Evolutionary processes involved in the diversification of chelonian and mammal polystomatid parasites (Platyhelminthes, Monogenea, Polystomatidae) revealed by palaeoecology of their hosts

Molecular Phylogenetics and Evolution 92 (2015) 1–10

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Evolutionary processes involved in the diversification of chelonian and mammal polystomatid parasites (Platyhelminthes, Monogenea, Polystomatidae) revealed by palaeoecology of their hosts q Laurent Héritier a,b, Mathieu Badets c, Louis H. Du Preez c, Martins S.O. Aisien d, Fan Lixian e, Claude Combes f, Olivier Verneau a,b,c,⇑ a

Univ. Perpignan Via Domitia, CEntre de Formation et de Recherche sur les Environnements Méditerranéens, UMR 5110, F-66860 Perpignan, France CNRS, CEntre de Formation et de Recherche sur les Environnements Méditerranéens, UMR5110, F-66860 Perpignan, France c School of Environmental Sciences and Development, North-West University, Potchefstroom Campus, Private Bag X6001, Potchefstroom 2520, South Africa d Laboratory of Parasitology Research, Department of Animal and Environmental Biology, Faculty of Life Sciences, University of Benin, P.M.B. 1154, Benin City, Nigeria e School of Life Sciences, Yunnan Normal University, Yunnan 650500, China f Univ. Perpignan Via Domitia, F-66860 Perpignan, France b

a r t i c l e

i n f o

Article history: Received 8 April 2015 Revised 27 May 2015 Accepted 29 May 2015 Available online 10 June 2015 Keywords: Polystomatidae Amphibians Chelonians Hippopotamus Phylogeny Molecular dating

a b s t r a c t Polystomatid flatworms (Platyhelminthes) are monogenean parasites that infect exclusively aquatic or semi-aquatic sarcopterygians such as the Australian lungfish, amphibians, freshwater turtles and the African common hippopotamus. Previous studies on the phylogenetic relationships of these parasites, excluding Oculotrema hippopotami infecting common hippos, showed a global coevolution between hosts and their parasites at a macroevolutionary scale. These studies also demonstrated a strong correlation between the diversification of early neobatrachian polystomes and Gondwana breakup in the Mesozoic period. However the origin of chelonian polystomes is still in question as a switch from presumably primitive aquatic amniotes to turtles at the time of their first appearance, or soon after during their radiation, was assumed. In order to resolve this sticking point, we extended the phylogeny of polystomes with broader parasite sampling, i.e. 55 polystome species including Nanopolystoma tinsleyi a polystome infecting caecilians and O. hippopotami, and larger set of sequence data covering two nuclear and two mitochondrial genes coding for the ribosomal RNA 18S and 28S, the Cytochrome c Oxidase I and the ribosomal RNA 12S, respectively. The secondary structure of nuclear rRNAs genes (stems and loops) was taken into account for sequence alignments and Bayesian analyses were performed based on the appropriate models of evolution selected independently for the four designed partitions. Molecular calibrations were also conducted for dating the main speciation events in the polystome tree. The phylogenetic position of chelonian parasites that are phylogenetically closer to N. tinsleyi than all other amphibian polystomes and molecular time estimates suggest that these parasites originated following a switch from caecilians, at a geological period when primitive turtles may already have adapted to an aquatic life style, i.e. at about 178 Million years ago, or a little later when the crown group of extant turtles have already diversified, i.e. at about 152 Mya. Similarly, because O. hippopotami constitutes the sister group of chelonian parasites, proposing that an African caecilian could be the ancestral host for this polystome species seems at this stage the most likely hypothesis to explain its occurrence within the common hippo. Regardless of the scenario that may be predicted to explain the origin of polystomes within aquatic or semi-aquatic amniotes, their presence and evolution are indicative of early aquatic ecological habits within ancestral lineages. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction q

This paper was edited by the Associate Editor N. Blackstone.

⇑ Corresponding author at: Univ. Perpignan Via Domitia, CEntre de Formation et de Recherche sur les Environnements Méditerranéens, UMR 5110, F-66860 Perpignan, France. E-mail address: [email protected] (O. Verneau). http://dx.doi.org/10.1016/j.ympev.2015.05.026 1055-7903/Ó 2015 Elsevier Inc. All rights reserved.

The Platyhelminthes, namely the flatworms, are a diverse phylum of aquatic and terrestrial invertebrates that include the primarily free-living turbellarians and the parasitic monophyletic Neodermata (Katayama et al., 1996; Littlewood et al., 1999;

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L. Héritier et al. / Molecular Phylogenetics and Evolution 92 (2015) 1–10

Rohde, 1994). The latter clade encompasses three classes, among them the Monogenea, which have a direct life-cycle and are largely ectoparasites of chondrichthyan and actinopterygian fishes, although some may be found within internal cavities of sarcopterygians. Monogeneans are divided into two subclasses, the Polyonchoinea and the Heteronchoinea (Boeger and Kritsky, 2001), which are epithelial and blood feeding parasites, respectively (Perkins et al., 2010). The Heteronchoinea are further subdivided into two infra-subclasses, the Oligonchoinea, which are mainly fish parasites and the Polystomatoinea, which infect exclusively aquatic or semi-aquatic sarcopterygian hosts (Boeger and Kritsky, 2001). The Polystomatoinea include one single family, the Polystomatidae sensu Sinnappah et al. (2001), with 25 genera infecting the Australian lungfish, lissamphibians, freshwater turtles and the African common hippopotamus. Polystomes are mostly host and site specific (see Verneau, 2004) and, unlike fish monogeneans which parasitize the gills or skin of their host, are typically found on gills or oral cavity of the Dipnoi (Pichelin et al., 1991); in the urinary bladder of lissamphibian adult hosts (see Prudhoe and Bray, 1982); in the bladder, conjunctival sacs and pharyngeal cavity of chelonians (see Morrison and Du Preez, 2011) and under the eyelid in the common hippopotamus (Thurston and Laws, 1965). With the exception of one single species, i.e. Concinnocotyla australensis Pichelin et al., 1991 of the Australian lungfish, polystomes are the only known monogeneans having colonized terrestrial vertebrate hosts, namely tetrapods, with ecological aquatic or semi-aquatic life habits. Because C. australensis occupies a basal position within the phylogenetic tree of polystomes, like its host within sarcopterygians, Verneau et al. (2002) assumed a very ancient origin for the Polystomatidae, which could have been related to the divergence between actinopterygians and sarcopterygians about 425 Million years ago (Mya) based on the palaeontological syntheses of Janvier (1998) and Ahlberg (1999). Verneau et al. (2002) also illustrated a sister group relationship between parasites of the Batrachia (frogs and salamanders) and freshwater chelonians, that was well correlated to the separation of extant lissamphibians from amniotes. Because the dichotomy was dated to about 353 Mya, it was postulated that following the split between lissamphibians and amniotes, polystomes would have coevolved with amphibians on one hand and primitive amniotes on the other. Polystomes would have secondarily switched to chelonians, i.e. at about 200 Mya when turtles originated or soon after when they adapted to freshwater environments (see Verneau et al., 2002). As the direct life-cycle of these parasites involves an obligatory infective aquatic larval stage, i.e. an oncomiracidium, this hypothesis implies that some primitive amniotes could have been adapted to an aquatic lifestyle, which was stated as plausible by Verneau et al. (2002) based on the fossil record of amniotes (see Motani et al., 1998; Reisz, 1997; Rieppel, 1999). Two polystome species were recently reported by Du Preez et al. (2008) from South American amphibians of the family Caeciliidae. Based on their morphology, Du Preez et al. (2008) placed both species in a new genus, i.e. Nanopolystoma, which was the first record of polystomes in caecilians (Gymnophiona). A thorough analysis of the morphological features of these parasites indicated strong similarities with chelonian polystomes, which led Du Preez et al. (2008) to suggest a close relationship between Nanopolystoma and the three known genera infecting freshwater turtles, i.e. Polystomoidella, Polystomoides and Neopolystoma. Finally a non-sanguinivorous diet was reported for Nanopolystoma (Du Preez et al., 2014), a biological characteristic that it also shares with all other chelonian polystomes. Because of the close resemblances between Nanopolystoma and turtle parasites, caecilians could represent the ancestral hosts for chelonian

