Mathieu Badets

Ian Whittington

Fabrice Lalubin

Jean-François Allienne

Jean-Luc Maspimby

Sophie Bentz

Louis H Du Preez

Diane Barton

Hideo Hasegawa

Veena Tandon

Rangpenyubai Imkongwapang

Annemarie Ohler

Claude Combes

Olivier Verneau
email: verneau@univ-perp.fr.

Correlating Early Evolution of Parasitic Platyhelminths to Gondwana Breakup

Keywords: Amphibia, codivergence, coevolution, cophylogeny, Gondwana breakup, neobatrachia, platyhelminthes, polystomatidae, vicariant biogeography

Investigating patterns and processes of parasite diversification over ancient geological periods should involve comparisons of host and parasite phylogenies in a biogeographic context. It has been shown previously that the geographical distribution of host-specific parasites of sarcopterygians was guided, from Palaeozoic to Cainozoic times, mostly by evolution and diversification of their freshwater hosts. Here, we propose phylogenies of neobatrachian frogs and their specific parasites (Platyhelminthes, Monogenea) to investigate coevolutionary processes and historical biogeography of polystomes and further discuss all the possible assumptions that may account for the early evolution of these parasites. Phylogenetic analyses of concatenated rRNA nuclear genes (18S and partial 28S) supplemented by cophylogenetic and biogeographic vicariance analyses reveal four main parasite lineages that can be ascribed to centers of diversity, namely Australia, India, Africa, and South America. In addition, the relationships among these biogeographical monophyletic groups, substantiated by molecular dating, reflect sequential origins during the breakup of Gondwana. The Australian polystome lineage may have been isolated during the first stages of the breakup, whereas the Indian lineage would have arisen after the complete separation of western and eastern Gondwanan components. Next, polystomes would have codiverged with hyloid sensu stricto and ranoid frog lineages before the completion of South American and African plate separation. Ultimately, they would have undergone an extensive diversification in South America when their ancestral host families diversified. Therefore, the presence of polystome parasites in specific anuran host clades and in discrete geographic areas reveals the importance of biogeographic vicariance in diversification processes and supports the occurrence and radiation of amphibians over ancient and recent geological periods.

Investigating patterns and processes of parasite evolution over geological time involves comparison of host and parasite phylogenies to document the history of the association (Page 1993a). Because of the scarcity of the fossil record, especially within organisms like platyhelminths with poor instances of softbodied tissue preservation [START_REF] Poinar | A rhabdocoel turbellarian (Platyhelminthes, Typhloplanoida) in Baltic amber with a review of fossil and sub-fossil platyhelminths[END_REF], measuring the extent of congruence between host and parasite phylogenetic branching patterns provides valuable information to assess past geographical, climatic, and ecological features that may have shaped their evolution. In this context, the approach of cophylogeny mapping [START_REF] Paterson | Analytical approaches to measuring cospeciation of host and parasites: through a glass, darkly[END_REF]) evaluates all kinds of evolutionary events that produced speciation and/or diversification of parasites. Processes can be divided into four main categories: cospeciation (evolution by descent), host switching (evolution by colonization), duplication (intrahost divergence), and extinction (sorting events) [START_REF] Page | Trees within trees: phylogeny and historical associations[END_REF][START_REF] Johnson | When do parasites fail to speciate in response to host speciation?[END_REF][START_REF] Banks | Multi-host parasite species in cophylogenetic studies[END_REF]. On the other hand, host phylogeny supplies a powerful timeframe to inspect parasite evolution, even more so when the host fossil record is well calibrated. With the advance of cophylogenetic tools (reviewed in [START_REF] Stevens | Computational aspects of host-parasite phylogenies[END_REF], various host-parasite associations have been investigated in the past two decades to explore the evolutionary processes of parasite diversification, among them viruses, bacteria, protozoans, crustaceans, insects, platyhelminths, and their vertebrate or invertebrate hosts [START_REF] Paterson | Have chondracanthid copepods cospeciated with their teleost hosts?[END_REF][START_REF] Clark | Cospeciation between bacterial endosymbionts (Buchnera) and a recent radiation of aphids (Uroleucon) and pitfalls of testing for phylogenetic congruence[END_REF][START_REF] Ricklefs | Diversification and host switching in avian malaria parasites[END_REF][START_REF] Jackson | A cophylogenetic perspective of RNA-virus evolution[END_REF][START_REF] Huyse | Comparing host and parasite phylogenies: Gyrodactylus flatworms jumping from goby to goby[END_REF]Volckaert 2005, Banks et al. 2006;[START_REF] Takiya | Cocladogenesis spanning three phyla: leafhoppers (Insecta: Hemiptera: Cicadellidae) and their dual bacterial symbionts[END_REF][START_REF] Hughes | Multiple cophylogenetic analyses reveal frequent cospeciation between pelecaniform birds and Pectinopygus lice[END_REF][START_REF] Light | Cophylogeny and disparate rates of evolution in sympatric lineages of chewing lice on pocket gophers[END_REF]. Within the phylum Platyhelminthes, for instance, only three genera of the class Monogenea, Lamellodiscus (see [START_REF] Desdevises | Coevolution between Lamellodiscus (Monogenea: Diplectanidae) and Sparidae (Teleostei): the study of a complex host-parasite system[END_REF], Dactylogyrus (see [START_REF] Simkova | Molecular phylogeny of congeneric monogenean parasites (Dactylogyrus): a case of intrahost speciation[END_REF], and Gyrodactylus (see [START_REF] Huyse | Comparing host and parasite phylogenies: Gyrodactylus flatworms jumping from goby to goby[END_REF], have been investigated. Results revealed numerous occurrences of duplication and host-switching events and very few events of cospeciation shaping the evolution of these strictly teleostean fish parasites. Cophylogenetic studies thus constitute a fundamental tool to determine how parasites have evolved and radiated during host evolution. On the other hand, parasites may provide additional data that can, in some cases, be very helpful to investigate the evolutionary ecology of the host [START_REF] Whiteman | Using parasites to infer host population history: a new rationale for parasite conservation[END_REF][START_REF] Nieberding | Parasites: proxies for host genealogy and ecology?[END_REF]. This is the case, for instance, when two divergent and nonsympatric host species are infected by two sister parasite species, which indicates that donor and receiving host lineages must have occurred in the same area at some point in time.

Anurans (frogs and toads) form the largest group of extant amphibians [START_REF] Frost | The amphibian tree of life[END_REF]. Although studied extensively during the past decade using molecular approaches (e.g., [START_REF] Hay | Phylogenetic relationships of amphibian families inferred from DNA sequences of mitochondrial 12S and ribosomal RNA genes[END_REF][START_REF] Ruvinsky | Phylogenetic relationships among bufonoid frogs (Anura: Neobatrachia) inferred from mitochondrial DNA sequences[END_REF][START_REF] Feller | Molecular evidence for the early history of living amphibians[END_REF][START_REF] Bossuyt | Amphibians as indicators of early tertiary "Out-of-India" dispersal of vertebrates[END_REF][START_REF] Biju | New frog family from India reveals an ancient biogeographical link with the Seychelles[END_REF][START_REF] Vences | Multiple overseas dispersal in amphibians[END_REF][START_REF] Hoegg | Phylogeny and comparative substitution rates of frogs inferred from sequences of three nuclear genes[END_REF][START_REF] Mauro | Initial diversification of living amphibians predated the breakup of Pangaea[END_REF]; [START_REF] Van Der Meijden | A previously unrecognized radiation of ranid frogs in Southern Africa revealed by nuclear and mitochondrial DNA sequences[END_REF][START_REF] Bossuyt | Phylogeny and biogeography of a cosmopolitan frog radiation: late Cretaceous diversification resulted in continent-scale endemism in the family Ranidae[END_REF], numerous issues are still in question, including phylogenetic relationships of the basal frog lineages within the Neobatrachia (see [START_REF] Biju | New frog family from India reveals an ancient biogeographical link with the Seychelles[END_REF][START_REF] Mauro | Initial diversification of living amphibians predated the breakup of Pangaea[END_REF][START_REF] Frost | The amphibian tree of life[END_REF][START_REF] Roelants | Global patterns of diversification in the history of modern amphibians[END_REF][START_REF] Wiens | Global patterns of diversification and species richness in amphibians[END_REF]. At this stage, the most relevant phylogenetic studies that address frogs in major families of the Neobatrachia reveal five main lineages whose biogeographic and phylogenetic patterns may reflect disintegration of the Gondwana supercontinent in the Mesozoic era (see [START_REF] Biju | New frog family from India reveals an ancient biogeographical link with the Seychelles[END_REF]): (i) the Hyloidea sensu stricto with the Bufonidae, Hylidae, Leptodactylidae, and South American families; (ii) the Ranoidea with the Ranidae, Rhacophoridae, and major African and Madagascan families; (iii) the Australian hyloids with the Myobatrachidae and two related taxa from southernmost South America (i.e., Telmatobufo venustus and Caudiverbera caudiverbera); (iv) the South African Heleophrynidae; and (v) a clade associating the Sooglossidae and Nasikabatrachidae, which are restricted to the Seychelles and India, respectively. Such a correlation between plate tectonics and neobatrachian frog relationships is of particular interest, not only for knowledge of amphibian evolution but also for subsequent dating of major speciation events within the Neobatrachia. However, in the absence of high resolution within basal groups of the Neobatrachia, their phylogenetic relationships are still viewed as a polytomy (San [START_REF] Mauro | Initial diversification of living amphibians predated the breakup of Pangaea[END_REF]. This may be explained by the relatively fast speciation processes that led to the major frog lineages in the Middle/Late Jurassic and Early Cretaceous periods [START_REF] Biju | New frog family from India reveals an ancient biogeographical link with the Seychelles[END_REF].

