1 A quantum leap in the evolution of platyhelminths: host-switching from turtles to hippopotamuses

2 illustrated from a phylogenetic meta-analysis of polystomes (Monogenea, Polystomatidae)

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4 Olivier Verneau<sup>a, b, c,*</sup>, Gerald R. Johnston<sup>d</sup>, Louis Du Preez<sup>c, e</sup>

5 <sup>a</sup>University of Perpignan Via Domitia, Centre de Formation et de Recherche sur les Environnements Méditerranéens, UMR 5110, F-66860, Perpignan, France

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7 <sup>b</sup>CNRS, Centre de Formation et de Recherche sur les Environnements Méditerranéens, UMR5110, F-66860, Perpignan, France

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9 <sup>c</sup>Unit for Environmental Sciences and Management, North-West University, Potchefstroom campus, Private Bag X6001, Potchefstroom 20520, South Africa

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11 <sup>d</sup>Department of Natural Sciences, Santa Fe College, Gainesville, Florida 32606, USA

12 <sup>e</sup>South African Institute for Aquatic Biodiversity, Private Bag 1015, Grahamstown 6140, South Africa

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14 *Corresponding author. University of Perpignan Via Domitia, Centre de Formation et de Recherche sur les Environnements Méditerranéens, UMR 5110, F-66860, Perpignan, France.

16 E-mail address: verneau@univ-perp.fr (O. Verneau).

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ABSTRACT

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While monogenean worms are mainly parasites of the gills and skin of fish, and to a lesser extent
parasites of the oral cavity, urinary bladder, and/or conjunctival sacs of amphibians and freshwater
turtles, *Oculotrema hippopotami* Stunkard, 1924 is the single monogenean polystome reported from
a mammal, the common hippopotamus (*Hippopotamus amphibius* Linnaeus). Several hypotheses
have been suggested in the last decade to explain the origin of this enigmatic parasite which infects
the conjunctival sacs of *H. amphibius*. Based on a molecular phylogeny inferred from nuclear (28S
and 18S) and mitochondrial (12S and COI) sequences of *O. hippopotami* and chelonian polystomes,
we found a sister group relationship between *O. hippopotami* and *Apaloneotrema moleri* (Du Preez
& Morrison, 2012). This result suggests lateral parasite transfer between freshwater turtles and
hippopotamuses, thus likely reflecting one of the most exceptional known examples of host-switching
in the course of vertebrate evolution. It also demonstrates that the proximity in the ecological habitat
of parasites within host species is an important feature for their speciation and diversification.
Because *A. moleri* and its host, the Florida softshell turtle (*Apalone ferox* (Schneider)), are restricted
to the USA, we suggest that an ancestral stock of parasites may have been isolated on primitive
African trionychids after they diverged from their American relatives, and then switched to
hippopotamuses or anthracotheres in Africa.

Keywords: Common hippopotamus; *Oculotrema hippopotami*; Chelonian; Trionychidae; *Apaloneotrema moleri*; *Apalone ferox*
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# 1. Introduction

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*Oculotrema hippopotami* Stunkard, 1924 is likely one of the most intriguing and fascinating monogenean parasites infecting a vertebrate host. Monogeneans are parasitic platyhelminths typically found on the gills and skin of chondrichthyan and teleostean fishes (Whittington et al., 2000; Poulin, 2002). However, members of the Gyrodactylidae, Lagarocotylidae, and Iagotrematidae of the subclass Monopisthocotylea and the Polystomatidae sensu Sinnappah et al. (2001) of the subclass Polyopisthocotylea are parasites of rhipidistians (Bentz et al., 2003). The Polystomatidae is the most speciose of these families, parasitizing amphibians, freshwater turtles, and the Australian lungfish (*Neoceratodus forsteri* Krefft) (Verneau et al., 2002). One polystome (*O. hippopotami*) has also been reported from a mammal (Sunkard, 1924), the common hippopotamus (*Hippopotamus amphibius* Linnaeus) hereafter referred to as hippo. Polystomes of tetrapods are usually found in the urinary bladder, the pharyngeal cavity, or the conjunctival sacs of their host. Amphibians typically host a single polystome species in the urinary bladder, but polystomes of the genera *Polystoma* Zeder, 1800 and *Metapolystoma* Combes, 1976 have also been documented on the gills of anuran tadpoles (Combes, 1966; Maeder, 1973; Murith et al., 1977; Murith, 1981). Polystomes can also be found on the gills or skin of American salamanders (Alvey, 1933, 1936; Hughes and Moore, 1943) and in the phallodeum of caecilians (Du Preez et al., 2008). Within chelonians, up to three distinct species can occur sympatrically within the same chelonian host species, occupying different sites of infestation (Morrison and Du Preez, 2011). Finally, the single polystome species infecting hippos occurs within the conjunctival sacs and on the surface of the eye.