polystomes and to some extent the missing link between batrachians and chelonians in the course of polystomatid dispersal and evolution. The phylogenetic position of Nanopolystoma within the Polystomatidae could also provide new insights regarding the origin of the enigmatic parasite Oculotrema hippopotami Stunkard, 1924, which is the single polystome species infecting a mammal, i.e. the common hippopotamus. With the exception of the fully aquatic mammals, namely cetaceans and sirenians, most mammals are terrestrial. However some are semi-aquatic, like pinnipeds, and others have aquatic habits, like hippos. Hippos are recognized as the closest living relatives of whales (Gatesy, 1997; Montgelard et al., 1997; Ursing and Arnason, 1998), from which they would have diverged at about 53 Mya (Arnason et al., 2004; Montgelard et al., 1997). Hippos and cetaceans are also believed to have diverged from extant Artiodactyla at about 66.4–62.5 Mya (Bajpai and Gingerich, 1998). Regarding ecological preferences of hippos and their phylogenetic position within the tree of life, it is highly unlikely that the ancestors of O. hippopotami evolved within early mammals that may have originated in the Early or Middle Jurassic, at about 180 Mya (Rowe, 1999). Because O. hippopotami shows numerous morphological and some biological similarities with Nanopolystoma (Du Preez et al., 2008) and chelonian polystomes (Du Preez and Moeng, 2004), it may have derived from caecilian or chelonian parasites in recent geological times when hippos colonized swampy habitats. In this study, we propose to extend the phylogeny of Verneau et al. (2002) with a more complete sampling, including C. australensis, worldwide chelonian and amphibian polystomes with one representative of the Nanopolystoma genus and O. hippopotami. The parasite phylogeny will be generated from the combination of four genes, two nuclear genes coding for ribosomal RNA 18S and 28S, and two partial mitochondrial genes coding for the Cytochrome c Oxydase I (COI) and ribosomal RNA 12S. Patterns and processes of parasite evolution will be discussed in the light of the evolutionary ecology of their hosts in order to determine origins of polystomes parasitizing chelonians and the common hippo. 2. Materials and methods 2.1. Parasite sampling Fifty three polystome species used in the current study were collected from amphibian and chelonian hosts of Africa, Australia, Eurasia, South and North America. In addition O. hippopotami and C. australensis were sampled from the African common hippopotamus and the Australian lungfish, respectively, and the two fish monogeneans used as outgroups were collected from France (Table 1). Specimens of O. hippopotami were recovered from the eyes of hippos following a culling operation that was undertaken in October 1996 by parks board officials from the Ndumo Game Reserve in Northern KwaZulu-Natal of South Africa. All parasites were stored and preserved in 70% ethanol before molecular processing. 2.2. DNA extraction, PCR amplification and sequencing Parasites were lyophilized to remove all traces of alcohol and crushed with the help of a pestle. They were resuspended in about 150 ll of Chelex 10% and proteinase K at final concentration of 1 mg/ml for tissue digestion. The mixture was heated at 55 °C for 1 h and placed at 100 °C for 15 min to stop enzymatic reaction. DNA extracts were then kept at 4 °C until PCR amplification. The complete 18S rRNA and the partial 28S rRNA genes were amplified and sequenced with specific primers reported in Sinnappah et al. (2001) and Verneau et al. (2009b). Another reverse

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L. Héritier et al. / Molecular Phylogenetics and Evolution 92 (2015) 1–10 Table 1 List of parasite species investigated with their host species and family, geographical origin and GenBank accession numbers for complete 18S and partial 28S, 12S and COI. Parasite species

Host species

Polystomes from amphibians Diplorchis ranae Glandirana rugosa Diplorchis shilinensis Babina pleuraden Eupolystoma alluaudi Bufo sp. Eupolystoma vanasi Schismaderma carens Kankana manampoka Platypelis pollicaris Madapolystoma sp. [B.w.] Blommersia wittei Metapolystoma cachani Ptychadena longirostris Nanopolystoma tinsleyi Typhlonectes compressicauda Neodiplorchis scaphiopi Spea bombifrons Parapolystoma bulliense Litoria gracilenta Polystoma cuvieri Physalaemus cuvieri Polystoma dawiekoki Ptychadena anchietae Polystoma dianxiensis Rana chaochiaoensis Polystoma floridana Hyla cinerea Polystoma gallieni Hyla meridionalis Polystoma indicum Rhacophorus maximus Polystoma integerrimum Rana temporaria Polystoma lopezromani Trachycephalus venulosus Polystoma marmorati Hyperolius marmoratus Polystoma naevius Smilisca baudinii Polystoma nearcticum Hyla versicolor Polystoma pelobatis Pelobates cultripes Polystoma testimagna Strongylopus fasciatus Polystoma sp. [R.o.] Rhacophorus omeimontis Polystoma sp. [R.a.] Rhacophorus arboreus Polystoma sp. [R.v.] Rhacophorus viridis Protopolystoma xenopodis Xenopus laevis Protopolystoma occidentalis Xenopus muelleri Pseudodiplorchis Scaphiopus couchii americanus Pseudopolystoma Onychodactylus japonicus dendriticum Sphyranura oligorchis Necturus maculosus Sundapolystoma Hylarana chalconota chalconotae Wetapolystoma almae Rhinella margaritifera

Host family

Origin

Infection site

Accession numbers 18S

28S

12S

COI

U. U. U. U. U. U. U. U.

bladder bladder bladder bladder bladder bladder bladder bladder

AM157184 KR856123a AM051066 AM157185 HM854292 FM897290 FM897280 KR856124a

AM157198 KR856141a AM157199 AM157200 HM854293 FM897273 FM897262 KR856142a

KR856070a KR856071a KR856072a KR856073a KR856074a KR856075a KR856076a KR856077a

JF699304 KR856162a FR667558 FR667559 JF699307 JF699308 KR856163a KR856164a

Scaphiopodidae Hylidae Leptodactylidae Ptychadenidae Ranidae Hylidae Hylidae Rhacophoridae Ranidae Hylidae Hyperoliidae Hylidae Hylidae Pelobatidae Pyxicephalidae Rhacophoridae Rhacophoridae Rhacophoridae Pipidae Pipidae Scaphiopodidae

Japan China Togo South Africa Madagascar Madagascar Nigeria French Guiana USA Australia Paraguay South Africa China USA France India France Paraguay South Africa Costa Rica USA France South Africa China Japan Japan South Africa Togo USA

U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U. U.

bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder bladder

AM051067 AM157186 AM051068 AM051069 KR856125a AM157188 AM051070 AM157193 AM051071 AM051072 AM051073 AM157187 AM051074 AM051076 AM157194 AM157189 AM157190 AM157191 AM051078 AM051077 AM051079

AM157201 AM157202 AM157203 AM157204 KR856143a AM157211 AM157205 AM157216 AM157206 AM157207 AM157208 AM157209 AM157210 KR856144a AM157217 AM157212 AM157213 AM157214 AM157218 KR856160a AM157219

KR856078a KR856079a KR856080a KR856081a KR856082a KR856083a KR856084a KR856085a KR856086a KR856087a KR856088a KR856089a KR856090a KR856091a KR856092a KR856093a KR856094a KR856095a KR856096a KR856121a KR856097a

KR856165a KR856166a AM913862 AM913856 KR856167a AM913870 JF699305 JF699303 JF699306 AM913863 AM913858 AM913864 AM913865 KR856168a AM913860 KR856169a KR856170a KR856171a KR856172a KR856179a KR856173a

Hynobiidae

Japan

U. bladder

FM992700

FM992707

KR856122a

KR856180a

Proteidae Ranidae

USA Malaysia

Skin and gills U. bladder

FM992701 AM051080

FM992708 KR856161a

KR856098a –

KR856174a –

Bufonidae

French Guiana

U. bladder

AM051081

AM157220

KR856099a

AM913866

Polystomes from chelonians Neopolystoma chelodinae Chelodina longicollis Neopolystoma euzeti Mauremys leprosa Neopolystoma liewi Cuora amboinensis

Chelidae Geoemydidae Geoemydidae

Australia Algeria Malaysia

KR856126a KR856127a KR856128a

KR856145a KR856146a KR856147a

KR856100a KR856101a KR856102a

Z83005 FR822587 FR822530

Neopolystoma orbiculare Neopolystoma palpebrae

Chrysemys picta marginata Pelodiscus sinensis

Emydidae Trionychidae

USA Vietnam

KR856129a FM992696

KR856148a AF382065

KR856103a KR856104a

FR822531 FR822601

Neopolystoma spratti

Chelodina longicollis

Chelidae

Australia

AJ228788

FM992702

KR856105a

Z83007

Neopolystoma sp. [A.s.] Neopolystoma sp. [C.s.]

Apalone spinifera Chelydra serpentina

Trionychidae Chelydridae

USA USA

KR856130a KR856131a

KR856149a KR856150a

KR856106a KR856107a

FR822527 FR822529

Neopolystoma sp. [G.p.]

Emydidae

USA

KR856132a

KR856151a

KR856108a

FR822553

Neopolystoma sp. [K.l.]