The Monogenea (Platyhelminthes) includes tens of thousands of parasite species that infest primarily chondrichthyan and actinopterygian fish but also to a lesser extent sarcopterygians, such as the Australian lungfish, amphibians, freshwater turtles, and the African hippopotamus. Among Monogenea, the Polystomatidae is the most diverse family with about 150 species classified in 23 genera of unequal diversity, Polystoma of anurans being the most diversified genus. Like all other monogeneans, polystomatids display a direct life cycle, which facilitates parasite transmission from host to host in aquatic environments. They are generally host and site specific [START_REF] Murith | Identite des larves de polystomes (Monogenea) para-´ sites du tetard deˆ Dicroglossus occipitalis (Gunther) en Cote-d'Ivoire[END_REF][START_REF] Preez | Supporting evidence of host-specificity among southern African polystomes (Polystomatidae: Monogenea)[END_REF][START_REF] Tinsley | Platyhelminth parasite reproduction: some general principles derived from monogenans[END_REF]). In fact, although they can be found either in the urinary bladder, the pharyngeal cavity, or the conjunctival sacs of their chelonian hosts, polystomes of amphibians are mainly found in the urinary bladder. A few reports have mentioned the presence of more than one polystome species within the same amphibian host species [START_REF] Bourgat | Polystoma lamottei n. sp. et P. aeschlimanni n. sp., deux polystomes (Monogenes) de la m` eme espece d'amphibien: Ptychadena pumilio (Boulenger, 1920)[END_REF][START_REF] Preez | Syntopic occurrence of new species of Polystoma and Metapolystoma (Monogenea: Polystomatidae) in Ptychadena porosissima in South Africa[END_REF], but in most cases, a single parasite species is found per host species. Finally, they show a large diversity of reproductive strategies that range from ovoviviparity to developmental plasticity depending on host ecology [START_REF] Kearn | Evolutionary expansion of the Monogenea[END_REF][START_REF] Rohde | The origins of parasitism in the Platyhelminthes[END_REF][START_REF] Whittington | Reproduction and host-location among the parasitic Platyhelminthes[END_REF][START_REF] Cribb | Diversity in Monogenea and Digenea: does lifestyle matter?[END_REF][START_REF] Tinsley | Platyhelminth parasite reproduction: some general principles derived from monogenans[END_REF][START_REF] Badets | Origin and evolution of alternative developmental strategies in amphibious sarcopterygian parasites (Platyhelminthes, Monogenea, Polystomatidae)[END_REF].

It has been established that phylogenetic relationships of parasites of the Polystomatidae are linked with key events in host evolution, such as the vertebrates' transition to land, the lissamphibians' origin, and the freshwater turtles' diversification [START_REF] Verneau | A view of early vertebrate evolution inferred from the phylogeny of polystome parasites (Monogenea: Polystomatidae)[END_REF]. Subsequently, it was shown that the present day geographical distribution of Polystoma of anurans was guided mostly by past dispersals affecting their hyloid hosts during the Tertiary period [START_REF] Bentz | Historical biogeography of amphibian parasites, genus Polystoma (Monogenea: Polystomatidae)[END_REF]).

Given all these findings and the worldwide distribution of polystomes of the Neobatrachia, we aimed to investigate whether evolution of this unique group of parasites may be correlated with the early evolution of their amphibian host species as well as to plate tectonics in the Early Jurassic. The parasite phylogeny was generated from analysis of nuclear 18S and partial 28S rRNA genes, whereas the host phylogeny was inferred from analysis of nuclear rhodopsin and mitochondrial 12S and 16S rRNA genes because these markers were shown to be the most suitable for frogs (e.g., [START_REF] Vences | Multiple overseas dispersal in amphibians[END_REF][START_REF] Hoegg | Phylogeny and comparative substitution rates of frogs inferred from sequences of three nuclear genes[END_REF][START_REF] Van Der Meijden | A previously unrecognized radiation of ranid frogs in Southern Africa revealed by nuclear and mitochondrial DNA sequences[END_REF]. Cophylogenetic and biogeographic vicariance analyses were conducted to investigate evolutionary processes of polystome diversification, which in turn may provide valuable insights about host evolutionary ecology.

MATERIALS AND METHODS

Parasite Sampling

Three main reasons explain our relatively small parasite sampling: (i) amphibians are declining more rapidly than any other vertebrate group, which complicated the ability to secure collection permits for frogs that are threatened with extinction and therefore their parasites; (ii) in contrast to the high diversity of frog species (more than 5000 described species, see [START_REF] Frost | The amphibian tree of life[END_REF], very few host anuran species (i.e., no more than 100) are currently known to be infected by polystomes (see [START_REF] Verneau | Origine et evolution des monog´ enes Polystomatidae, parasites d'amphibiens et de cheloniens d'eau douce[END_REF]; and (iii) prevalence of infected hosts is usually very low, rarely >20%. Thus, our molecular data set (Table 1) integrated 20 polystome species from hyloid and ranoid host species, sampled from all continental regions including India, and three other polystome species infecting pelobatid and pipid frogs from the basal archaeobatrachian anurans for outgroup comparisons. Sampling also incorporated the polystome species from the Australian lungfish (i.e., Concinnocotyla australensis), the most basal species within the Polystomatidae and two monogenean parasite species of the Infrasubclass Oligonchoinea infecting teleost fishes (see [START_REF] Boeger | Phylogenetic relationships of the Monogenoidea[END_REF][START_REF] Verneau | A view of early vertebrate evolution inferred from the phylogeny of polystome parasites (Monogenea: Polystomatidae)[END_REF], all of which were used in a global phylogenetic analysis for molecular dating.

Host Sampling

Sequences of all frog species infected by polystomes investigated in the present study were recovered from GenBank (Table 1). We first selected complete or partial 12S and 16S mitochondrial genes that were sequenced for almost all the infected frog species. We also obtained data for part of exon 1 of the nuclear rhodopsin gene that was sequenced for twothirds of the host taxa. Because several host species had not been sequenced for all three genes, some species were replaced by phylogenetically closely related species according to the classification of [START_REF] Frost | The amphibian tree of life[END_REF] (Table 1). Hence, seven frog species were substituted in the rhodopsin and two in the (12S + 16S) data sets to produce the most comprehensive data sets. This strategy of exchanging host species with close relatives had no impact on the resultant scenarios because phylogenies inferred from both mitochondrial and nuclear data sets were congruent and because we only examined evolutionary processes at the earliest stages of evolution in this host-parasite association. Thus, subsequent cophylogenetic analyses conducted with TreeMap, version 2.02β [START_REF] Charleston | TreeMap 2.0β. A Macintosh R program for the analysis of how dependent phylogenies are related, by cophylogeny mapping[END_REF], were simply done with the host phylogeny inferred from the nuclear rhodopsin data set.

Molecular Experiments

All methods used for DNA extraction, amplification, and sequencing are described elsewhere [START_REF] Bentz | Historical biogeography of amphibian parasites, genus Polystoma (Monogenea: Polystomatidae)[END_REF]). The complete 18S rRNA gene was amplified in one round with primers F18, 5'-ACCTGGTTGATCCT GCCAGTAG-3' and IR5, 5'-TACGGAAACCTTGTTAC GAC-3', yielding a polymerase chain reaction (PCR) product of about 2 kb that was subsequently sequenced with the same primers and also the following internal primers: 18F1, 5'-GTTGTGTCGTGTTGACTCTG-3'; 18F2, 5'-GGAGGGCAAGTCTGGTGCCAG-3'; 18F3, 5'-GGACGGCATGTTTACTTTGA-3'; 18RA, 5'-GCCCGCGGGGACGATATGTAC-3'; 18RB, 5'-TGCTTTGAGC-ACTCAAATTT-3'; 18RC, 5'-TACGAGCTTTTTAACTGCAG-3'; and 18RG, 5'-CTCTCTTAACCATTACTTCGG3'. The partial 28S rRNA gene corresponding to the 5' terminal end was amplified with primers LSU5', 5'TAGGTCGACCCGCTGAAYTTAAGCA-3' and LSU3', 5'-TAGAAGCTTCCTGAGGGAAACTTCGG-3' [START_REF] Snyder | Evolutionary relationships among the Schistosomatidae (Platyhelminthes: Digenea) and an Asian origin for Schistosoma[END_REF], yielding a PCR product of about 1.4 kb that was subsequently sequenced with the same primers and also the following internal primers: IF13, 5'AGCAAACAAGTACCGTGAGGG-3'; IF15, 5'-GTCTGT GGCGTAGTGGTAGAC-3'; IR13, 5'-GTCGTGGCTTAC ACCCTGAGG-3'; and IR14, 5'-CATGTTAAACTCCTT GGTCCG-3'.