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The earliest original description of polystomes dates to the middle of the 18th century when, in 1758, Roesel von Rosenhof discovered the first monogenean from the common frog (*Rana temporaria* Linnaeus) (Prudhoe and Bray, 1982). Zeder (1800) reported this parasite as *Polystoma integerrimum* (Fröhlich, 1791). In the 19th century, only five new species were recorded from
amphibian and chelonian hosts of the USA, Australia, and Europe. When *O. hippopotami* was described by Stunkard (1924), there were no more than 15 reported and described polystome species in the world and just a single species from Africa. Because this parasite was the first polystome recorded from the conjunctival sacs of its host, and because it was described from a few specimens that were preserved with the label "from the eye of hippopotamus" with doubtful geographical origins (Stunkard, 1924), the validity of the species was questioned until redescription (Thurston and Laws, 1965). Thurston and Laws (1965) re-examined several polystome specimens that were collected from hippos of north-eastern shores of Lake Edward in Western Uganda and concluded that *O. hippopotami* was valid. Subsequently, *O. hippopotami* has been reported from hippos in the Letaba River of the Kruger National Park of South Africa (McCully et al., 1967) and in the Kazinga Channel and Lake Edward in the Queen Elizabeth National Park of Western Uganda (Thurston, 1968a, b). *Oculotrema hippopotami* was further redescribed from parasites collected during a hippo culling operation in the KwaZulu-Natal Province of South Africa (Du Preez and Moeng, 2004).

Although *O. hippopotami* is taxonomically distinct in its general morphology that shows a haptor with six well-developped suckers (Du Preez and Moeng, 2004), numerous questions have arisen regarding the uniqueness of this enigmatic parasite. For example, which ancestral parasite lineage or species gave rise to *O. hippopotami* during the course of vertebrate evolution, and where and when did speciation occur? Following comparisons of ciliated cells on the tegument of oncomiracidia between *O. hippopotami* and several species of *Protopolystoma* Bychowsky, 1857 infecting the urinary bladder of amphibians and *Polystomoides* Ward, 1917 infecting the urinary bladder or the oral / pharyngeal cavities of chelonians, Tinsley (2013) tentatively concluded an origin of *O. hippopotami* from species of *Neopolystoma* Price, 1939 infecting the conjunctival sacs. Nevertheless, he did not reject an alternative hypothesis considering an origin of *O. hippopotami* from representatives of *Polystomoides* occurring in the anterior sites of their host. Regardless of the
scenario proposed to explain the presence of *O. hippopotami* on hippos, it is difficult to draw a
conclusion without establishing the evolutionary relationships among species in a phylogenetic
framework where representatives of the main polystome genera have been investigated and
compared. Tinsley (2013), however, based on the relatively well documented origins of hippos,
considered that *Oculotrema* Stunkard, 1924 could have arisen quite recently in the Hippopotaminae.
He also did not exclude a more ancient origin of the genus considering that *O. hippopotami* could be
a relict of a more widespread species that colonized mammals with a wider geographical range.