Graptemys pseudogeographica Kinosternon leucostomum

Kinosternidae

Costa Rica

KR856133a

KR856152a

KR856109a

KR856175a

Neopolystoma sp. [R.p.]

Rhinoclemmys pulcherrima

Geoemydidae

Costa Rica

KR856134a

KR856153a

KR856110a

FR822555

a

KR856135 AJ228792 FM992697 FM992699

a

KR856154 FM992704 FM992703 FM992706

a

KR856111 KR856112a KR856113a KR856114a

FR828360 Z83011 Z83009 FR822604

Polystomoides sp. [T.s.s.] Polystomoides malayi Polystomoides asiaticus Polystomoides siebenrockiellae Polystomoides oris Polystomoides tunisiensis Polystomoides sp. [P.n.] Polystomoides sp. [P.s.] Polystomoides sp. [P.d.]

Ranidae Ranidae Bufonidae Bufonidae Microhylidae Mantellidae Ptychadenidae Typhlonectidae

Trachemys scripta sripta Cuora amboinensis Cuora amboinensis Siebenrockiella crassicollis

Emydidae Geoemydidae Geoemydidae Geoemydidae

USA Malaysia Malaysia Malaysia

U. bladder U. bladder Conjunct. sacs U. Bladder Conjunct. sacs Conjunct. sacs Pharyng. cav. Conjunct. sacs Conjunct. sacs Conjunct. sacs Conjunct. sacs Pharyng. cav. U. bladder Pharyng. cav. U. bladder

Chrysemys picta marginata Mauremys leprosa Pseudemys nelsoni Pelomedusa subrufab Pelusios castaneus

Emydidae Geoemydidae Emydidae Pelomedusidae Pelomedusidae

USA Algeria USA Nigeria Nigeria

Pharyng. cav. Pharyng. cav. Pharyng. cav. U. bladder U. bladder

FM992698 KR856136a KR856137a KR856138a KR856139a

FM992705 KR856155a KR856156a KR856157a KR856158a

KR856115a KR856116a KR856117a KR856118a KR856119a

FR822534 FR822570 FR822603 KR856176a KR856177a

Hippopotamidae

South Africa

Conjunct. sacs

KR856140a

KR856159a

KR856120a

KR856178a

Polystome from the common hippo Oculotrema hippopotami Hippopotamus amphibius

(continued on next page)

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Table 1 (continued) Parasite species

Host species

Host family

Origin

Infection site

Accession numbers 18S

28S

12S

COI

Polystome from the Australian lungfish Concinnocotyla australensis Neoceratodus forsteri

Neoceratodontidae

Australia

Skin and gills

AM157183

AM157197

–

–

Monogeneans from teleosts Microcotyle erythrinii Pagellus erythrinus Pseudaxine trachuri Trachurus trachurus

Sparidae Carangidae

France France

Gills Gills

AM157195 AM157196

AM157221 AM157222

– –

– –

– Indicates that no sequence was available. Abbreviations used: U. bladder = Urinary bladder; Conjunct. sacs = Conjunctival sacs; Pharyng. cav. = Pharyngeal cavity. a This study. b According to the systematic revision of the African helmeted terrapin, P. subrufa in Nigeria should be considered as P. olivacea (Petzold et al., 2014).

primer IR16 (50 -ATTCACACCCATTGACTCGCG-30 ) was also used instead of IR14 to amplify the 28S (Verneau et al., 2009b). For the 12S rRNA gene amplification, one forward 12SpolF1 (50 -YVGT GMCAGCMRYCGCGGYYA-30 ) and two reverse 12SpolR1 (50 -TACCR TGTTACGACTTRHCTC-30 ) and 12SpolR9 (50 -TCGAAGATGACGGGCG ATGTG-30 ) were first designed from an alignment of complete 12S sequences selected among 15 very divergent platyhelminth parasite species (Polyonchoinea, Heteronchoinea, Digenea and Cestoda) extracted from GenBank (Accession Numbers AB269235, AB374543, AB731761, NC002546, NC008815, NC009055, NC009680, NC009682, NC010976, NC011127, NC012147, NC014291, NC014591, NC06057 and NC016950). 12SpolF1 primer was used with either 12SpolR1 or 12SpolR9. Both resulting PCR products of about 500 and 470 bp, respectively, were sequenced with corresponding amplification primers. For the COI gene amplification, forward L-CO1p and reverse H-Cox1p2 primers (see Littlewood et al., 1997) were used in combination for all polystomes. When necessary, a newly designed reverse H-Cox1R primer (50 -AACAACAAACCAAGAATCATG-30 ) was used in combination with L-CO1p. Both resulting PCR products of about 440 and 415 bp, respectively, were sequenced with relevant amplification primers. The procedure for amplifying the various genes was identical regardless of the gene of interest or primers used: one initial step of 50 at 95 °C for long denaturation; 30 cycles of 10 at 94 °C for denaturation, 10 at 55 °C for annealing and 20 at 72 °C for elongation; one final step of 100 at 72 °C for terminal elongation. Each PCR reaction was run twice and independently in a final volume of 25 ll comprising Buffer 1X, MgCl2 1.5 mM, dNTPs 0.2 mM, primers 0.4 mM, GoTaq Polymerase 0.75 unit (Promega, France) and DNA (2 ll). PCR products were then pooled and sent to a commercial company (Genoscreen, Lille, France) for purification and sequencing. Sequences were finally edited using the software Geneious (Saint Joseph, Missouri, USA) to check chromatograms.

complete data set were aligned using DCSE v2.6 software (De Rijk and De Wachter, 1993). COI and 12S sequences were edited with MEGA version 5 (Tamura et al., 2011) and then aligned with Clustal W under default parameters (Thompson et al., 1994). Regarding the 12S data set, the most variable region was deleted as homologous characters were too difficult to determine unambiguously. 2.4. Phylogenetic analysis Because it was impossible to amplify regions of interest in the COI and 12S genes for two polystome species, i.e. C. australensis and Sundapolystoma chalconotae Lim & Du Preez, 2001, and the two outgroups, namely Microcotyle erythrinii Van Beneden & Hesse, 1863 and Pseudaxine trachuri Parona & Perugia, 1889 (See Table 1), these regions were treated as missing data in a global alignment comprising concatenated 18S, 28S, COI and 12S sequences obtained from the 57 platyhelminth species. Four partitions were specified for the Bayesian analysis regarding genes and/or structure. Ribosomal nuclear sequences were partitioned into stem and loop regions that were analyzed taking into account a doublet model for the first partition, as recommended by Telford et al. (2005), and a GTR + I + C model for the second. A TVM + I + C model was selected independently for the COI and 12S partitions following the Akaike Information Criterion (AIC) implemented in Modeltest 3.06 (Posada and Crandall, 1998). A GTR + I + C was therefore designated for both mitochondrial partitions, and evolutionary parameters were estimated independently for all partitions. The Bayesian analysis was run using MrBayes 3.04b (Huelsenbeck and Ronquist, 2001), with four chains running for one million generations and sampled every 100 cycles. The consensus tree was then drawn after removing the first 1000 trees (10%) as the burn-in phase. 2.5. Molecular calibration

2.3. Sequence alignment The rRNA secondary structure (stems and loops) of 18S sequences was determined for Polystomoides malayi Rohde, 1963, Nanopolystoma tinsleyi Du Preez et al., 2014 and O. hippopotami taking into account whenever possible the secondary structure of rRNAS previously determined for some amphibian polystomatids and C. australensis (see Badets et al., 2011). Otherwise, it was inferred with the aid of the Mfold software using default parameters (Zucker, 2003). This concerned in most case helices E10_1, 12, E23_1, E23_2, E23_5, E23_6, E23_7, 43 and 49 (see Van de Peer et al., 1999 for the nomenclature of rRNA secondary structures). The same procedure was followed for the 28S sequences. However the C and D5 regions were not constrained due to the high level of variability within polystomes and the lack of common motifs after Mfold reconstructions. These two regions were therefore treated as loops. Finally, 18S and 28S sequences for the

BEAUti v1.8.2 (Drummond et al., 2012) was used to import concatenated nuclear and mitochondrial aligned sequences. Four partitions were thus selected as stated above with the same substitution models. Gamma and invariant sites parameters were estimated independently for each partition. A Log normal relaxed clock (uncorrelated) was selected to take into account molecular rate variations within the Polystomatidae and the speciation model of Yule was preferred (Gernhard, 2008). A substitution rate with its standard deviation (0.002 ± 2.4104) and temporal calibrations (see below) were specified a priori. Fifty millions iterations were performed following the MCMC approach using BEAST v.1.8.2 (Drummond et al., 2012) and sampled every 1000 cycles. Results were imported in Tracer v1.6 to estimate the burn-in phase and to check estimates during iterations. TreeAnnotator v1.8.2 was then used to substract the burn-in phase and to summarize all the information produced by BEAST onto a single consensus tree.