Phylogenetic Analyses

Parasite tree reconstructions.-The secondary structure of the small subunit (SSU) ribosomal RNA of Calicophoron calicophorum (L06566) was first recovered from the European Ribosomal RNA database (http://www.psb.ugent. be/rRNA/) and aligned with the Polystoma gallieni sequence using DCSE v2.6 software [START_REF] Rijk | DCSE, an interactive tool for sequence alignment and secondary structure research[END_REF]. Although most stems and loops in P. gallieni were inferred from conserved aligned regions, a few in hyper variable and insertion regions were determined from a search of common motifs in the most distant polystome species with the aid of Mfold software, using default parameters (http://www.bioinfo.rpi.edu/) [START_REF] Zuker | Mfold web server for nucleic acid folding and hybridization prediction[END_REF]. This concerned helices E10 1, 11, 12, E23 1, E23 2, E23 5, E23 6, E23 7, 43, and 49 (see [START_REF] Van De Peer | Database on the structure of small subunit ribosomal RNA[END_REF] for the nomenclature of RNA secondary structures). The 18S sequences of all other parasite species were aligned subsequently according to the structural constraints of the P. gallieni sequence. The large subunit (LSU) ribosomal RNA structure of P. gallieni was inferred following the same procedure as described above with regard to the RNA secondary structure of Caenorhabitis elegans (X03680) and Dugesia tigrina (U78718). The 28S sequences of all other parasite species were thus aligned according to the structural constraints of the P. gallieni sequence. The C and D5 regions were not constrained due to the high level of divergence within polystomes and the lack of common motifs after Mfold reconstructions. They were therefore treated as loop regions in phylogenetic analyses.

The incongruence between 18S and 28S data sets was first measured by the incongruence length difference (ILD) test implemented in PAUP* 4.0b9 [START_REF] Swofford | PAUP*: phylogenetic analysis using parsimony (* and other methods)[END_REF]. Because no conflicting signal was observed (P = 0.515; 1000 replicates), genes were combined for subsequent phylogenetic analyses. The combined data set, including 4160 characters from both nuclear rRNA genes of the 23 anuran polystome species, was partitioned into stem and loop regions for the Bayesian analysis. The Xstem software [START_REF] Telford | Consideration of RNA secondary structure significantly improves likelihoodbased estimates of phylogeny: examples from the Bilateria[END_REF]) was used to extract the RNA secondary structure information of the DCSE alignment and to convert it into a nexus file format. A GTR + I + Γ model was selected for the loop regions by the Akaike Information Criterion (AIC) implemented in the program Modeltest 3.06 [START_REF] Posada | MODELTEST: testing the model of DNA substitution[END_REF]. A doublet model was preferred for the stem partitions as recommended by [START_REF] Telford | Consideration of RNA secondary structure significantly improves likelihoodbased estimates of phylogeny: examples from the Bilateria[END_REF]. Bayesian analysis was conducted using the software MrBayes 3.04b [START_REF] Huelsenbeck | MRBAYES: Bayesian inference of phylogenetic trees[END_REF], with four chains running for million generations, sampling each 100 cycles. Bayesian posterior probabilities were computed after removing the first 1000 trees as the burn-in phase. Bayesian Inferences (BI) were run three times independently to assess for convergence, using the Tracer software available at http://tree.bio.ed.ac.uk/software. The maximum-likelihood (ML) analysis was performed on 3125 characters without partitioning and with gaps excluded. A search for the best ML tree was done using the GTR + I + Γ model selected by the AIC in Modeltest and following a heuristic procedure under the Tree Bisection and reconnection (TBR) branch swapping option with PAUP* 4.0b9. ML bootstrap support values were calculated with the same model of sequence evolution under the Nearest Neighbor Interchange (NNI) branch swapping option using 1000 replicates. The maximum parsimony (MP) analysis was performed with PAUP* 4.0b9 [START_REF] Swofford | PAUP*: phylogenetic analysis using parsimony (* and other methods)[END_REF] following a branch-and-bound search on 593 equally weighted informative characters, with gaps considered as missing data. Percentage support values were calculated following heuristic search and stepwise addition with 1000 bootstrap replicates. Finally, a global phylogenetic analysis was conducted on all polystomatid and fish monogenean species to produce the topology used as support for molecular calibrations (26 taxa).

Host tree reconstructions.-Rhodopsin sequences were aligned with Clustal W [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequencealignments through sequence weighting, position specific gap penalties and weight matrix choice[END_REF], which is implemented in MEGA version 4 [START_REF] Tamura | MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0[END_REF]. Alignment revealed 282 characters, of which 89 were parsimony informative (20 taxa). Because rhodopsin is a protein-coding gene, the complete alignment was partitioned for Bayesian analysis and ML according to codon Positions 1, 2, or 3 that may evolve at different evolutionary rates. The most appropriate models of evolution were selected independently for each position categories using the AIC in Modeltest. BI was obtained using a GTR model for Positions 1 and 2 allowing rate variation across sites, and a GTR + I + Γ model for Position 3, after assessing convergence with the aid of the software Tracer. Bayesian posterior probabilities were thus computed after removing the first 1000 trees as the burn-in phase. ML analysis was conducted with a GTR model allowing rate variation across Partitions 1, 2, and 3. The best ML tree was recovered following a heuristic search under the TBR branch swapping option, and ML bootstrap support values were calculated under the NNI branch swapping option, using 1000 bootstrap replicates. The MP analysis was performed following a branch-and-bound search on all equally weighted informative characters and the percentage support values were calculated following the same procedure with 1000 bootstrap replicates. The procedure of host tree reconstruction from mitochondrial markers is available as Supplementary Methods (see Supplementary material available from http://www.sysbio.oxfordjournals.org). Assuming that actual and substituted host species may be interchanged, the ILD test was also performed between rhodopsin and (12S + 16S) data sets. Once phylogenetic incongruence was excluded (P = 0.87; 1000 replicates), a Bayesian analysis was conducted on the complete combined data set using the different models of evolution as selected for nuclear (rhodopsin) and mitochondrial (12S and 16S) partitions.

Cophylogeny

The mathematical procedure developed by [START_REF] Charleston | Jungles: a new solution to the host/parasite phylogeny reconciliation problem[END_REF], which is implemented in the algorithm of TreeMap, version 2.02β [START_REF] Charleston | TreeMap 2.0β. A Macintosh R program for the analysis of how dependent phylogenies are related, by cophylogeny mapping[END_REF], explores all the possibilities of mapping an associate tree onto a host tree, by minimizing the number of noncodivergence events (i.e., duplication, horizontal transfer, and sorting events). A statistical test is implemented to test whether the most parsimonious reconstructions are significantly better than reconstructions inferred from 1000 randomized associate phylogenies [START_REF] Charleston | Preferential host switching by primate lentiviruses can account for phylogenetic similarity with the primate phylogeny[END_REF]. Regarding the phylogenetic congruence between rhodopsin and (12S + 16S) host trees, the cophylogenetic analysis was conducted only from comparisons of nuclear host and parasite phylogenies. Host and parasite trees inferred from ML analyses were thus reconciled, after having excluded the most divergent taxa in both phylogenetic trees, namely archaeobatrachian hosts and their parasites that were used only for tree rooting. Due to uncertainties in nuclear and mitochondrial host trees, we computed separate TreeMap analyses on nine alternative topologies within host assemblage following invertion of the three main groups within the Hyloidea and Ranoidea lineages, respectively. Consequently, the cophylogenetic structure was evaluated for each of the nine combinations without the use of the branch length option. Costs for the different processes were settled by setting default at zero for cospeciation and at one for all three other types of events. Preliminary analyses on this host-parasite association revealed that host-switching events were negatively correlated with the number of extinction and duplication events (data not shown). Taking biological features of polystomes into consideration (i.e., a single polystome species per host species in most cases), assuming duplication therefore seems less likely than host switching. The search of the most parsimonious reconstructions was thus performed constraining the maximum number of host-switching events to four due to computing limitations.

The ParaFit approach [START_REF] Legendre | A statistical test for hostparasite coevolution[END_REF] was also used to assess coevolution between hosts and their parasites. This method which is implemented in Copycat [START_REF] Meier-Kolthoff | COPYCAT: cophylogenetic analysis tool[END_REF]) tests the significance of a global hypothesis of coevolution within host-parasite assemblages from the patristic distances among host and parasite species. This test was computed over three host-parasite combinations, the first resulting from distance comparisons of nuclear ML host and Bayesian parasite trees (17 taxa per tree) and the second and the third resulting from distance comparisons of mitochondrial ML host and nuclear Bayesian parasite trees, with 17 and 19 taxa per tree, respectively.

Vicariant Biogeography

To investigate the historical biogeography of neobatrachian polystomes, and to more precisely explore links between the parasite phylogenetic tree and the Earth's history at the stage of Gondwana breakup, two kinds of procedures, a posteriori and a priori methods, were pursued.