From the most comprehensive phylogenetic study covering 55 species from the main genera
of polystomatids, Héritier et al. (2015) showed a sister group relationship between *O. hippopotami*
and the chelonian polystome lineage, with a basal position of *Nanopolystoma* Du Preez, Wilkinson
& Huyse, 2008 infecting amphibian caecilians. Integrating phylogenetic relationships of
polystomatids and molecular clock calibrations, Héritier et al. (2015) concluded that a parasite's
switch from caecilians to freshwater turtles approximately 178 million years ago (Mya) would best
explain the origin of chelonian polystomes, thus implying that some primitive stem turtles could have
already been adapted to aquatic ecosystems in ancient times. On the contrary, because the occurrence
of *O. hippopotami* within extant hippos could not be unambiguously explained from its phylogenetic
position, Héritier et al. (2015) suggested several scenarios to account for its origins. The first
hypothesis considered a very deep origin of the hippo's parasite lineage dating back to approximately
152 Mya, following a switch from the stem branch of chelonian polystomes. It was nonetheless
discarded by Héritier et al. (2015) regarding the Miocene origin of hippopotamids (Boisserie et al.,
2011; Pickford, 2011). The second hypothesis considered a parasite's switch from an ancestral stock
of turtles, when they may have originated, to early mammals already adapted to aquatic environments.
This implied that polystomes would have survived and evolved within extant aquatic mammals such
as cetaceans and sirenians before switching to extant hippos, which has never been documented. The
third hypothesis considered a switch from extant African aquatic turtles to hippos. However, despite a thorough sampling of chelonian polystomes in Nigeria and South Africa where turtles and hippos occur sympatrically, the only two polystome species found today infecting African freshwater turtles are not closely related to *O. hippopotami*. Therefore hypotheses two and three were also ruled out by Héritier et al. (2015). The fourth hypothesis considered a parasite's switch from extant African caecilians to hippos. Because caecilian polystomes have only been reported and described from South America, from two host species of unknown localities (Du Preez et al., 2008) and from a third species of French Guiana (Du Preez et al., 2014), one may expect that African caecilian polystomes, if they actually exist, will break the long branch of *O. hippopotami*. This is why Héritier et al. (2015) considered the last hypothesis as the most likely to account for the origins of this parasite.

Following extensive polystome sampling in North Carolina and Florida in the USA, Du Preez and Verneau (2020) revised the classification of chelonian polystomes. According to a molecular phylogeny that included 28 distinct polystome species infecting freshwater turtles, and following reexamination of morphological characters of relevant polystome specimens, Du Preez and Verneau (2020) described new genera within the Polystomatidae. In addition to the five previously recognized genera of chelonian polystomatids (*Neopolystoma*, *Polystomoidella* Price, 1939, *Polystomoides sensu Tinsley* (2017), *Uropolystomoides*, Tinsley & Tinsley, 2016 and *Uteropolystomoides* Tinsley, 2017), three genera (*Apaloneotrema* Du Preez & Verneau, 2020, *Aussietrema* Du Preez & Verneau, 2020 and *Fornixtrema* Du Preez & Verneau, 2020) were erected from redescription of polystomes that were previously considered members of *Neopolystoma*. Despite the fact that these parasites all share the same ecological niche, namely the conjunctival sacs of their hosts, these genera were divided into three unrelated phylogenetic lineages with *Apaloneotrema* the most basal taxon within chelonian polystomatids (Du Preez and Verneau, 2020). Based on morphological similarities between *Oculotrema* and *Apaloneotrema*, Du Preez and Verneau (2020) claimed that *Apaloneotrema* could be
the closest relative of *O. hippopotami*. This hypothesis was supported by the confinement of reproductive and digestive organs to the anterior third of the body within *Apaloneotrema*, *O. hippopotami*, and *Fornixtrema* but not in *Aussietrema*, and by long eggs with more rounded ends within *Apaloneotrema* and *O. hippopotami*, unlike the fusiform eggs of *Aussietrema* and *Fornixtrema*. Finally, Du Preez et al. (2022) erected two new genera, namely *Manotrema* Du Preez, Domingues & Verneau, 2022 and *Pleurodirotrema* Du Preez, Domingues & Verneau, 2022, while Chaabane et al. (2022) redefined outlines of *Polystomoides* sensu Du Preez et al. (2022) which led to the suppression of *Neopolystoma*.

The phylogenetic position of *Apaloneotrema* and its morphological resemblance to *O. hippopotami* (Du Preez and Verneau, 2020) suggests a possible sister group relationship between these two taxa. The objective of this study was thus to infer a complete phylogeny of chelonian polystomes including representatives of almost all genera and *O. hippopotami*. We then discuss the origins of this singular species within hippos in light of its phylogenetic position.