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FigTree v1.4.2 was selected to depict the consensus tree and to illustrate divergence time estimates with their 95% confidence intervals. Seven nodes were calibrated into the polystome tree based on tectonic events, palaeobiogeography and the vertebrate fossil record. Following lines of parallel evolution between hosts and their parasites (see Verneau et al., 2002), the divergence between oligonchoinean and polystomatoinean parasites can be correlated to the actinopterygian – sarcopterygian split, which is dated at about 421.75–419 Mya (Benton and Donoghue, 2007; Benton et al., 2009; Botella et al., 2007; Yu et al., 2010; Zhu et al., 2009). Similarly, the separation between C. australensis and all other polystomes can be dated to about 419–415 Mya according to the divergence time estimate between Dipnoi and Tetrapoda (Lu et al., 2012; Zhu and Fan, 1995; Zhu and Yu, 2002; Zhu et al., 2009). Following plate tectonics in the Middle Jurassic, the initial divergences of the Australian and Indian polystome lineages from the ancestral African/South American lineage could be linked to the separation between eastern and western Gondwanan components (see Badets et al., 2011). This geological event, which is dated to approximately 165–162 Mya after McLoughlin (2001), was then used to calibrate these two speciations. Lastly, the Polystoma sensu stricto lineage (i.e. Wetapolystoma, Metapolystoma and Polystoma to the exception of Asian Polystoma species) would have dispersed from South America to North America twice (Bentz et al., 2006), at a geological period, i.e. in the Paleocene, when amphibian dispersal could be assumed (see Gayet et al., 1992). These two events were therefore dated to about 66–56 Mya. Because one of the two resultant North American polystome lineages would have subsequently colonized Africa via Eurasia, i.e. in the Miocene epoch (see Bentz et al., 2001) when vertebrate dispersal was allowed (see Rage, 1988), this final dispersal was dated to about 25–5 Mya.

3. Results A total of 111 new sequences were obtained among which 18 from 18S, 21 from 28S, 53 from 12S and 19 from COI. Other sequences used for the phylogenetic reconstructions were retrieved from GenBank. Parasite species with host species and family, locality, infection sites and GenBank Accession numbers are reported in Table 1. Phylogenetic analyses were conducted on a data set comprising 57 species and 5469 characters. The resulting consensus tree inferred from the Bayesian analysis is depicted in Fig. 1. It shows with strong Bayesian Posterior Probabilities (BPP = 0.98) a basal position of C. australensis after rooting the tree with the two fish monogenean parasites. Two major monophyletic groups are evidenced, the first one, which we named ‘‘Polbatrach’’, includes all polystomes of batrachian hosts (caudatans and anurans), the second one, which we called ‘‘Polchelon’’, associates all polystomes of chelonians, but also N. tinsleyi of the caecilian host and O. hippopotami of the common hippopotamus (BPP = 0.99 for both clades). The ‘‘Polbatrach’’ clade shows a basal polytomy with Sphyranura oligorchis Alvey, 1933 parasitizing a caudatan host, Protopolystoma infecting archaeobatrachian pipids and a robust lineage including all remaining polystomes (BPP = 0.99). Within the latter lineage, Pseudopolystoma dendriticum (Ozaki, 1948), which also infects a salamander, is well nested within anuran polystomes (BPP = 0.90). It is actually intermediate between a basal group that associates Neodiplorchis and Pseudodiplorchis genera that each infect archaeobatrachian scaphiopodids and a robust clade (BPP = 0.99) that includes only polystomes of the Neobatrachia (Polystoma, Diplorchis, Parapolystoma etc., see Fig. 1) to the exception of Polystoma pelobatis (Euzet & Combes, 1966), another

5

parasite of the Archaeobatrachia. That clade can be subdivided in four lineages (Neobat-1 to Neobat-4) that were shown to be ascribed to discrete centers of diversity, namely India, Australia, Africa and South America, respectively (see Badets et al., 2011). The five polystome species infecting archaeobatrachians do not form a monophyletic assemblage, Protopolystoma being the most basal taxon within anuran polystomes and P. pelobatis being nested within neobatrachian polystomes. The ‘‘Polchelon’’ clade shows a strong basal position for N. tinsleyi (BPP = 0.98). O. hippopotami appears as sister species of a robust group that associates all chelonian polystomes (BPP = 0.98). Within the latter clade, phylogenetic relationships reveal three main lineages. The most basal lineage (Chelon-1, BPP = 1.00) includes only parasites located in the urinary bladder. Two polystomes infect African pleurodires of the Pelomedusidae while the two others infect Malaysian cryptodires of the Geoemydidae. The second lineage (Chelon-2, BPP = 0.99) comprises polystomes that infect either the urinary bladder, or the pharyngeal cavity of cryptodires. Polystomes of the urinary bladder are from North African Geoemydidae and North American Emydidae. Polystomes of the pharyngeal cavity are from Malaysian and North African Geoemydidae and from North American Emydidae and Trionychidae. All polystomes of the third clade (Chelon-3, BPP = 0.99) infect the conjunctival sacs of their hosts, except one species which is found in the urinary bladder. Some parasites are from Australian pleurodires of the Chelidae, the others from cryptodires of Asian Geoemydidae and Trionychidae and South and North American Chelydridae, Emydidae, Geoemydidae and Kinosternidae. Lineages Chelon-2 and Chelon-3 present a strong sister group relationship (BPP = 0.97). The tree used for molecular dating (Fig. 2), shows very similar branching patterns to those depicted in the phylogenetic tree of Fig. 1. In that tree, S. oligorchis appears as a sister taxon to Protopolystoma. Divergence time estimates inferred from molecular calibrations indicate a split between the ‘‘Polbatrach’’ and ‘‘Polchelon’’ clades at about 334 Mya. Within the ‘‘Polchelon’’ clade, the divergence between N. tinsleyi and all other polystomes of chelonians plus O. hippopotami is estimated at about 178 Mya. It is about 152 Mya between O. hippopotami and chelonian polystomes and about 131 Mya for the early diversification of chelonian polystomes.

4. Discussion 4.1. Host-parasite coevolution at a macroevolutionary scale Regarding the phylogenetic structure of polytomes, C. australensis being the most basal taxon within the Polystomatidae and intermediate between monogeneans of fish and tetrapods (Fig. 1), we may argue that polystomes evolved from fish parasites that colonized terrestrial hosts during the ecological transition of vertebrates close to lungfish (Brinkmann et al., 2004; George and Blieck, 2011), in the Late Devonian (Carroll, 2001), or even earlier in the early Middle Devonian (Niedz´wiedzki et al., 2010). Although polystomes of amphibians do not constitute a monophyletic group, i.e. the polystomes of chelonians form a clade that is nested within amphibian polystomes, we conclude that polystomes of amphibians coevolved with their hosts, as originally proposed by Verneau et al. (2002). The molecular dating estimate for the crown group of polystomes infecting tetrapods suggests indeed that these parasites originated in the ancestor of the Lissamphibia, which is plausible considering the sister group relationship between the ‘‘Polbatrach’’ and ‘‘Polchelon’’ lineages and the monophyly of extant amphibians. The age estimate for the deepest node within polystomes of tetrapods, i.e. about 334 Mya, is actually

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L. Héritier et al. / Molecular Phylogenetics and Evolution 92 (2015) 1–10

0.99

1.00 0.99

1.00

«Polbatrach» clade

1.00 1.00

0.99 0.90

0.99 0.91 1.00 1.00

0.99

0.98

1.00 0.99

0.99

1.00 1.00 1.00

0.99

1.00 0.99 0.99 1.00 0.91 0.99 0.99 0.99

1.00 1.00

«Polchelon» clade

1.00

0.98

0.99 0.99 0.98

0.98

0.99 0.97 0.99 0.99

0.99 1.00 1.00

Microcotyle erythrinii Pseudaxine trachuri Concinnocotyla australensis Sphyranura oligorchis Protopolystoma xenopodis Protopolystoma occidentalis Neodiplorchis scaphiopi Pseudodiplorchis americanus Pseudopolystoma dendriticum Polystoma sp. [R.a.] Neobat-1 Polystoma sp. [R.o.] Polystoma sp. [R.v.] Polystoma indicum Diplorchis ranae Neobat-2 Parapolystoma bulliense Diplorchis shilinensis Sundapolystoma chalconotae Madapolystoma sp. [B.w.] Kankana manampoka Neobat-3 Eupolystoma alluaudi Eupolystoma vanasi Polystoma lopezromani Polystoma naevius Polystoma nearcticum Polystoma floridana Polystoma cuvieri Wetapolystoma almae Neobat-4 Polystoma integerrimum Polystoma dianxiensis Polystoma gallieni Polystoma pelobatis Polystoma testimagna Polystoma marmorati Polystoma dawiekoki Metapolystoma cachani Nanopolystoma tinsleyi Oculotrema hippopotami Polystomoides malayi Chelon-1 Polystomoides siebenrockiellae Polystomoides sp. [P.d.] Polystomoides sp. [P.s.] Neopolystoma sp. [A.s.] Neopolystoma orbiculare Polystomoides sp. [T.s.s.] Chelon-2 Polystomoides oris Polystomoides sp. [P.n.] Polystomoides asiaticus Polystomoides tunisiensis Neopolystoma euzeti Neopolystoma sp. [K.l.] Neopolystoma liewi Chelon-3 Neopolystoma palpebrae Neopolystoma sp. [R.p.] Neopolystoma sp. [G.p.] Neopolystoma sp. [C.s.] Neopolystoma spratti Neopolystoma chelonidae

