T. querquedulae, T. physellae and cercariae isolated on Radix auricularia and Radix peregra (in mm). T. franki T. franki auricularia T. franki peregra T. querquedulae T. physellae Reference Müller and Kimmig (1994) This paper This paper Brant and Loker (2009) McMullen and Beaver (1945) Brant and Loker (2009) Snail host R. auricularia R. auricularia R. peregra Physa gyrina P. parkeri, P. gyrina P. gyrina Number Total length Ant. end-eye Ant. end-acetabulum Head organ length Head organ width Acetabulum Tail stem length Tail stem width Furca length Furca width from 0.07 to 1.6%, according to the different sites. The phylogenetic trees based on ITS-2 sequences of snail isolates (Fig. 4), rooted on L. stagnalis, show four different clades, as reported by Bargues et al. (2001, 2003): the first is formed by R. labiata, the second by R. ampla and R. lagotis, the third by Radix sp. and R. auricularia (including six haplotypes from France with the following sequences RSFO1, RSFO4, RSBE1, RSBE2, RSBE13, RSBE67 and RAN77) and the fourth by R. balthica (=R. peregra, ovata) with three haplotypes (including isolates RAN 79 and RAN80 from France, and IS1F, IS2F, IS3F, IS4F, IS5F and IS6F, from Iceland). Although the haplotypes of ITS-2 from snails showed diversity, all snails emitting the furcocercariae we had found were included in the R. auricularia or the R. balthica (=peregra, ovata) branches which are well supported by the data. 4. Discussion As previously shown, morphological identification of snails of the genus Radix based on the external features (shell size and shape) is very complicated because of the continuous variability and plasticity of these characteristics depending on environmental conditions (Pfenninger et al., 2006). According to Bargues et al. (2001, 2003) and Pfenninger et al. (2006) the molecular approach might be effective for identifying species of this genus. In the future, identification of snails belonging to this genus needs to be confirmed by molecular analyses. In our study, ITS-2 was chosen for phylogenetic analysis. In the genus Radix Montfort, 1810, six valid species are recognized: R. labiata (Rossmaessler, 1835), R. ampla (Hartmann, 1821), R. lagotis (Schrank, 1803), Radix sp., R. auricularia (Linnaeus, 1758), R. balthica (Linnaeus, 1758) (=R. peregra (Müller, 1774), =R. ovata (Draparnaud, 1805)). In our study, the molluscs collected in France and Iceland belong to two species: R. balthica (= peregra, ovata) and R. auricularia. In the past, only three Trichobilharzia species were recognized and molecularly identified in Europe (Horák et al., 2002). On the basis of these species, furcocercariae isolated from L. stagnalis, R. auricularia and R. peregra were identified as pertaining to the species T. szidati (=ocellata), T. franki and T. regenti, respectively, by the nature of their intermediate host. This specific relationship between intermediate host and larval stages of bird schistosomes, formerly used as a criterion for differentiation of the cercariae at the specific level, was subsequently questioned by the discovery of several parasitic species in the same intermediate host (Jouet et al., 2008). This phenomenon could be explained by mechanisms of pressure of selection and adaptation of the parasite to a new host. Following this view, cercariae with similar molecular sequences and isolated from snails of different species (R. auricularia and R. peregra) were included in the same complex T. franki. Today, the recent morphological and molecular description of new species, particularly in North America (Brant and Loker, 2009), and the more precise characterization of the intermediate hosts by molecular biology have allowed us to question the haplotypes described in the past. In our study, we showed that the parasites isolated from R. auricularia and R. peregra seem not to belong to the single species T. franki, but to two different clades that we consider to represent distinct species. The molecular variations observed between these two haplotypes thus do not correspond to intraspecific variations within the same species, but rather to interspecific variations between two distinct species. According to the principle of priority, we need to take into account that the species T. franki was described by Müller and Kimmig (1994) from R. auricularia. It thus appears that the haplotypes isolated from R. auricularia in the past should still be considered as belonging to this species. However, it is important to note that, in the future, the identification of a snail host belonging to the Radix genus will have to be confirmed by molecular analysis, due to the difficulties encountered in morphological identification. This molecular identification of snails and cercariae allowed us to show the presence on a same site, the Annecy Lake, of the two species T. franki and T. franki peregra (haplotypes EAN77 and EAN79, EAN80). Concerning the recently described American species, molecular analyses show significant interspecific variations between T. querquedulae, T. physellae and T. franki peregra, thus excluding this haplotype from membership of these two species. This separation between the European and American species is confirmed by the differences concerning the life cycle of the parasites: European species use lymnaeid snails as intermediate hosts, whereas T. querquedulae and T. physellae use physid snails. Consequently, it seems important for us to revise the membership of the species T. franki for the haplotypes isolated from R. peregra. Our results showed that, molecularly, they differed from T. franki, but also from T. querquedulae and T. physellae, for different domains such as the D2 and ITS of the ribosomal DNA, but also for the COX1 domain of the mitochondrial DNA. This seems to argue in favour of their membership of a new species. However, it is impossible to define them as such on the simple basis of the morphology of the cercariae or their molecular analysis, or on the description of a specific host. Only a search for adults flukes corresponding to these haplotypes can finally clarify our assumption and thus lead to the description of a new species of Trichobilharzia, and, therefore, explain the complete life cycle of this schistosome, to avoid any confusion in the future.

6 [(Fig._1)TD$FIG] D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) Fig. 1. Phylogenetic tree based on the D2 domain of rdna of ocellate furcocercariae and sequences of bird schistosomes (genera Bilharziella, Gigantobilharzia, Dendritobilharzia and Trichobilharzia) constructed using the maximum likelihood method in Phyml online (HKY+G model of substitution). The scale shows the number of nucleotide substitutions per site between DNA sequences. Sequences of B. polonica were set as outgroup. The node support is given in neighbour-joining, maximum likelihood and maximum parsimony bootstraps.

7 [(Fig._2)TD$FIG] 1224 D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) Fig. 2. Phylogenetic tree based on the ITS of rdna of ocellate furcocercariae constructed using the maximum likelihood method in Phyml online (HKY+G model of substitution). The scale shows the number of nucleotide substitutions per site between DNA sequences. Sequences of Trichobilharzia szidati were set as outgroup. The node support is given in neighbour-joining and maximum likelihood bootstraps.

8 [(Fig._3)TD$FIG] D. Jouet et al. / Infection, Genetics and Evolution 10 (2010) Fig. 3. Phylogenetic tree based on the COX1 domain of mtdna of ocellate furcocercariae constructed using the maximum likelihood method in Phyml online (HKY+G model of substitution). The scale shows the number of nucleotide substitutions per site between DNA sequences. Sequences of Trichobilharzia szidati were set as outgroup. The node support is given in neighbour-joining and maximum likelihood bootstraps. Table 5 Estimation of pairwise distance between species T. franki haplotypes auricularia and peregra, T. physellae, T. querquedulae, T. regenti and T. szidati for the D2 and ITS region of the rdna and the COX1 domain of the mtdna. D2 ITS COX T. franki auricularia 2 T. franki peregra T. physellae T. querquedulae T. regenti T. szidati

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