The first approach, DIVA for Dispersal-Vicariance Analysis [START_REF] Ronquist | Dispersal-vicariance analysis: a new approach to the quantification of historical biogeography[END_REF], is based on a posteriori assumptions. It minimizes dispersal and extinction events and allows multiple and reticulate relationships among areas [START_REF] Ronquist | Dispersal-vicariance analysis: a new approach to the quantification of historical biogeography[END_REF]). Thus, ancestral distributions of organisms are hypothesized without constraining geographical area relationships a priori. In that context, costs are assigned to each kind of speciation event and a three-dimensional cost matrix is built to reconstruct the most parsimonious ancestral distributions of taxa [START_REF] Ronquist | Dispersal-vicariance analysis: a new approach to the quantification of historical biogeography[END_REF]. As different equally parsimonious distributions may be inferred for specific nodes within the tree, the optimal ancestral distributions must be considered a posteriori, according to the current scenarios of plate tectonics (see for instance [START_REF] Bossuyt | Phylogeny and biogeography of a cosmopolitan frog radiation: late Cretaceous diversification resulted in continent-scale endemism in the family Ranidae[END_REF]. In order to reduce hypotheses of ancestral distributions within the polystome tree, we considered the lineage including European and African Polystoma species to have originated in Eurasia [START_REF] Bentz | Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods[END_REF]. Similarly, we considered the lineage including Polystoma species from Central and North America to be of South American origin [START_REF] Bentz | Historical biogeography of amphibian parasites, genus Polystoma (Monogenea: Polystomatidae)[END_REF]. DIVA version 1.1 [START_REF] Ronquist | DIVA 1.1. User's manual[END_REF] was run on a Macintosh and costs were set at zero for allopatric (i.e., biogeographic vicariance) and sympatric speciation and at one for dispersal and extinction.

The second approach we used to reconstruct ancestral distributions within the polystome tree was TreeMap, version 2.02β [START_REF] Charleston | TreeMap 2.0β. A Macintosh R program for the analysis of how dependent phylogenies are related, by cophylogeny mapping[END_REF]. All its principles are the same as those described above for cophylogenetic studies, but host phylogeny is substituted by an area cladogram. In that context, it is based on a priori assumptions of plate tectonic relationships. The most parsimonious reconstructions are then inferred and likewise, one statistical test based on 1000 randomizations of the associate phylogeny gives the significance for the results. For our analyses, we considered for the same reasons as mentioned above the clade of European and African Polystoma species of Eurasian origin [START_REF] Bentz | Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods[END_REF]) and the group associating Central and North American Polystoma species of South American origin [START_REF] Bentz | Historical biogeography of amphibian parasites, genus Polystoma (Monogenea: Polystomatidae)[END_REF]. Costs were set at zero by default for biogeographic vicariance (similar to cospeciation) and at one for sympatric speciation (similar to duplication), dispersal (similar to host switching), and extinction. Contrary to cophylogenetic analysis, no constraint was fixed for the number of dispersal events. The polystome tree was then reconciled to two successive area cladograms, according to the current hypotheses of plate tectonics. The first area cladogram considers a sister relationship between South American and African plates and Indian and Australian plates. Indeed, it is well accepted that the landmasses of Gondwana began to separate in the Early Jurassic, approximately 180-160 Ma, leading to western (South America and Africa) and eastern (India, Madagascar, Seychelles, Antarctica, and Australia) Gondwanan components (Storey 1995, Chatterjee and[START_REF] Chatterjee | The breakup of Gondwana and the evolution and biogeography of the Indian plate[END_REF][START_REF] Lawver | A tight fit-early Mesozoic Gondwana, a plate reconstruction perspective[END_REF][START_REF] Briggs | The biogeographic and tectonic history of India[END_REF]. During subsequent stages of rifting in the Early Cretaceous, the block uniting the Seychelles, Madagascar, and India separated from Antarctica-Australia about 130 Ma [START_REF] Briggs | The biogeographic and tectonic history of India[END_REF], and South America may have diverged from Africa at about the same time [START_REF] Storey | The role of mantle plumes in continental breakup: case histories from Gondwanaland[END_REF][START_REF] Lawver | A tight fit-early Mesozoic Gondwana, a plate reconstruction perspective[END_REF][START_REF] Macdonald | Mesozoic break-up of SW Gondwana: implications for regional hydrocarbon potential of the southern South Atlantic[END_REF]. The second area cladogram considers the Australian plate as the most basal, the Indian plate being more closely related to the South American and African continents. Indeed, though the first stages of Gondwana breakup were initiated following seafloor spreading in the Somali, Mozambique, and Weddell Sea basins [START_REF] Storey | The role of mantle plumes in continental breakup: case histories from Gondwanaland[END_REF], it has been proposed from analysis of the seismic structure and sediment distribution in the western Weddell Sea that this region was the site of the initial breakup of Gondwana [START_REF] Rogenhagen | The sedimentary structure in the western Weddell Sea[END_REF]. Afterwards, the Australian plate could have first diverged in its austral part from the South American and African plates at a time when India was still joined to the western part of Gondwana and Australia. Because the area cladogram (5 "taxa") is smaller than the parasite phylogeny (20 taxa), randomizing tests may seriously increase the significance of outputs and lead to subjective conclusions. Hence, to test for the reliability of results, analyses were also conducted with both area cladograms and a phylogenetic representation of a restricted subsample of parasite species, which was deduced from the parasite ML tree. The selection of eight polystome species was made according to their phylogenetic position within the phylogenetic tree (i.e., at least one species was selected from the main lineages and sublineages) and their occurence in specific biogeographic areas.

Molecular Calibrations

Because the constancy of the molecular clock was rejected within the Polystomatidae (see [START_REF] Verneau | A view of early vertebrate evolution inferred from the phylogeny of polystome parasites (Monogenea: Polystomatidae)[END_REF], we used estbranches and multidivtime implemented in the multidistribute package [START_REF] Thorne | Estimating the rate of evolution of the rate of molecular evolution[END_REF][START_REF] Thorne | Divergence time and evolutionary rate estimation with multilocus data[END_REF] to infer divergence time estimates within polystomes of the Neobatrachia. The instructions described in the manual of [START_REF] Rutschmann | Bayesian molecular dating using PAML/multidivtime. A step-by-step manual[END_REF] were followed to perform the molecular dating. The topology used as a constraint for molecular calibrations is a global phylogenetic tree including all polystomatid species, fish monogenean species being used as an outgroup (for a total of 26 taxa). It was inferred from a Bayesian analysis after selecting a doublet model for the stem regions and, by the AIC, a GTR + I + Γ model for the loop regions. ML parameters were estimated for that topology under the F84 + Γ model with BASEML [START_REF] Yang | PAML: a program package for phylogenetic analysis by maximum likelihood[END_REF]. ML branch lengths and their variance-covariance matrix were estimated with the estbranches program [START_REF] Thorne | Estimating the rate of evolution of the rate of molecular evolution[END_REF]. Rates of molecular evolution and divergence time estimations with their 95% confidence intervals were calculated with multidivtime using the MCMC approach [START_REF] Thorne | Estimating the rate of evolution of the rate of molecular evolution[END_REF][START_REF] Kishino | Performance of a divergence time estimation method under a probabilistic model of rate evolution[END_REF]. Before running the analyses, the following parameters were fixed regarding the origin of the Polystomatidae and according to authors' recommendations: (i) The mean of the prior distribution for the time separating the ingroup root from the present largest window of time that separates the Actinopterygii(rttm) was set to 437 Ma (sd = 12). It corresponds to the and Sarcopterygii [START_REF] Janvier | Forerunners of four legs[END_REF][START_REF] Ahlberg | Something fishy in the family tree[END_REF]) and, consequently, their respective monogenean parasites under hypothesis of coevolution [START_REF] Verneau | A view of early vertebrate evolution inferred from the phylogeny of polystome parasites (Monogenea: Polystomatidae)[END_REF]. (ii) The mean rate of molecular evolution (rtrate) was estimated from the distance tree computed with estbranches and set at 0.0004819. Finally, (iii) brownmean and brownsd were set at 0.0048 following advice in [START_REF] Rutschmann | Bayesian molecular dating using PAML/multidivtime. A step-by-step manual[END_REF] and big time, which corresponds to the maximum expected origin of the ingroup, was set to 450 Ma.

Three calibration procedures depending on selected calibration points were then used to infer divergence time estimates. Procedure 1 attempts to fix two calibration points onto the global phylogenetic tree. The separation between C. australensis and the polystomes of tetrapods at about 410-415 Ma [START_REF] Verneau | A view of early vertebrate evolution inferred from the phylogeny of polystome parasites (Monogenea: Polystomatidae)[END_REF] corresponds to the divergence time between Dipnoi and tetrapods [START_REF] Janvier | Forerunners of four legs[END_REF][START_REF] Zhu | A primitive sarcopterygian fish with an eyestalk[END_REF]. The colonization of Africa by Polystoma at about 5-25 Ma corresponds to the largest window of time for vertebrate dispersal between Southern Europe and North Africa during the Miocene [START_REF] Rage | Gondwana, Tethys, and terrestrial vertebrates during the Mesozoic and Cainozoic[END_REF]. Indeed, though [START_REF] Bentz | Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods[END_REF] assumed that ancestors of Pelobates frogs could have been potential hosts that carried polystomes from Eurasia to Africa about 5 Ma, via a terrestrial dispersal route on the western extremity of the Mediterranean Basin by the Upper Miocene (see [START_REF] Rage | Gondwana, Tethys, and terrestrial vertebrates during the Mesozoic and Cainozoic[END_REF], another temporal scheme could have been considered because a dispersal route for vertebrates was also invoked between Eurasia and Africa in the eastern Mediterranean area by the Lower Miocene, about 25 Ma [START_REF] Rage | Gondwana, Tethys, and terrestrial vertebrates during the Mesozoic and Cainozoic[END_REF]. Procedures 2 and 3 reestimate these divergence times with one single calibration point, either the separation between C. australensis and the polystomes of tetrapods at about 410-415 Ma or the colonization of Africa by Polystoma at about 5-25 Ma. This allows the detection of whether multidivtime is wrongly influenced by one of the two calibration points.