Fig. 1. Polystome tree inferred from the Bayesian analysis of four concatenated nuclear and mitochondrial genes. Red rectangles indicate the main polystome lineages within amphibians (Neobat-1 to 4) and chelonians (Chelon-1 to 3). Numbers at nodes indicate Bayesian Posterior Probabilities. Weakly supported branches (values <0.90) were collapsed into polytomies. See Table 1 for abbreviations in brackets. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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L. Héritier et al. / Molecular Phylogenetics and Evolution 92 (2015) 1–10 Microcotyle erythrinii Pseudaxine trachuri Concinocotyla australensis Sphyranura oligorchis Protopolystoma xenopodis Protopolystoma occidentalis Neodiplorchis scaphiopi

420 [419, 422]

275 [223, 330]

Pseudodiplorchis americanus Pseudopolystoma dendriticum Polystoma sp. [R.a.] Polystoma sp. [R.o.] Polystoma sp. [R.v.] Polystoma indicum

417 [415, 419]

Parapolystoma bulliense Diplorchis ranae

164 [163, 166]

Diplorchis shilinensis Sundapolystoma chalconotae Madapolystoma sp. [B.w.] Kankana manampoka

163 [161, 164]

Eupolystoma alluaudi Eupolystoma vanasi Polystoma lopezromani

59 [54, 64]

Polystoma naevius

334 [278, 386]

Polystoma nearcticum Polystoma floridana Polystoma cuvieri Wetapolystoma almae Polystoma integerrimum

61 [56, 67]

Polystoma dianxiensis Polystoma gallieni Polystoma pelobatis

25 [18, 32]

Polystoma testimagna Polystoma marmorati Polystoma dawiekoki Metapolystoma cachani Nanopolystoma tinsleyi Oculotrema hippopotami Polystomoides malayi

178 [120, 247]

Polystomoides siebenrockiellae

69 [37, 109]

Polystomoides sp. [P.d.] Polystomoides sp. [P.s.]

152 [105, 209]

Neopolystoma sp. [A.s.] Neopolystoma orbiculare Neopolystoma sp. [T.s.s.] Polystomoides oris

131 [93, 180]

Polystomoides sp. [P.n.]

54 [33, 81]

Polystomoides asiaticus Polystomoides tunisiensis

27 [11, 51]

Neopolystoma euzeti Neopolystoma sp. [K.l.]

115 [79, 158]

Neopolystoma liewi Neopolystoma palpebrae Neopolystoma sp. [R.p.] Neopolystoma sp. [G.p.]

98 [66, 136]

Neopolystoma sp. [C.s.] Neopolystoma spratti

58 [25, 95]

400

300

200

100

Neopolystoma chelonidae

0

Fig. 2. Divergence time estimates with their 95% confidence intervals (blue bars) inferred from the BEAST analysis of the four concatenated genes. Red bars indicate divergence time estimates with their 95% confidence intervals used for molecular calibrations. Scale in Million years. See Table 1 for abbreviations in brackets. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

consistent with palaeontological evidences (328–335 Mya) (Anderson et al., 2008) and molecular time estimates of the basal divergence in the Lissamphibia (i.e. the divergence between the Batrachia and the Gymnophiona lineages), although molecular estimates within amphibians vary from 316 Mya to 369 Mya depending on taxon sampling, genes and methods of calibration (Hugall et al., 2007; Igawa et al., 2008; Roelants et al., 2007; San Mauro, 2010; San Mauro et al., 2005; Zhang and Wake, 2009; Zhang et al., 2005). The ‘‘Polbatrach’’ lineage would have subsequently coevolved with anuran frogs assuming that S. oligorchis and P. dendriticum, which infect both caudatan hosts, evolved following host switching. Indeed, whereas other scenarios that suppose parasite duplications within ancestral host lineages followed by extinction processes can be retained, duplication events seem very unlikely according to Badets et al. (2011). This result thus implies that: (i) the molecular estimate for the crown group of batrachian polystomes, i.e. about 275 Mya, reveals the early divergence within the Anura rather than the early divergence within the Batrachia that was calibrated from 264 Mya to 358 Mya (Hugall et al., 2007; Igawa et al., 2008; Roelants et al., 2007; San Mauro, 2010; San Mauro et al., 2005; Zhang and Wake, 2009; Zhang et al., 2005); (ii) the sister group relationship between S. oligorchis and Protopolystoma reflects host switching from archaeobatrachian pipids to caudatans; (iii) the nested phylogenetic position of P. dendriticum within anuran polystomes involves host switching from either archaeobatrachian or neobatrachian hosts to caudatans. Finally, the phylogenetic pattern of anuran polystomes would illustrate phylogenetic relationships of their hosts (see Fig. 3), which show basal pipids and a sister group relationship between pelobatoids and neobatrachians (Frost et al., 2006; Pyron and Wiens, 2011; Roelants et al., 2007; San Mauro et al., 2005). Indeed, polystomes of archaeobatrachian pipids and pelobatoids, with the exception of P. pelobatis, are basal to a group that includes all polystomes of the Neobatrachia. On the opposite, the nested

position of P. pelobatis within neobatrachian polystomes implicates host switching from neobatrachian hosts to Pelobates cultripes (Cuvier, 1829), as suggested earlier by Bentz et al. (2001). On the other side, because primitive amniotes were mostly adapted to terrestrial environments, we must envisage the possibility that the ‘‘Polchelon’’ lineage would have coevolved with caecilians, which is illustrated by the occurrence of N. tinsleyi within Typhlonectes compressicauda (Duméril & Bibron, 1841). Polystomes would have secondarily invaded aquatic and semi-aquatic amniotes, namely turtles and hippos, at most recent times when some vertebrates reinvaded aquatic habitats. 4.2. Origin and evolution of chelonian polystomes According to our molecular calibrations, the crown divergence within the ‘‘Polchelon’’ clade is estimated at about 178 Mya, while the separation between O. hippopotami and chelonian polystomes is estimated at about 152 Mya. Based on fossil record, the oldest known turtle, Odontochelys semitestacea Li et al., 2008 of the Late Triassic, is dated at about 220 Mya (Li et al., 2008). Its basal phylogenetic position within fossil and extant turtles and added to the fact that it was found among marine deposits, led Li et al. (2008) to suggest that the earliest turtles may possibly have originated within aquatic habitats. Although several stem turtle fossils, such as Proganochelys, were originally considered as animals with aquatic preferences (see Rougier et al., 1995), new investigations on forelimbs morphometry and shell bone histology of extinct turtles shed new light on palaeoecology of basal turtles (Anquetin, 2011; Anquetin et al., 2009; Joyce and Gauthier, 2004; Li et al., 2008; Scheyer and Sander, 2007). Because many fossils have been interpreted or re-interpreted as terrestrial forms, like Proganochelys, Palaeochersis, Proterochersis and Kayentachelys, the ecological origin of turtles can be questioned. However Anquetin (2011) concluded that stem turtles were probably ecologically more diverse than

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L. Héritier et al. / Molecular Phylogenetics and Evolution 92 (2015) 1–10 Mantellidae

Rhacophoridae

Ranidae

Pyxicephalidae

Ranoidea

Ptychadenidae

Hyperoliidae

Neobatrachia

Microhylidae

Anura Bufonidae

Hyloidea

Leptodactylidae

Hylidae

Pelobatidae

Scaphiopodidae

Pipidae

Proteidae

Caudata Hynobiidae

Typhlonectidae

Gymnophiona

Outgroup

Fig. 3. Schematic phylogeny of infected amphibian families. Redraw from the skeletal representation of amphibian families and subfamilies relationships, after Pyron and Wiens (2011).