## 2. Materials and methods

### 2.1. *Selection of genetic data*

All the sequences used during this study were retrieved from GenBank (see Table 1). Most were obtained following sequencing of nuclear (18S and 28S) and mitochondrial (12S and COI) genes of several described and undescribed chelonian polystome species classified in the genera *Apaloneotrema*, *Aussietrema*, *Fornixtrema*, *Pleurodirotrema*, *Polystomoidella*, *Polystomoides* sensu Chaabane et al. (2022), *Uropolystomoides*, and *Uteropolystomoides*. In total, we processed 28 of the 65 chelonian polystome species known to occur in the urinary bladder, the pharyngeal cavity, or the conjunctival sacs of freshwater turtles worldwide (see Du Preez et al., 2023). Thus, our data sampling
included eight more species than those investigated in Héritier et al. (2015) from the same genetic markers. Other sequences were obtained from *O. hippopotami* of the hippo and from *Neopolystoma tinsleyi* Du Preez, Badets & Verneau, 2014 of the Cayenne caecilian (*Typhlonectes compressicauda* (Duméril & Bibron)). We used sequences from *Polystoma gallieni* Price, 1939 of the stripeless tree frog (*Hyla meridionalis* Boettger) and *Concinnocotyla australensis* (Reichenbach-Klinke, 1966) of the Australian lungfish for outgroup comparisons (Héritier et al., 2015). All sequences listed in Table 1 were actually used for different purposes (Littlewood et al., 1997; Bentz et al., 2006; Badets and Verneau, 2009; Badets et al., 2011; Raharivololoniaina et al., 2011; Verneau et al., 2011; Héritier et al., 2015; Du Preez et al., 2017; Du Preez and Verneau, 2020).

## 2.2. Phylogenetic analyses

18S and 28S sequences were edited and aligned according to the secondary structure (stems and loops) of rRNAs that was determined earlier in Badets et al. (2011) and Héritier et al. (2015). 12S and COI sequences were aligned using Clustal W implemented in MEGA version 7 (Kumar et al., 2016) under default parameters (Thompson et al., 1994). All sequences were concatenated in a single alignment for Bayesian, parsimony and distance analyses. For the Bayesian approach, nuclear and mitochondrial ribosomal genes were treated as three distinct partitions of 2291, 1484 and 548 characters, while the COI gene was subdivided in three other partitions of 132 characters each related to a codon position. Following the Akaike Information Criterion (AIC) implemented in Modeltest 3.06 (Posada and Crandall, 1998), a GTR + I + G model was selected independently for each ribosomal partition. We therefore applied six types of substitutions and invariable-gamma rates with four gamma rate categories for each of these partitions. Concerning the COI partitions, six types of substitutions and four gamma rate categories were applied to first and second positions following the AIC test whereas six types of substitutions and invariable-gamma rates with four gamma rate
categories were applied to the third position. Evolutionary parameters for all six partitions were estimated independently. Finally, the Bayesian analysis was run using MrBayes 3.04b (Huelsenbeck and Ronquist, 2001), with four chains running for five million generations and sampled every 100 cycles. The Bayesian consensus tree was drawn after removing the first 5,000 trees as the burn-in phase and opened with TreeGraph 2 (Stöver and Müller, 2010). It was converted into a phylogram and nodes supported by less than 0.9 posterior probabilities were collapsed. For the parsimony approach, a bootstrap test (5,000 replicates) was applied with heuristic search under PAUP* Version 4.0 b10 (Swofford, 2002) on the 975 parsimony-informative characters, gaps being treated as missing data. The tree-bisection-reconnection (TBR) branch swapping option was used to find trees of minimal length. Only bootstrap proportions higher than 75% were indicated next to the branches on the phylogram depicted in Fig. 1. For the distance analysis, evolutionary distances were computed under the pairwise deletion option using the Kimura 2-parameter method (Kimura, 1980). There were a total of 4,719 positions in the final dataset. The evolutionary history was inferred using the Minimum Evolution (ME) method (Rzhetsky and Nei, 1992). A bootstrap test (5,000 replicates) was applied and for each replication the ME tree was searched using the Close-Neighbor-Interchange (CNI) algorithm (Nei and Kumar, 2000) at a search level of 1. The Neighbor-joining algorithm (Saitou and Nei, 1987) was used to generate the initial tree. Finally, only bootstrap proportions higher than 75% were reported next to the branches on the phylogram depicted in Fig. 1.