RESULTS

Alignment of Nuclear Ribosomal Genes

All monogenean species used in this study are listed in Table 1, and new sequences of the complete 18S and partial 28S rRNA genes are deposited in GenBank under accession numbers AM157183 to AM157222. Secondary structures of the complete SSU and partial LSU ribosomal sequences of P. gallieni are shown as Supplementary Appendices 1 and 2, respectively. These models served as a template to shape the secondary structure of all other polystomatids and fish monogeneans. This allowed us to refine an alignment according to structural constraints (i.e., loops and stems).

Polystomatid Phylogeny

The MP analysis, which was conducted on the 23 taxa, yields a single parsimonious tree (tree length = 1135 ; consistency index = 0.67), this being identical to the Bayesian consensus and ML trees (ML tree score = 13266.3028). For this reason, we only present the ML tree with bootstrap values inferred from ML and MP analyses and Bayesian posterior probabilities (Fig. 1). Phylogenetic relationships of polystomes reveal four main lineages, whose distributions can be ascribed to restricted geographical areas. The first group associates two species, Parapolystoma bulliense and Diplorchis ranae, found, respectively, on hylid frogs (Pelodryadinae) of Australia (i.e., Litoria gracilenta) and ranid frogs of Japan (i.e., Rana rugosa). The second lineage encompasses four species of Polystoma. All are recovered from Rhacophorus species in India, China, and Japan. The third clade is composed of two species of Eupolystoma sampled from African bufonids and the last lineage unites Wetapolystoma almae and most of the Polystoma species. These parasites, which are found from both hyloid sensu stricto and ranoid frog lineages, are distributed worldwide except in Australia and Madagascar. For convenience, we will call the monophyletic group that includes W. almae and all species of Polystoma infecting non-Rhacophorus hosts, the Polystoma sensu stricto lineage. Results also reveal that American polystomes do not form a clade, the strict Central and North American polystome group being the most basal taxon among the Polystoma sensu stricto lineage. Concerning the phylogenetic relationships between the four main parasite lineages, the "Parapolystoma/Diplorchis" lineage appears most basal among neobatrachian polystomes and the lineage including Indian and South-eastern Asian rhacophorid polystomes is related to the stem branch of the closely related Eupolystoma and Polystoma sensu stricto lineages (Fig. 1). Finally, the global phylogeny that includes 26 taxa confirms the monophyly of the Polystomatidae, with C. australensis being the most basal taxon within polystomes (results not shown).

Frog Phylogeny

Because no incongruence is revealed between rhodopsin and (12S + 16S) data sets, we only present the ML tree inferred from analysis of the rhodopsin alignment (Fig. 2), with Bayesian posterior probablities and branch support values provided from ML and MP analyses. Posterior probabilities obtained from the Bayesian analysis of the complete combined data set (12S, 16S, and rhodopsin) are also indicated. Nuclear and mitochondrial host trees are very similar to each other and to the most recent phylogenetic hypotheses of amphibian families and subfamilies [START_REF] Darst | Novel relationships among hyloid frogs inferred from 12S and 16S mitochondrial DNA sequences[END_REF][START_REF] Pauly | The history of a nearctic colonization: molecular phylogenetics and biogeography of the nearctic toads (Bufo)[END_REF][START_REF] Wiens | Hylid frog phylogeny and sampling strategies for speciose clades[END_REF][START_REF] Roelants | Global patterns of diversification in the history of modern amphibians[END_REF]). The Hyloidea sensu stricto and Ranoidea lineages are indeed monophyletic; the Hylinae, Bufonidae, Ranidae, and the Rhacophoridae are each monophyletic; and the two latter families are sister taxa (Fig. 2).

Cophylogeny

TreeMap analysis.-For the cophylogenetic analyses, we retain the ML host tree inferred from the rhodopsin data set (without outgroups) because it allows comparisons of host-parasite relationships that each results from nuclear gene analysis. As TreeMap requires fully resolved phylogenies, we considered nine topologies within the host tree by exchanging branching patterns within Hyloidea (i.e., between Leptodactylus fuscus (A), Hylinae (B), and Bufonidae (C)) and within Ranoidea (i.e., between the group uniting Strongylopus fasciatus, Ranidae and Rhacophoridae (X), Hyperolius viridiflavus (Y), and Ptychadena mascareniensis (Z)) (see Fig. 2). All of these arrangements were successively compared with the polystome topology after having excluded P. gallieni, W. almae, and outgroups (Fig. 1). A total of 162 significant cophylogenetic reconstructions, depending on the host cladogram selected for TreeMap analysis, are recovered differing by their global cost, the direction, and number of host-switching events and the number of codivergence, duplication, and extinction events. A tanglegram showing frog and polystome phylogenetic relationships and their interactions is shown in Figure 3 based on the host phylogeny in Figure 2 and on the parasite phylogeny in Figure 1. Among the five significant reconstructions (P < 0.01), the most parsimonious regarding the cost scheme designed for the different processes (duplication, horizontal transfer, and sorting events) involves a total cost of 30 (Fig. 4). It suggests four host switching, 22 codivergence, 10 duplication, and 16 extinction events and illustrates one of the numerous scenarios that may account for the evolution of polystomes within amphibians. Host-switching events would have occured from L. fuscus to the ancestral stock of Ranoidea, from L. fuscus to Physalaemus cuvieri, from L. fuscus to Phrynohyas venulosa, and from H. viridiflavus to S. fasciatus (Fig. 4).

ParaFit analysis.-Among the three host-parasite combinations that were tested, none of them is significant (P > 0.05), suggesting that hosts and polystomes could be randomly associated. These results match well with TreeMap reconstructions which propose either a large number of ancient and recent duplications followed by extinction events or numerous cases of host-switching events, to reconcile host and parasite phylogenies.

Vicariant Biogeography

DIVA, like TreeMap, requires fully resolved phylogenies. Thus, we used the polystome phylogeny depicted in Figure 1 as input for analysis and considered Polystoma sp. of Rhacophorus viridis and R. omeimontis as sister species. Similarly, we also assumed a Eurasian origin for the lineage including European and African Polystoma species and a South American origin for the clade including Polystoma species from Central and North America (see [START_REF] Bentz | Historical biogeography of amphibian parasites, genus Polystoma (Monogenea: Polystomatidae)[END_REF]).

DIVA analysis.-This gives one reconstruction with several hypotheses of distribution for the two most basal nodes (Fig. 5). Five different geographical distributions are hypothesized for the ancestral polystome lineage: one corresponding to Gondwana and the other four to blocks associating Eurasia with three of the four remaining plates (Fig. 5). Similarly, six distributions are hypothesized for the ancestral group uniting Eupolystoma, Polystoma, and W. almae, three of them associating Eurasia with two of the four other plates to the exclusion of Australia and the three others associating India to South America, India to Africa, and India to the "South American-African" block. On the other hand, only one geographical distribution is proposed for all other nodes (Fig. 5). If, then, 30 different scenarios can be proposed to account for the distribution of ancestral stocks of polystomes, most of them can be rejected due to the current knowledge of plate tectonics and reticulate relationships among areas.

TreeMap analysis.-This was conducted using two different area cladograms. In the first scenario (see Tanglegram of Fig. 6), when a sister relationship between, respectively, South American and African plates and Indian and Australian plates is considered, six different reconstructions are proposed (P < 0.01). Five of them suggest a Pangaean polystome origin and differ from each other by the number of dispersals and extinctions. In addition, three of those five reconstructions suggest duplication events in the most ancestral distribution (i.e., in Pangaea). Only the sixth reconstruction (Fig. 7) proposes a different origin for polystomes that would be centred on the "Indian-Australian" block (i.e., in the eastern part of Gondwana). After divergence of the Australian and Indian polystomes, following plate tectonics, dispersal would have occurred from the "Indian" plate to the "South American-African" block (i.e., to the western part of Gondwana) and from the "Australian" plate to Eurasia. A third polystome dispersal would have occurred from South America to Eurasia after codivergence of African and South American polystomes with their respective continents. In the second scenario (see Tanglegram of Fig. 8), if the Australian plate is considered as the most basal and the Indian plate intermediate between closely related South American and African continents, five different reconstructions are proposed (P < 0.01). Four of them suggest a Pangaean origin for polystomes and differ from each other by the number of dispersals and extinctions. In addition, all of them suggest duplication events (two to eight) in Pangaea. On the other hand, the fifth reconstruction suggests a polystome origin in Gondwana. Six codivergence events are proposed (biogeographic vicariance) following separation of geographical blocks and three dispersal events, respectively, from Australia, India, and South America to Eurasia (Fig. 9). When a phylogenetic representation of a restricted subsample of polystome species (species marked with an asterisk in Fig. 1) is compared with both area cladograms, the same parsimonious reconstructions are found. Whereas ten of them are not significant (P > 0.2), the single significant reconstruction (P < 0.05) also suggests a Gondwanan origin for polystomes and vicariance patterns following plate tectonics (Supplementary Appendix 3).