previously though as Odontochelys, the most primitive turtle, and Eileanchelys, a most recent fossil, were aquatic. Regardless of the primitive habitat for the earliest turtles, i.e. aquatic versus terrestrial environments, it is now well accepted that the ancestor of all living turtles was aquatic (Joyce and Gauthier, 2004). Their parasites, i.e. polystomes, are obligate parasitic flatworms with a direct life-cycle. Mature worms lay eggs in water that, after three to four weeks of development, give rise to the infective larval stage, i.e. the oncomiracidium. These parasites are then able to attach and infect a new host quite rapidly (see Verneau et al., 2009a). Because water is a prerequisite for egg development and larval transmission, host switching from aquatic or semi-aquatic amphibians to turtles must have occurred when chelonians were already adapted to aquatic environments. Since caecilians have stayed dependent on water to reproduce over evolutionary times, a possible switch from caecilians to freshwater turtles at about 178 Mya cannot be ruled out, which suggests that some primitive stem turtles could have already been adapted to an aquatic life style. Because that molecular estimate corresponds roughly to the age of the turtle crown (Joyce et al., 2013a,b; Lourenço et al., 2012; Sterli et al., 2013), that switch would have occurred to the ancestor of pleurodires and cryptodires that likely lived in aquatic environments (Joyce and Gauthier, 2004). However, if we consider a switch from caecilians to hippos at about 152 Mya to explain the occurrence of polystomes within aquatic mammals (see Section 4.3), then chelonian polystomes would find their origins later than about 178 Mya, namely after the early divergence of Testudines. Another possibility is to consider a younger date for the crown chelonian divergence (see Chiari et al., 2012), which would reconcile molecular datings of host and parasite assemblages. Chelonian polystomes (Neopolystoma, Polystomoides and Polystomoidella) are globally dispersed among pleurodires and cryptodires of distinct families (Table 1). They have colonized discrete ecological niches within the host, i.e. the urinary bladder, the pharyngeal cavity and the conjunctival sacs (Table 1), and are highly host and site specific, except when turtles are translocated in new environments (see Meyer et al., 2015; Verneau et al., 2011). Regarding our phylogeny that comprises 20 species collected from 16 distinct turtles (Fig. 1), it is actually extremely

difficult to unravel processes that have shaped the evolutionary history of extant chelonian polystomes. The crown divergence, dated at about 131 Mya, indicates that chelonian polystomes might have radiated when modern turtles were already differentiated in the Late Jurassic, i.e. after the split between pleurodires and cryptodires. Regardless of the temporal scheme adopted for host and parasite divergences, how do we explain such discordant phylogenetic relationships between hosts and their parasites? Neopolystoma spratti Pichelin, 1995 and N. chelodinae (MacCallum, 1918) are for instance two sister polystome species infecting the same host Chelodina longicollis (Shaw, 1794) in Australia, the first being found in the conjunctival sacs, the second in the urinary bladder. Because their divergence is estimated at about 58 Mya, intra host duplication in C. longicollis is very unlikely. The same interrogation arises for N. euzeti Combes & Ktari, 1976 and P. tunisiensis Gonzales & Mishra, 1977, which are two sister parasite species infecting the same host species, i.e. Mauremys leprosa (Schweigger, 1812) of Algeria, and whose divergence dates back to about 27 Mya. Similarly, the divergence between two pleurodire and cryptodire parasite sublineages (N. spratti and N. chelodinae versus N. liewi Du Preez & Lim, 2000, N. palpebrae Strelkov, 1950, Neopolystoma sp. [R.p.], Neopolystoma sp. [G.p.], Neopolystoma sp. [C.s.] and Neopolystoma sp. [K.l.]), is estimated at about 98 Mya, which is long after the split between the two host lineages, Pleurodira versus Cryptodira. All these examples raise numerous interrogations about precise evolutionary scenarios that may account for non-concordant host and parasite phylogenetic patterns. Is evolution of chelonian polystomes the consequence of plate tectonics, as illustrated for early neobatrachian polystomes (see Badets et al., 2011), or the result of codivergences, host dispersal followed by host switching, intra host speciation or a mixture of all these events? These questions remain to be clarified using more diverse polystome sampling, especially from pleurodire turtles. 4.3. Origin of Oculotrema hippopotami O. hippopotami is clearly nested within the ‘‘Polchelon’’ clade, and can be regarded as the sister taxon of chelonian polystomes, from which it would have diverged at about 152 Mya according

L. Héritier et al. / Molecular Phylogenetics and Evolution 92 (2015) 1–10

to our molecular calibrations (Fig. 2). The common hippo, Hippopotamus amphibious Linnaeus, 1758, and the pygmy hippo, Choeropsis liberiensis Leidy, 1852, are the only two representatives of extant hippopotamids that would have diverged from each other at about 5.7 Mya (Montgelard et al., 1997). Whereas hippopotamids may have originated in the Early Miocene, Orliac et al. (2010) considered an origin at about 21 Mya, while Pickford (2011) argued for their first appearance at about 16.5 Mya. Regardless of the exact timing of their origin, hippopotamids may have adopted semi-aquatic habits at least as early as the latest Miocene (Boisserie et al., 2011). So, if we assume that O. hippopotami originated from the stem branch of chelonian polystomes, it would suppose a switch at about 152 Mya, which is impossible regarding the Miocene origin of hippopotamids. If we however consider that the ancestral host for O. hippopotami may be found within the clade of extant turtles, it should be sought among African turtles, which is also very unlikely regarding the polystome sampling that includes 20 chelonian parasite species among which two were collected from two distinct host species in the Afrotropic ecozone, namely Pelomedusa subrufa (Bonnaterre, 1789) and Pelusios castaneus (Schweigger, 1812) of Nigeria. Furthermore polystomes have never been found among South African turtles despite a thorough sampling the past seven years (Du Preez and Verneau, unpublished observations). Another possibility is to consider a switch from stem turtles to earliest mammals, which are suspected to have originated within the interval 162.5–191.1 Mya after Benton and Donoghue (2007). Indeed, whether mammals probably arose as terrestrial forms (see Rowe, 1999), convergent adaptations to aquatic environments happened repeatedly along their evolution. However, this hypothesis implies that polystomes should also be found within certain extant aquatic mammals, like cetaceans and sirenians, which is actually not the case. Finally considering that O. hippopotami may originate from a presumably extinct chelonian polystome lineage is extremely difficult to validate, or invalidate, without evidence of parasite fossil. Though the presence of polystomes within marine vertebrates cannot be excluded, these parasites are presently known only from freshwater environments. Therefore the evolutionary scenario, which is at this stage the most likely to explain the occurrence of O. hippopotami, is to consider a switch from another extant caecilian to common hippos. In comparison to most anuran and chelonian polystomes (see Verneau, 2004), caecilian polystomes were described only recently by Du Preez et al. (2008, 2014). We can therefore expect a larger polystome diversity within caecilians that are mainly distributed in the Southern tropical parts of the world, especially in South America, South-East Asia and Africa (see Wilkinson et al., 2011). The three known caecilian polystome species are from South American hosts, Caecilia pachynema Günther, 1859 and Caecilia gracilis Shaw, 1802 of the Caeciliidae and T. compressicauda of the Typhlonectidae. According to Zhang and Wake (2009), Caecilia of the paraphyletic Caeciliidae diverged from its closest relative Typhlonectidae at about 106 Mya while African Caeciliidae and Scolecomorphidae diverged from South American Caeciliidae and Typhlonectidae at about 175–185 Mya. If one assumes that African caecilian polystomes do exist on one hand, and that an African caecilian could be the ancestral host for O. hippopotami on the other, those polystomes should be sought among hosts of African caecilian families, which are primarily distributed in Kenya, Tanzania and Ghana, where hippos also occur.