## 2.3. Data accessibility

The complete sequence alignment is accessible at Mendeley Data, V1, doi: 10.17632/52427m4m96.1.
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Fig. 1 depicts the phylogenetic relationships of the polystomes infecting chelonians, hippos, and caecilians. Five of the polystome genera infecting turtles are each represented by a single species, i.e. *Apaloneotrema*, *Aussietrema*, *Pleurodirotrema*, *Polystomoidella*, and *Uteropolystomoides*, while the other three include at least four species. The tree clearly shows a nested position of *O. hippopotami* within chelonian polystomes, being closely related to *A. moleri* with high confidence values. For clarity, the tree was subdivided into six well differentiated lineages A to F and sublineages A1, A2 and C1–C4 whose inter-relationships are moderately resolved. Lineage A includes parasites of the pharyngeal cavity and urinary bladder of the two genera *Polystomoides* and *Uteropolystomoides*, illustrating the paraphyly of *Polystomoides* (see Chaabane et al., 2022). Lineage B includes two parasites of *Aussietrema* and *Pleurodirotrema* that infect the conjunctival sacs and the urinary bladder of their hosts, respectively. Lineage C includes all parasites of *Fornixtrema* infecting the conjunctival sacs of their hosts. Lineage D contains a single species of *Polystomoidella* that is found in the urinary bladder of its host. Lineage E includes all parasites of *Uropolystomoides* infecting the urinary bladder of their hosts. Finally, lineage F includes two parasites of *Apaloneotrema* and *Oculotrema* that both infect the conjunctival sacs of their hosts.

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When considering more specifically the ecological niche of parasites, the polystomes of the urinary bladder fall into the four lineages A, B, D, and E. Those of the pharyngeal cavity fall into a single lineage, i.e. lineage A, whereas those of the conjunctival sacs fall into the three lineages B, C, and F, with *O. hippopotami* being included in lineage F. Regarding the geographical distribution of all these parasites, African chelonian polystomes fall into the two lineages A and E; Asian chelonian polystomes fall into the three lineages A, C, and E; Australian chelonian polystomes fall into the lineage B; North American chelonian polystomes fall into the four lineages A, C, D, and F; and South American chelonian polystomes fall into the two lineages A and C.
Finally, if we look at the range of host species, seven of the investigated chelonians are infected by at least two polystomes: the Southeast Asian box turtle (*Cuora amboinensis* (Daudin)) of Malaysia is infected by three polystomes that occur, respectively, in the pharyngeal cavity, conjunctival sacs, and urinary bladder of the lineages A, C, and E; the pond slider (*Trachemys scripta* (Thunberg)) of the USA is infected by three polystomes that occur, respectively, in the pharyngeal cavity and conjunctival sacs of the lineages A and C, with two distinct unrelated parasite species in lineage C; the painted turtle (*Chrysemys picta* (Schneider)) of the USA is infected by two polystomes that occur, respectively, in the pharyngeal cavity and urinary bladder of the same lineage A; the spotted-legged turtle (*Rhinoclemmys punctularia* (Daudin)) of French Guiana is infected by two polystomes that occur, respectively, in the urinary bladder and conjunctival sacs of the lineages A and C; the Mediterranean pond turtle (*Mauremys leprosa* (Schweigger)) of Algeria is infected by two polystomes that occur, respectively, in the pharyngeal cavity and urinary bladder of the same lineage A; the common snake-necked turtle (*Chelodina longicollis* (Shaw)) of Australia is infected by two polystomes that occur, respectively, in the urinary bladder and conjunctival sacs of the same lineage B; the striped mud turtle (*Kinosternon baurii* Garman) of the USA is infected by two polystomes that occur, respectively, in the urinary bladder and conjunctival sacs of the lineages C and D.