Molecular Dating

We kept a tree for molecular dating whose branching patterns within neobatrachian polystomes are identical to the one depicted in Figure 1. The lungfish parasite, namely C. australensis, is placed at the base of the Polystomatidae, and the archaeobatrachian polystomes are considered as a monophyletic sister group of neobatrachian polystomes. Calibration procedure 1 produces more recent estimates than procedures 2 and 3 (Table 2). Because procedures 2 and 3 present related estimates, it appears that multidivtime gives consistent estimates using either calibration point individually. Thus, molecular estimates inferred from procedure 1 can be considered for molecular dating. These estimates show an age of about 194 Ma for the split between "Parapolystoma/Diplorchis" and "Eupolystoma/Polystoma/ W. almae" lineages, an age of about 177 Ma for the divergence between the rhacophorid polystomes and the "Eupolystoma/Polystoma sensu stricto" clade, an age of about 156 Ma for the divergence between Eupolystoma and Polystoma sensu stricto lineages, an age of about 71-79 Ma for the American polystomes diversification, an age of about 106 Ma for the split between Parapolystoma and Diplorchis, and finally an age of about 86 Ma for the Indian-South-eastern Asian rhacophorid polystome divergence.

DISCUSSION

Coevolution

The cophylogenetic approach based on the comparison of phylogenetic trees of hosts and their parasites has been largely developed by [START_REF] Page | Component analysis: a valiant failure?[END_REF]Page ( , 1993b[START_REF] Page | Maps between trees and cladistic analysis of historical associations among genes, organisms, and areas[END_REF][START_REF] Page | Parallel phylogenies: reconstructing the history of host-parasite assemblages[END_REF] and expanded from the study of pocket gophers and their chewing lice [START_REF] Hafner | Molecular phylogenies and hostparasite cospeciation: gophers and lice as a model system[END_REF][START_REF] Page | Trees within trees: phylogeny and historical associations[END_REF]. Whereas cospeciation has been globally hypothesized for vertebrates and their strict host-specific ectoparasites (i.e., lice) with a direct lifecycle (e.g., [START_REF] Hafner | Phylogenetic trees support the coevolution of parasites and their hosts[END_REF][START_REF] Hafner | Disparate rates of molecular evolution in cospeciating hosts and parasites[END_REF]Page et al. 1998;[START_REF] Clayton | Linking coevolutionary history to ecological process: doves and lice[END_REF][START_REF] Banks | Cophylogenetic relationships between penguins and their chewing lice[END_REF]), it appears to be less prevalent within monogeneans and their fish host species [START_REF] Desdevises | Coevolution between Lamellodiscus (Monogenea: Diplectanidae) and Sparidae (Teleostei): the study of a complex host-parasite system[END_REF][START_REF] Simkova | Molecular phylogeny of congeneric monogenean parasites (Dactylogyrus): a case of intrahost speciation[END_REF][START_REF] Huyse | Comparing host and parasite phylogenies: Gyrodactylus flatworms jumping from goby to goby[END_REF]. Polystomes are globally distributed hostspecific monogenean mesoparasites and are, in most cases, represented by a single parasite species per host species [START_REF] Murith | Identite des larves de polystomes (Monogenea) para-´ sites du tetard deˆ Dicroglossus occipitalis (Gunther) en Cote-d'Ivoire[END_REF]Du Preez andKok 1997, Tinsley 2004;[START_REF] Verneau | Origine et evolution des monog´ enes Polystomatidae, parasites d'amphibiens et de cheloniens d'eau douce[END_REF]. They also have a direct life cycle involving free swimming larvae in aquatic environments. Therefore, investigating the coevolutionary processes that account for the intimate amphibian and polystome relationships may provide relevant insights about parasite diversification and host evolutionary ecology.

According to our cophylogenetic analyses on different combinations of host topology, the most parsimonious reconstructions (i.e., those which show the minimal total cost) imply three or four host-switching events in most cases. Unfortunately, it is impossible to find other solutions involving more host switching due to computation limitations. Furthermore, most reconstructions show ancient duplication events that appear to have occurred within the ancestral stock of neobatrachians and/or within ancestral hyloid and ranoid hosts (Fig. 4). Several duplication events within ancestral neobatrachian lineages imply that some divergent parasite lineages should co-occur within extant host species, which is actually not the case. A few reports have indeed mentioned the presence of more than one polystome species within the same host species [START_REF] Bourgat | Polystoma lamottei n. sp. et P. aeschlimanni n. sp., deux polystomes (Monogenes) de la m` eme espece d'amphibien: Ptychadena pumilio (Boulenger, 1920)[END_REF][START_REF] Preez | Syntopic occurrence of new species of Polystoma and Metapolystoma (Monogenea: Polystomatidae) in Ptychadena porosissima in South Africa[END_REF]Ibikounle M., Sakiti N., and Verneau O., unpublished observations). However, the three last authors showed that this parasite diversity was the result of recent duplications in the host (unpublished data). Even though extinction processes cannot be rejected, either for host and or parasite lineages, assuming the occurrence of several polystome duplication events within the ancestral frog lineage or within ancestral hyloids, seems very unlikely. Similarly, if Diplorchis and Parapolystoma genera originated in ancestral neobatrachian frogs, they must have arisen in Gondwana, which is considered the centre of origin for neobatrachians [START_REF] Zhang | Mitogenomic perspectives on the origin and phylogeny of living amphibians[END_REF]. Thus, how can we explain that the survivors of these two polystome lineages are only found in South-eastern Asia and Australia, respectively (see [START_REF] Verneau | Origine et evolution des monog´ enes Polystomatidae, parasites d'amphibiens et de cheloniens d'eau douce[END_REF])? The same question arises with the clade of rhacophorid polystomes, which is considered of the same geographical and temporal origin, but is only distributed in India and Southeastern Asia. On the other hand, host-switching events from hyloid lineages to an ancestral stock of ranoids are highlighted for every most parsimonious cophylogenetic reconstruction (see Fig. 4). Such events imply that extant African Polystoma species have originated in ancestral ranoids on Gondwana, thus challenging the Eurasian origin hypothesis inferred from phylogenetic analyses [START_REF] Bentz | Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods[END_REF].

Regardless of the coevolutionary processes invoked to explain the polystome diversification across host evolution (i.e., ancestral duplication and host-switching events), neither process accounts for the biology and recent evolutionary history of polystomes. [START_REF] Weckstein | Biogeography explains cophylogenetic patterns in toucan chewing lice[END_REF] showed within the toucan (bird)-chewing louse (insect) association that biogeography could be more important than host associations in structuring parasite populations and species. In contrast, polystomes are mesoparasites that parasitize the urinary bladder of frogs. Therefore, polystome diversification has necessarily been constrained by host evolutionary ecology. Thus, two nonexclusive hypotheses may be advanced at this stage to explain the nonreliability of such cophylogenetic scenarios. (i) There is a bias in our parasite sampling that does not reflect the overall diversity of neobatrachian polystomes. Because only 20 frog species were surveyed, new polystome species may be expected to be found, especially in poorly studied geographic areas like Asia and Australia, which will reinforce the proposed cophylogenetic scenarios. (ii) Some codivergence events may have been hidden by subsequent ancestral parasite and/or host extinctions. Due to the poor evidence in the fossil record for platyhelminths [START_REF] Poinar | A rhabdocoel turbellarian (Platyhelminthes, Typhloplanoida) in Baltic amber with a review of fossil and sub-fossil platyhelminths[END_REF], this hypothesis may be extremely difficult to demonstrate.