5. Conclusions Our results clearly show that polystomes of amphibians originated very early, during the ecological transition from fish to tetrapod in the Middle-Late Devonian, and further coevolved with their

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hosts in the Mesozoic and Cenozoic periods. Because the life-cycle of this monogenean parasite requires water for larval development, hosts must be adapted to aquatic environments for infestation. If this holds for extant amphibians, aquatic chelonians and semi-aquatic common hippos, the presence and evolution of polystomes among these groups of vertebrates are indicative of early aquatic ecological habits within some ancestral lineages. Acknowledgments Partial financial support for this research was provided by grants from the CNRS. This study is part of the Master’s degree of L.H. Authors also thank two anonymous reviewers for helpful comments. References Ahlberg, P.E., 1999. Palaeontology: something fishy in the family tree. Nature 397, 564–565. Anderson, J.S., Reisz, R.R., Scott, D., Fröbisch, N.B., Sumida, S.S., 2008. A stem batrachian from the Early Permian of Texas and the origin of frogs and salamanders. Nature 453, 515–518. Anquetin, J., 2011. Evolution and palaeoecology of early turtles: a review based on recent discoveries in the Middle Jurassic. Bull. Soc. géol. Fr. 182, 235–244. Anquetin, J., Barrett, P.M., Jones, M.E.H., Moore-Fay, S., Evans, S.E., 2009. A new stem turtle from the Middle Jurassic of Scotland: new insights into the evolution and palaeoecology of basal turtles. Proc. Roy. Soc. B – Biol. Sci. 276, 879–886. Arnason, U., Gullberg, A., Janke, A., 2004. Mitogenomic analyses provide new insights into cetacean origin and evolution. Gene 333, 27–34. Badets, M., Whittington, I., Lalubin, F., Allienne, J.-F., Maspimby, J.-L., Bentz, S., Du Preez, L.H., Barton, D., Hasegawa, H., Tandon, V., Imkongwapang, R., Ohler, A., Combes, C., Verneau, O., 2011. Correlating early evolution of parasitic platyhelminths to Gondwana breakup. Syst. Biol. 60, 762–781. Bajpai, S., Gingerich, P.D., 1998. A new Eocene archaeocete (Mammalia, Cetacea) from India and the time of origin of whales. Proc. Natl. Acad. Sci. USA 95, 15464–15468. Benton, M.J., Donoghue, P.C.J., 2007. Paleontological evidence to date the tree of life. Mol. Biol. Evol. 24, 26–53. Benton, M., Donoghue, P.C.J., Asher, R.J., 2009. Calibrating and constraining molecular clocks. In: Hedges, S.B., Kumar, S. (Eds.), The Timetree of Life. Oxford University Press, pp. 35–86. Bentz, S., Leroy, S., Du Preez, L., Mariaux, J., Vaucher, C., Verneau, O., 2001. Origin and evolution of African Polystoma (Monogenea: Polystomatidae) assessed by molecular methods. Int. J. Parasitol. 31, 697–705. Bentz, S., Sinnappah-Kang, N.D., Lim, L.-H.S., Lebedev, B., Combes, C., Verneau, O., 2006. Historical biogeography of amphibian parasites, genus Polystoma (Monogenea: Polystomatidae). J. Biogeogr. 33, 742–749. Boeger, W.A., Kritsky, D.C., 2001. Phylogenetic relationships of the Monogenoidea. In: Littlewood, D.T.J., Bray, R.A. (Eds.), Interrelationships of the Platyhelminthes. Taylor & Francis London, pp. 92–102. Boisserie, J.-R., Fisher, R.E., Lihoreau, F., Weston, E.M., 2011. Evolving between land and water: key questions on the emergence and history of the Hippopotamidae (Hippopotamoidea, Cetancodonta, Cetartiodactyla). Biol. Rev. 86, 601–625. Botella, H., Blom, H., Dorka, M., Ahlberg, P.E., Janvier, P., 2007. Jaws and teeth of the earliest bony fishes. Nature 448, 583–586. Brinkmann, H., Venkatesh, B., Brenner, S., Meyer, A., 2004. Nuclear protein-coding genes support lungfish and not the coelacanth as the closest living relatives of land vertebrates. Proc. Natl. Acad. Sci. USA 101, 4900–4905. Carroll, R.L., 2001. The origin and early radiation of terrestrial vertebrates. J. Paleont. 75, 1202–1213. Chiari, Y., Cahais, V., Galtier, N., 2012. Phylogenomic analyses support the position of turtles as the sister group of birds and crocodiles (Archosauria). BMC Biol. 10, 65. De Rijk, P., De Wachter, R., 1993. DCSE, an interactive tool for sequence alignment and secondary structure research. Bioinformatics 9, 735–740. Drummond, A.J., Suchard, M.A., Xie, D., Rambaut, A., 2012. Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol. Biol. Evol. 29, 1969–1973. Du Preez, L.H., Moeng, I.A., 2004. Additional morphological information on Oculotrema hippopotami Stunkard, 1924 (Monogenea: Polystomatidae) parasitic on the African hippopotamus. Afr. Zool. 39, 225–233. Du Preez, L.H., Wilkinson, M., Huyse, T., 2008. The first record of polystomes (Monogenea: Polystomatidae) from caecilian hosts (Amphibia: Gymnophiona), with the description of a new genus and two new species. Syst. Parasitol. 69, 201–209. Du Preez, L.H., Badets, M., Verneau, O., 2014. Assessment of platyhelminth diversity within amphibians of French Guiana revealed a new species of Nanopolystoma (Monogenea: Polystomatidae) in the caecilian Typhlonectes compressicauda. Folia Parasit. 61, 537–542. Frost, D.R., Grant, T., Faivovich, J., Bain, R.H., Haas, A., Haddad, C.F.B., De Sa, R.O., Channing, A., Wilkinson, M., Donnellan, S.C., Raxworthy, C.J., Campbell, J.A., Blotto, B.L., Moler, P., Drewes, R.C., Nussbaum, R.A., Lynch, J.D., Green, D.M.,

10

L. Héritier et al. / Molecular Phylogenetics and Evolution 92 (2015) 1–10

Wheeler, . The amphibian tree of life. Bull. Am. Mus. Nat. Hist. 297, 1–291. Gatesy, J., 1997. More DNA support for a Cetacea/hippopotamidae clade: the bloodclotting protein gene !-fibrinogen. Mol. Biol. Evol. 14, 537–543. Gayet, M., Rage, J.-C., Sempere, T., Gagnier, P.-Y., 1992. Modalités des échanges de vertébrés continentaux entre l’Amérique du Nord et l’Amérique du Sud au Crétacé supérieur et au Paléocène. Bull. Soc. géol. France 6, 781–791. George, D., Blieck, A., 2011. Rise of the earliest tetrapods: an early Devonian origin. PLoS ONE 6, e22136. Gernhard, T., 2008. The conditioned reconstructed process. J. Theor. Biol. 253, 769– 778. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754–755. Hugall, A.F., Foster, R., Lee, M.S.Y., 2007. Calibration choice, rate smoothing, and the pattern of tetrapod diversification according to the long nuclear gene RAG-1. Syst. Biol. 56, 543–563. Igawa, T., Kurabayashi, A., Usuki, C., Fujii, T., Sumida, M., 2008. Complete mitochondrial genomes of three neobatrachian anurans: a case study of divergence time estimation using different data and calibration settings. Gene 407, 116–129. Janvier, P., 1998. Forerunners of four legs. Nature 395, 748–749. Joyce, W.G., Gauthier, J.A., 2004. Palaeoecology of Triassic stem turtles sheds new light on turtle origins. Proc. Roy. Soc. B – Biol. Sci. 271, 1–5. Joyce, W.G., Parham, J.F., Lyson, T.R., Warnock, R.C.M., Donoghue, P.C.J., 2013a. A divergence dating analysis of turtles using fossil calibrations: an example of best practices. J. Paleont. 87, 612–634. Joyce, W.G., Schoch, R.R., Lyson, T.R., 2013b. The girdles of the oldest fossil turtles, Proterochersis robusta, and the age of the turtle crown. BMC Evol. Biol. 13, 266. Katayama, T., Nishioka, M., Yamamoto, M., 1996. Phylogenetic relationships among turbellarian orders inferred from 18S rDNA sequences. Zool. Sci. 13, 747–756. Li, C., Wu, X.-C., Rieppel, O., Wang, L.-T., Zhao, L.-J., 2008. An ancestral turtle from the Late Triassic of southwestern China. Nature 456, 497–501. Littlewood, D.T.J., Rohde, K., Clough, K.A., 1997. Parasite speciation within or between host species? Phylogenetic evidence from site-specific polystome monogeneans. Int. J. Parasitol. 27, 1289–1297. Littlewood, D.T.J., Rohde, K., Bray, R.A., Herniou, E.A., 1999. Phylogeny of the Platyhelminthes and the evolution of parasitism. Biol. J. Linn. Soc. Lond. 68, 257–287. Lourenço, J.M., Claude, J., Galtier, N., Chiari, Y., 2012. Dating cryptodiran nodes: Origin and diversification of the turtle superfamily Testudinoidea. Mol. Phylogenet. Evol. 62, 496–507. Lu, J., Zhu, M., Long, J.A., Zhao, W., Senden, T.J., Jia, L., Qiao, T., 2012. The earliest known stem-tetrapod from the Lower Devonian of China. Nat. Commun. 3, 1160. McLoughlin, S., 2001. The breakup history of Gondwana and its impact on preCenozoic floristic provincialism. Aust. J. Bot. 49, 271–300. Meyer, L., Du Preez, L., Bonneau, E., Héritier, L., Quintana, M.F., Valdeón, A., Sadaoui, A., Kechemir-Issad, N., Palacios, C., Verneau, O., 2015. Parasite host-switching from the invasive American red-eared slider, Trachemys scripta elegans, to the native Mediterranean pond turtle, Mauremys leprosa, in natural environments. Aquat. Invasions 10, 79–91. Montgelard, C., Catzeflis, F.M., Douzery, E., 1997. Phylogenetic relationships of artiodactyls and cetaceans as deduced from the comparison of cytochrome b and 12S rRNA mitochondrial sequences. Mol. Biol. Evol. 14, 550–559. Morrison, C., Du Preez, L., 2011. Turtle polystomes of the world. Neopolystoma, Polystomoidella & Polystomoides, VDM Verlag Dr. Muller, Saarbrücken. Motani, R., Minoura, N., Ando, T., 1998. Ichthyosaurian relationships illuminated by new primitive skeletons from Japan. Nature 393, 255–257. Niedz´wiedzki, G., Szrek, P., Narkiewicz, K., Narkiewicz, M., Ahlberg, P.E., 2010. Tetrapod trackways from the early Middle Devonian period of Poland. Nature 463, 43–48. Orliac, M., Boisserie, J.-R., MacLatchy, L., Lihoreau, F., 2010. Early Miocene hippopotamids (Cetartiodactyla) constrain the phylogenetic and spatiotemporal settings of hippopotamid origin. Proc. Natl. Acad. Sci. USA 107, 11871–11876. Perkins, E.M., Donnellan, S.C., Bertozzi, T., Whittington, I.D., 2010. Closing the mitochondrial circle on paraphyly of the Monogenea (Platyhelminthes) infers evolution in the diet of parasitic flatworms. Int. J. Parasitol. 40, 1237–1245. Petzold, A., Vargas-Ramirez, M., Kehlmaier, C., Vamberger, M., Branch, W.R., Du Preez, L., Hofmeyr, M.D., Meyer, L., Schleicher, A., Široky, P., Fritz, U., 2014. A revision of African helmeted terrapins (Testudines: Pelomedusidae: Pelomedusa), with descriptions of six new species. Zootaxa 3795, 523–548. Pichelin, S., Whittington, I., Pearson, J., 1991. Concinnocotyla (Monogenea: Polystomatidae), a new genus for the polystome from the Australian lungfish Neoceratodus forsteri. Syst. Parasitol. 18, 81–93. Pickford, M., 2011. Morotochoerus from Uganda (17.5 Ma) and Kenyapotamus from Kenya (13–11 Ma): implications for hippopotamid origins. Estud. Geol. Madrid 67, 523–540. Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14, 817–818. Prudhoe, S., Bray, R.A., 1982. Platyhelminth parasites of the Amphibia. Oxford University Press, London. Pyron, A.R., Wiens, J.J., 2011. A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians. Mol. Phylogenet. Evol. 61, 543–583.