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## 4. Discussion

Regarding the phylogenetic relationships of chelonian polystomes, their distribution across host species, and host biogeography, Héritier et al. (2015) concluded that their evolution could have been the consequence of several non-exclusive events such as plate tectonics, codivergences, host dispersal followed by host-switching, and intra-host speciation. With a larger sampling of chelonian polystomes in the present study, which comprises 28 species compared with the 20 species investigated earlier by Héritier et al. (2015), our phylogeny includes 13 polystome species infecting
the conjunctival sacs of their host rather than seven in Héritier et al. (2015). It also differs from the previous work in the number of polystome lineages, i.e., eight in the present study versus six in Héritier et al. (2015), and by the distribution of the polystomes of the conjunctival sacs that fall into three distinct lineages, B, C and F. Regarding our phylogeny, some postulated hypotheses can now be ruled out with confidence, such as intra-host duplication, which suggests parasite speciation within the host following specialization of parasites in distinct ecological niches or habitats of the same host species. Because some chelonian species, including *C. amboinensis*, *T. scripta*, *R. punctularia*, and *K. baurii* (see Table 1), are each infected by several phylogenetically distant polystome species of distinct parasite lineages (see Fig. 1), intra-host duplication is very unlikely and should be rejected. This conclusion is also strengthened by *C. picta* and *M. leprosa*, which are both infected by phylogenetically unrelated polystome species of the same parasite lineage and by *C. longicollis*, which is infected by sister polystome species of the same parasite lineage and whose divergence would date back to about 58 Mya (Héritier et al., 2015). Considering now that plate tectonics and codivergences would have been the main forces driving parasite speciation and diversification, close correspondence between host and parasite phylogenetic patterns should have been apparent, but it was not. Regarding sublineage A1 that includes only polystomes infecting hosts of the family Geoemydidae and sublineage A2 that is composed of polystomes infecting hosts of families Emydidae, Geoemydidae, and Trionychidae, there is no clear correspondence between parasite and host phylogenetic relationships (Guillon et al., 2012; Crawford et al., 2015; Pereira et al., 2017; Shaffer et al., 2017; Thomson et al., 2021). Similarly, while sublineages C1 and C2 are only represented by polystomes infecting hosts of the family Trionychidae and Geoemydidae, respectively, sublineage C3 includes polystomes of the Chelydridae, Emydidae, Geoemydidae, and Kinosternidae, and sublineage C4 includes polystomes of the Emydidae, Geoemydidae, and Kinosternidae. These patterns of polystome relationships also contradict expected patterns of host-
parasite relationships under the assumption of strict cospeciation (see Guillon et al., 2012; Crawford 285 et al., 2015; Pereira et al., 2017; Shaffer et al., 2017; Thomson et al., 2021 for turtle phylogenetic 286 relationships), which indicates that host evolution did not act as the main force driving parasite 287 evolution (Hoberg and Brooks, 2008; Garamszegi, 2009; Araujo et al., 2015; Horká et al., 2016). As 288 a consequence, polystome host-switching probably occurred frequently during chelonian evolution. 289 This hypothesis is highlighted, at least in part, by the patterns of phylogenetic relationships within 290 polystomes of lineages C and E. Lineage C, which corresponds to *Fornixtrema*, only includes 291 polystomes of the conjunctival sacs, and lineage E, which corresponds to *Uropolystomoides*, is only 292 composed of polystomes of the urinary bladder. Host-switching of chelonian polystomes across the 293 same ecological habitat could be best favored between sympatric host species of similar ecology 294 rather than between sympatric host species of close phylogenetic relatedness. Ultimately, host 295 dispersals over evolutionary time would have allowed parasite speciation and dispersal. 296

The sister group relationship between *O. hippopotami* and *Apaloneotrema moleri* (Du Preez 297 and Morrison, 2012) clearly indicates that the plesiomorphic host for the mammal parasite lineage 298 would have been a turtle, which contradicts Héritier et al. (2015) who favored a caecilian origin for 299 that parasite based on a smaller sample of chelonian polystomes than in the present study. The 300 proximity of hippos and freshwater turtles in aquatic environments could have enhanced the chances 301 of parasite transmission from one host to another. The close association between hippos and some 302 freshwater turtles can be illustrated by turtles basking on the back and head of a hippo (Fig. 2). While 303 host shifts between distantly related taxa have previously been documented (e.g., between birds and 304 bats following colonization by bats of ecological niches occupied by birds (Duval et al., 2007) and 305 between humans and domesticated animals in close physical contact (Ren et al., 2016)), the 306 probability of host switching usually decreases as the phylogenetic distance between donor and 307 recipient hosts increases. This has been termed the phylogenetic distance effect (Engelstaedter and 308
Fortuna, 2019). Because the sauropsid and synapsid clades that include modern turtles and mammals, respectively, may have diverged from each other approximately 312 Mya (Benton and Donoghue, 2007), the occurrence of a monogenean parasite within hippos might reflect one of the most exceptional switches in the course of vertebrate evolution. To our knowledge, such lateral transfer of platyhelminths across such distantly related host taxa has only been documented in digenean schistosomes from marine turtles to birds (Brant and Loker, 2005). Since digenean trematodes of the Microphallidae are mainly distributed within birds, the presence of *Longiductotrema tethepae* Kudlai, Cribb & Cutmore, 2016 (Digenea: Microphallidae) within the fish *Gymnothorax pseudothyrsoideus* (Bleeker) could also be the result of a parasite's switch from bird to fish, given that muraenid eels may share prey species with sea birds (Kudlai et al., 2016). In the same vein, because blood flukes of the family Aporocotylidae are fish parasites, the presence of *Cardicola dhangali* Hutson, Vaughan & Blair, 2019 (Digenea: Aporocotylidae) within the sirenian *Dugong dugon* (Muller) could also represent a parasite's switch from actinopterygian fish to mammal (Hutson et al., 2019). However, without phylogenetic evidence, these two hypotheses require further investigation before any conclusion can be made. Although Tinsley (2013) considered that some polystomes of the conjunctival sacs previously classified in *Neopolystoma* could be the best parasite candidate for host-switching between turtles and hippos, he did not exclude the possibility that some *Polystomoides* spp. of the pharyngeal cavities could represent the missing link. Our analysis suggests that the polystomes which switched from turtles to hippos were already adapted to the conjunctival sacs, demonstrating that the proximity in the ecological habitat of polystomes within host species is an important feature for parasite speciation and diversification, even more important than phylogenetic relatedness.