Historical Biogeography of Neobatrachian Polystomes

According to plate tectonic and reticulate relationships among areas, DIVA and TreeMap analyses show very similar results that indicate a Gondwanan origin for neobatrachian polystomes. Vicariance biogeography would be at the origin of the earliest divergences, and following subsequent continental drifting and colliding, terrestrial dispersal would have been possible in the new areas. Because Australia could have been isolated during the first stages of Gondwana breakup from South America and Africa at a geological period where India was still bound to the western part of Gondwana, the ancestral "Parapolystoma/Diplorchis" lineage would have originated in Australia and the ancestral "Eupolystoma/Polystoma/W. almae" lineage in the block associating India, Africa, and South America. This hypothesis that implies one biogeographic vicariance for the ancestral stock of polystomes instead of extra dispersal from plate to plate fits well with an Africa-India origin proposed for advanced frogs [START_REF] Zhang | Mitogenomic perspectives on the origin and phylogeny of living amphibians[END_REF]. It also concurs well with our molecular dating at approximately 194 Ma (Table 2), which corresponds to the period of intensive sea-floor spreading in the Somali, Mozambique, and Weddell Sea basins [START_REF] Storey | The role of mantle plumes in continental breakup: case histories from Gondwanaland[END_REF]) that could have isolated Australia from the western part of Gondwana [START_REF] Rogenhagen | The sedimentary structure in the western Weddell Sea[END_REF]. Next, the ancestral stock of the "Eupolystoma/Polystoma sensu stricto" lineage would have diverged from the ancestral stock of rhacophorid polystomes by vicariance, the first clade evolving in the western part of Gondwana and the second in India. This second codivergence is dated about 177 Ma, which is approximately the period when eastern (i.e., India, Madagascar, Seychelles, Antarctica, and Australia) and western (i.e., South America and Africa) Gondwanan components were fully separated [START_REF] Storey | The role of mantle plumes in continental breakup: case histories from Gondwanaland[END_REF][START_REF] Chatterjee | The breakup of Gondwana and the evolution and biogeography of the Indian plate[END_REF][START_REF] Lawver | A tight fit-early Mesozoic Gondwana, a plate reconstruction perspective[END_REF][START_REF] Briggs | The biogeographic and tectonic history of India[END_REF]. Finally, the ancestral distribution suggested for the Polystoma sensu stricto lineage, which is centred in the South American plate, is also in accordance with biogeographic vicariance. Following the separation of South America from Africa, the Polystoma sensu stricto and Eupolystoma lineages would have been isolated in South America and in Africa, respectively. This third codivergence is estimated about 156 Ma, which is more or less close to the separation of the South American and African plates [START_REF] Storey | The role of mantle plumes in continental breakup: case histories from Gondwanaland[END_REF][START_REF] Lawver | A tight fit-early Mesozoic Gondwana, a plate reconstruction perspective[END_REF][START_REF] Macdonald | Mesozoic break-up of SW Gondwana: implications for regional hydrocarbon potential of the southern South Atlantic[END_REF].

According to this proposed scheme for polystome evolution, several dispersals would have occurred. (i) From Australia to Eurasia, when Parapolystoma and Diplorchis diverged. The Parapolystoma-Diplorchis divergence dated at about 106 Ma suggests that Diplorchis dispersal could have taken place at this period of time or more recently. Because Australia and Asia are separated by a network of deep ocean trenches, dispersal from one continent to another, even from step to step, is not obvious even though it was illustrated for dispersal across the Wallace's and Huxley's lines for fanged frogs of the ranid Limnonectes (see [START_REF] Evans | Phylogenetics of fanged frogs: testing biogeographical hypotheses of the Asian and Australian faunal zones[END_REF]. On the other hand, to explain the distribution and phylogenetic relationships within microhylids, [START_REF] Van Bocxlaer | Late Cretaceous vicariance in Gondwanan amphibians[END_REF] suggested a dispersal route from Australia to Eurasia following a Late Cretaceous connection of Indo-Madagascar and Australia-New Guinea with Southeast Asia. Accordingly, assuming polystome dispersal from Australia to Eurasia is not unrealistic. (ii) From India to Southeast Asia when Indian and Southeastern Asian rhacophorid polystomes diverged. This dichotomy, which is dated about 86 Ma at a period near the collision of India with Asia [START_REF] Briggs | The biogeographic and tectonic history of India[END_REF], allows ample time for terrestrial polystome invasions. Finally, (iii) from South America to "Eurasia" in a stepwise manner. It would have started approximately from 79 to 71 Ma, which corresponds with a period of intensive diversification of polystomes in South America. Following land connections between South and North America via a central corridor in the Palaeocene and between North America and Eurasia via Beringia in the same period of time, polystomes would have first dispersed to Eurasia and next to Africa (Fig. 5; [START_REF] Bentz | Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods[END_REF][START_REF] Bentz | Historical biogeography of amphibian parasites, genus Polystoma (Monogenea: Polystomatidae)[END_REF].

In conclusion, the phylogenetic branching patterns and divergence time estimates presented here for polystomes support vicariant clades that probably arose during the breakup of Gondwana. Polystome lineages may therefore reflect rifting and drifting of ancient and present continents and coevolution between ancestral host lineages and their parasites.

Correlating Vicariance of Polystomatids to the Early Evolution of Frogs

Due to their direct life cycle which requires freshwater environments for egg laying, larval development, and infection process, polystome dispersal can only occur through amphibian dispersal. Hence, polystome vicariance necessarily implies host vicariance, a process which is similar to host-parasite codivergence following continental drift. One may question, therefore, which of the main stem lineages of the Neobatrachia carried polystomes to their definitive continents and subcontinents. When host and parasite phylogenetic branching patterns at lower levels are compared, some discrepancies are apparent. The Australian polystome lineage appears most basal in the parasite tree (Fig. 1), whereas the Australian frog subfamily Pelodryadinae from which Parapolystoma bulliense was recovered falls within the primarily South American hyloid lineage (Fig. 2; San [START_REF] Mauro | Initial diversification of living amphibians predated the breakup of Pangaea[END_REF][START_REF] Frost | The amphibian tree of life[END_REF][START_REF] Roelants | Global patterns of diversification in the history of modern amphibians[END_REF][START_REF] Wiens | Global patterns of diversification and species richness in amphibians[END_REF]. According to vicariance biogeographic evidence and molecular dating of polystomes, it seems very unlikely that pelodryadine hylids coevolved with Parapolystoma. Therefore, occurrence of the basal Australian polystome lineage within frogs of South American origin likely indicates ancestral host switching. It would have occurred either from uninvestigated extant Australian host taxa (see below) or extinct host species to pelodryadine frogs when the latter colonized Australia via an archipelago that is supposed to have connected the South America and Antarctica/Australia supercontinent (see [START_REF] Ruvinsky | Phylogenetic relationships among bufonoid frogs (Anura: Neobatrachia) inferred from mitochondrial DNA sequences[END_REF]. If extant Australian myobatrachids are those hosts, they should be basal within the neobatrachian tree, but instead, they seem to be related to South American hyloids [START_REF] Roelants | Global patterns of diversification in the history of modern amphibians[END_REF][START_REF] Wiens | Global patterns of diversification and species richness in amphibians[END_REF]. However, the early history of the Neobatrachia is still regarded as a polytomy by some authors (San [START_REF] Mauro | Initial diversification of living amphibians predated the breakup of Pangaea[END_REF] and probably warrants a more in depth study. Moreover, though no polystome species have so far been described from myobatrachids, it must be remembered that almost all anurans in Australia, that is, myobatrachid, pelodryadine, and microhylid frogs, have not been investigated for polystomes. An alternative hypothesis to reconcile the deepest host and parasite branching patterns is to consider gaps within the Australian fossil record. Finding older Australian frog fossils might indeed challenge current hypotheses about the early neobatrachian evolution, as has been the case following the recent discovery of a Late Cretaceous fossil in Madagascar that showed strong affinities with South American hyloids [START_REF] Evans | A giant frog with South American affinities from the late Cretaceous of Madagascar[END_REF]. Finally, hylids and ranids have an almost worldwide distribution but with a single exception (i.e., R. daemeli), ranids are absent from Australia. On the other hand if the Pelodryadinae is endemic to the Australo-Papuan region, its nearest relative, that is the Phyllomedusinae (see [START_REF] Wiens | Hylid frog phylogeny and sampling strategies for speciose clades[END_REF][START_REF] Wiens | Global patterns of diversification and species richness in amphibians[END_REF]), is distributed across South America. Thus, the ancestral host that disseminated Australian polystome species to Southeast Asia still remains in question. It could be microhylid frogs of the Asterophryinae that show strong phylogenetic affinities with Madagascan, Indian, and South-Eastern Dyscophinae and Microhylinae frog species.

Subsequently, the stem branch of the "Indian" frog lineage (i.e., the Nasikabatrachidae and Sooglossidae) would have carried ancestral polystomes to the "supercontinent" Madagascar-Seychelles-India that was still joined to the Antarctican-Australian block. Polystomes would have secondarily switched to rhacophorid hosts when their distant ancestors invaded India, probably from Africa or Madagascar (see [START_REF] Bossuyt | Phylogeny and biogeography of a cosmopolitan frog radiation: late Cretaceous diversification resulted in continent-scale endemism in the family Ranidae[END_REF], and would have invaded Asia after India collided with Southeast Asia. Indeed, it was suggested that rhacophorine tree frogs dispersed out of India and subsequently radiated outward in Southeast Asia [START_REF] Bossuyt | Amphibians as indicators of early tertiary "Out-of-India" dispersal of vertebrates[END_REF]. It should be noted that this hypothesis concurs well with the molecular dating proposed for the origin of the "Sooglossidae/ Nasikabatrachidae lineage" [START_REF] Biju | New frog family from India reveals an ancient biogeographical link with the Seychelles[END_REF] though the placement of the latter clade within the Neobatrachia is still disputed [START_REF] Biju | New frog family from India reveals an ancient biogeographical link with the Seychelles[END_REF][START_REF] Mauro | Initial diversification of living amphibians predated the breakup of Pangaea[END_REF][START_REF] Frost | The amphibian tree of life[END_REF][START_REF] Roelants | Global patterns of diversification in the history of modern amphibians[END_REF][START_REF] Wiens | Global patterns of diversification and species richness in amphibians[END_REF]. Thus, if our hypothesis about the earlier evolution of polystomes at the period of the breakup of Gondwana is correct, a survey of frogs of Myobatrachidae and Microhylidae in Australia, Nasikabatrachidae in India, and Sooglossidae in the Seychelles should reveal new polystome species intimately related to the Australian and Indian parasite lineages, respectively.