Rage, J.-C., 1988. Gondwana, Tethys, and terrestrial vertebrates during the Mesozoic and Cainozoic. In: Audley-Charles, M.G., Hallam, A. (Eds.), Gondwana and Tethys, Special Publications. Geological Society, London, pp. 255–273. Reisz, R.R., 1997. The origin and early evolutionary history of amniotes. Trends Ecol. Evol. 12, 218–222. Rieppel, O., 1999. Turtle origins. Science 283, 945–946. Roelants, K., Gower, D.J., Wilkinson, M., Loader, S.P., Biju, S.D., Guillaume, K., Moriau, L., Bossuyt, F., 2007. Global patterns of diversification in the history of modern amphibians. Proc. Natl. Acad. Sci. USA 104, 887–892. Rohde, K., 1994. Niche restriction in parasites: proximate and ultimate causes. Parasitology 109, 69–84. Rougier, G.W., de la Fuente, M.S., Arcucci, A.B., 1995. Late Triassic turtles from South America. Science 268, 855–858. Rowe, T., 1999. At the roots of the mammalian family tree. Nature 398, 283–284. San Mauro, D., 2010. A multilocus timescale for the origin of extant amphibians. Mol. Phylogenet. Evol. 56, 554–561. San Mauro, D., Vences, M., Alcobendas, M., Zardoya, R., Meyer, A., 2005. Initial diversification of living amphibians predated the breakup of Pangaea. Am. Nat. 165, 590–599. Scheyer, T.M., Sander, P.M., 2007. Shell bone histology indicates terrestrial palaeoecology of basal turtles. Proc. Roy. Soc. B – Biol. Sci. 274, 1885–1893. Sinnappah, N.D., Lim, L.H.S., Rohde, K., Tinsley, R., Combes, C., Verneau, O., 2001. A paedomorphic parasite associated with a neotenic amphibian host: phylogenetic evidence suggests a revised systematic position for Sphyranuridae within anuran and turtle Polystomatoineans. Mol. Phylogenet. Evol. 18, 189–201. Sterli, J., Pol, D., Laurin, M., 2013. Incorporating phylogenetic uncertainty on phylogeny-based palaeontological dating and the timing of turtle diversification. Cladistics 29, 233–246. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Telford, M.J., Wise, M.J., Gowri-Shankar, V., 2005. Consideration of RNA secondary structure significantly improves likelihood-based estimates of phylogeny: examples from the bilateria. Mol. Biol. Evol. 22, 1129–1136. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673–4680. Thurston, J.P., Laws, R.M., 1965. Oculotrema hippopotami (Trematoda: Monogenea) in Uganda. Nature 205, 1127. Ursing, B.M., Arnason, U., 1998. Analyses of mitochondrial genomes strongly support a hippopotamus-whale clade. Proc. Natl. Acad. Sci. USA 265, 2251– 2255. Van de Peer, Y., Robbrecht, E., de Hoog, S., Caers, A., De Rijk, P., De Wachter, R., 1999. Database on the structure of small subunit ribosomal RNA. Nucl. Acids Res. 27, 179–183. Verneau, O., 2004. Origine et Evolution des monogènes Polystomatidae, parasites d’amphibiens et de chéloniens d’eau douce. Habilitation à Diriger des Recherches, Université de Perpignan, Perpignan. Verneau, O., Bentz, S., Sinnappah, N.D., Du Preez, L., Whittington, I., Combes, C., 2002. A view of early vertebrate evolution inferred from the phylogeny of polystome parasites (Monogenea: Polystomatidae). Proc. Roy. Soc. B – Biol. Sci. 269, 535–543. Verneau, O., Du Preez, L., Badets, M., 2009a. Lessons from parasitic flatworms about evolution and historical biogeography of their vertebrate hosts. CR. Biol. 332, 149–158. Verneau, O., Du Preez, L.H., Laurent, V.R., Raharivololoniaina, L., Glaw, F., Vences, M., 2009b. The double odyssey of Madagascan polystome flatworms leads to new insights on the origins of their amphibian hosts. Proc. Roy. Soc. B – Biol. Sci. 276, 1575–1583. Verneau, O., Palacios, C., Platt, T., Alday, M., Billard, E., Allienne, J.-F., Basso, C., Du Preez, L.H., 2011. Invasive parasite threat: parasite phylogenetics reveals patterns and processes of host-switching between non-native and native captive freshwater turtles. Parasitology 138, 1778–1792. Wilkinson, M., San Mauro, D., Sherratt, E., Gower, D.J., 2011. A nine-family classification of caecilians (Amphibia: Gymnophiona). Zootaxa 2874, 41–64. Yu, X., Zhu, M., Zhao, W., 2010. The origin and diversification of osteichthyans and sarcopterygians: rare Chinese fossil findings advance research on key issues of evolution. Bull. Chin. Acad. Sci. 24, 71–75. Zhang, P., Wake, M.H., 2009. A mitogenomic perspective on the phylogeny and biogeography of living caecilians (Amphibia: Gymnophiona). Mol. Phylogenet. Evol. 53, 479–491. Zhang, P., Zhou, H., Chen, Y.-Q., Liu, Y.-F., Qu, L.-H., 2005. Mitogenomic perspectives on the origin and phylogeny of living amphibians. Syst. Biol. 54, 391–400. Zhu, M., Fan, J., 1995. Youngolepis from the Xishancun formation (Early Lochkovian) of Qujing, China. Geobios 19, 293–299. Zhu, M., Yu, X., 2002. A primitive fish close to the common ancestor of tetrapods and lungfish. Nature 418, 767–770. Zhu, M., Zhao, W., Jia, L., Lu, J., Qiao, T., Qu, Q., 2009. The oldest articulated osteichthyan reveals mosaic gnathostome characters. Nature 458, 469–474. Zucker, M., 2003. Mfold web server for nucleic acid folding and hybridization prediction. Nucl. Acids Res. 31, 3406–3415.