Despite good correspondence between the relationships of hyloid sensu stricto and ranoid frog lineages and the separation of South American and African plates, it is now considered that these two lineages have diverged about 150-160 Ma [START_REF] Biju | New frog family from India reveals an ancient biogeographical link with the Seychelles[END_REF][START_REF] Mauro | Initial diversification of living amphibians predated the breakup of Pangaea[END_REF][START_REF] Roelants | Global patterns of diversification in the history of modern amphibians[END_REF], most likely before complete separation of the plates and not at the time of their separation about 130 Ma as was suggested by [START_REF] Feller | Molecular evidence for the early history of living amphibians[END_REF]. According to molecular dating, the Polystoma sensu stricto and Eupolystoma lineages would have codiverged with hyloid sensu stricto and ranoid frogs, respectively. Eupolystoma would have secondarily undergone extinction within ranoids and switched to bufonoids, at a period close to the separation of the South American and African plates. Following the complete separation of the two continents, the Polystoma sensu stricto and Eupolystoma lineages would have been isolated in South America and Africa, respectively. Ultimately, the period of diversification of polystomes in South America, dated approximately about 71-79 Ma, relates well to the presumed diversification of their ancestral-specific host families [START_REF] Estes | The early fossil record of frogs[END_REF][START_REF] Sanchiz | An overview of the anuran fossil record[END_REF]. Indeed, studies of frog specimens close to extant genera from South America have extended fossil records of the Bufonidae, Hylidae, and Leptodactylidae in the Palaeocene of Brazil [START_REF] Estes | The early fossil record of frogs[END_REF]. These last results corroborate our findings on the early historical biogeography of neobatrachian polystomes because once again, they demonstrate the link between host and parasite diversification. Furthermore, they validate a scheme of dispersal by amphibians from South America to North America in the Palaeocene, which would have brought polystomes from South America, via North America, to Eurasia and later to Africa (Fig. 5; [START_REF] Bentz | Historical biogeography of amphibian parasites, genus Polystoma (Monogenea: Polystomatidae)[END_REF].

CONCLUSIONS

The close correspondence between the early polystome dichotomies and plate tectonics, substantiated by molecular dating, illustrates vicariance events within flatworm parasites. This implies ancestral host-parasite codivergence following continental drift, although such processes have not been identified from cophylogenetic analyses that suggest numerous ancestral duplication and host-switching events. Several hypotheses may be developed to explain the incongruence between cophylogenetic and biogeographic analyses. (i) The parasite phylogeny is not accurate because of the "limited parasite data set". This has been well documented with the improvement of taxon sampling through phylogenetic studies (e.g., Heath et al. 2008aHeath et al. , 2008b)). However, if this hypothesis is correct, the parasite branching patterns presented here would, by chance, reveal a strong relationship with the Earth's early history, which is very unlikely. (ii) There is a bias in our parasite sampling which would explain incongruent phylogenies between hosts and parasites at deeper levels. (iii) Codivergence events may have been hidden by subsequent ancestral parasite and/or host extinctions. Then the presence of polystome parasites in specific anuran host clades and in discrete geographic areas indicates the occurrence and radiation of amphibians over ancient and recent geological periods and thus provide promising biological tags for host evolutionary ecology in the Mesozoic period, especially when the fossil record is quite limited. Regarding these hypotheses, a more thorough parasite sampling should help to propose a more robust polystome phylogeny. However, regarding biogeographic scenarios, one may expect to find new polystome species from frogs of the Myobatrachidae and Microhylidae in Australia, Nasikabatrachidae in India, and Sooglossidae in the Seychelles.

Figure 4

Cophylogenetic reconstruction inferred from a TreeMap analysis (total cost = 30). Key to symbols: open squares and circles refer to duplication (10 events), filled black circles to codivergence (22 events), stars to extinction (16 events), and filled triangles to host switching (4 events). Asterisks indicate taxa substituted for actual host species. For abbreviations in brackets, refer to Figure 3. This figure is available in black and white in print and in color at Systematic Biology online.

Figure 9

One possible area-polystome historical reconstruction inferred from a analysis on the Tanglegram shown in Figure 8. Key to symbols: open squares and circles refer to sympatric speciation, filled circles to biogeographic vicariance, and filled triangles to dispersal. For abbreviations in brackets, refer to Figure 5.

Table 1

Parasite species investigated, their host species, geographical origin, and GenBank accession numbers (polystomes : 18S and partial 28S sequences ; frogs : partial Rhodopsin, 12S, and 16S sequences)

Table 2

Molecular dating estimates (million years) for the numbered speciation events shown in Figure 1, with their standard deviation and 95% confidence intervals

Figure 1

 1 Figure 1 Parasite ML tree inferred from analysis of 3125 characters (complete 18S and partial 28S). Abbreviations in brackets refer to host species, from top to bottom: R.t. = Rana temporaria; H.me. = Hyla meridionalis; P.a. = Ptychadena anchietae; S.f. = Strongylopus fasciatus; H.ma. = Hyperolius marmoratus; P.v. = Phrynohyas venulosa; B.m. = Bufo margaritifer; L.m. = Leptodactylus mystaceus; P.c. = Physalaemus cuvieri; S.ba. = Smilisca baudinii; H.c. = Hyla cinerea; H.v. = Hyla versicolor; B.sp. = Bufo sp.; S.c. = Schismaderma carens; R.m. = Rhacophorus maximus; R.a. = Rhacophorus arboreus; R.v. = Rhacophorus viridis; R.o. = Rhacophorus omeimontis; L.g. = Litoria gracilenta; R.r. = Rana rugosa; S.bo. = Spea bombifrons; S.c. = Scaphiopus couchii; X.l. = Xenopus laevis. Numbers at nodes indicate, from left to right, Bayesian posterior probabilities, MP, and ML percentage bootstrap support values with 1000 replicates. Labelled nodes 1-7 correspond to the speciation events discussed in the text (see Table2for molecular dating). Asterisks indicate species that were selected to build a restricted phylogenetic representation of polystomes used in vicariance analyses.

Figure 2

 2 Figure 2Amphibian ML tree inferred from analysis of 282 characters (nuclear rhodopsin). Numbers indicate from left to right Bayesian posterior probabilities, MP, and ML percentage bootstrap support values with 1000 replicates. Framed values correspond to Posterior probabilities resulting from the Bayesian analysis of the complete data set (combined 12S, 16S, and rhodopsin markers). Asterisks indicate taxa substituted for actual host species. Letters on the right-hand side show the six lineages that were inverted for the cophylogenetic analyses.

Figure 3

 3 Figure 3Tanglegram for frogs (left; from Fig.2) and their polystomes (right; from Fig.1). Parasites are linked to their specific or substituted hosts by lines. Asterisks indicate taxa substituted for actual host species. Abbreviations in brackets refer to host species from top to bottom: R.o. = Rhacophorus omeimontis; R.a. = Rhacophorus arboreus; L.m. = Leptodactylus mystaceus. This figure is available in black and white in print and in color at Systematic Biology online.

Figure 5

 5 Figure 5 All ancestral distributions inferred from DIVA analysis. A = Eurasia; B = Africa; C = South America; D = India; E = Australia. When several hypotheses for ancestral distributions are hypothesized, the most parsimonious is framed (see text for more details). Open circles at nodes refer to biogeographic vicariance, whereas coloured tree branches and matching arrows indicate dispersal from one area to the other. Dashed and solid lines on the graphic represent incomplete and complete fault lines between western and eastern Gondwanan components on one side and between South America and Africa on the other. Abbreviations in brackets refer to host species from top to bottom: L.m. = Leptodactylus mystaceus; R.v. = Rhacophorus viridis; R.a. = Rhacophorus arboreus; R.o. = Rhacophorus omeimontis

Figure 8

 8 Figure 8Tanglegram generated from TreeMap analysis for Gondwanan areas (left) and polystomes (right) when a basal position is assumed for Australia and an intermediate one for India. Parasites are linked to their geographical area by lines. For abbreviations in brackets, refer to Figure5. This figure is available in black and white in print and in color at Systematic Biology online.

ACKNOWLEDGMENTS

We are grateful to Dan Brooks and Salah Bouamer for providing us with Polystoma naevius collected in Costa Rica and to Takeshi Shimazu for polystomes collected in Japan. We also acknowledge Frank Rutschmann, Frederik Leliaert, and Remi Emans for their helpful advice´ as well as Jack Sullivan, Michael Charleston, and anonymous reviewers for their constructive remarks.

FUNDING

This study received financial support from the Centre National de la Recherche Scientifique and University of Perpignan Via Domitia.

SUPPLEMENTARY MATERIAL

Supplementary material can be found at http:// www.sysbio.oxfordjournals.org/. Alignments and trees for both host and parasite can also be retrieved under Accession Number 11352 in TreeBASE (http:// www.treebase.org/treebaseweb/home.html).