*Apaloneotrema moleri*, which has been identified as the sister species of *O. hippopotami*, was recovered from the Florida softshell turtle (*Apalone ferox* (Schneider)) (Fig. 3). Because *A. ferox* occurs in Alabama, Florida, Georgia, and South Carolina in the USA (Rhodin et al., 2021), assuming
a parasite's switch from that turtle species to hippos seems unlikely. Similarly, the hippo is widely distributed in wetlands and rivers of almost 40 countries in Africa (IUCN Red List of Threatened Species. https://www.iucnredlist.org/species/10103/18567364#geographic-range (accessed on 05 August 2022), but *Apalone* Rafinesque includes three species restricted to the USA. That genus therefore cannot be considered as the plesiomorphic host for *Oculotrema*. However, *A. ferox* belongs to the family Trionychidae which is one of the most widely distributed reptile groups in the world according to Li et al. (2017). Trionychid turtles comprise 34 species which are distributed in Africa, Greater Indonesia, North America, Southeastern Europe, and Southern Asia (Rhodin et al., 2021). They are subdivided into two subfamilies, i.e. the Cyclanorbinae and the Trionychinae, which are both monophyletic (Le et al., 2014), and 13 genera. From a phylogenetic study based on mitogenomes that was complemented with estimates of divergence times and ancestral area reconstructions, Li et al. (2017) showed that the softshell turtles would have originated in Asia approximately 108 Mya. They may have subsequently taken different routes of dispersal out of Asia (to Africa approximately 63 Mya, to insular Southeast Asia approximately 53 Mya, and to North America approximately 76 Mya). Regarding the occurrence of *A. moleri* in the Florida softshell turtle, the American *Apaloneotrema* lineage would have arisen following migration of trionychids across the Beringia landbridge during the Late Cretaceous – Early Eocene (Li et al., 2017). Therefore, it is possible that an ancestral stock of parasites may have been isolated on primitive trionychids before they dispersed to Africa, namely across western Asia (Li et al., 2017) where they could have further switched to hippos. According to the divergence time estimates, relationships, and biogeography of extant trionychids (Le et al., 2014; Li et al., 2017; Rhodin et al., 2021) as well as the sister group relationship between *O. hippopotami* and *A. moleri*, the ‘African’ parasite lineage may have arisen on primitive trionychids approximately 108 Mya if polystomes evolved on *Cyclanorbis* Gray, and / or *Cycloderma* Peters, or approximately 86 Mya if they evolved on *Trionyx* Geoffroy Saint-Hilaire. The
plesiomorphic host which has served for parasite dispersal to hippos should therefore be found among
one of these three genera. Regarding the actual distribution of hippos in Africa and evolutionary
history of hippopotamids (Montgelard et al., 1997; Orliac et al., 2010; Pickford, 2011), host switching
could have occurred in the time window 21 Mya to the present day as hippopotamids may have been
adapted to semi-aquatic habitats at least as early as the latest Miocene (Boisserie et al., 2011). That
polystomes may have switched earlier, from ancestral freshwater turtles to anthracotheres in the
African Paleogene, also seems plausible as certain primitive mammal forms already may have
adapted to aquatic environments (Lihoreau et al., 2015), which is a prerequisite for the polystome
life-cycle. A more thorough sampling across turtles of the family Trionychidae, at least in Africa (five
species distributed in three genera; see Rhodin et al., 2021), but also in Asia (26 species distributed
in nine genera; see Rhodin et al., 2021), is now needed to conclude from which host lineage or turtle
species that switch would have occurred and when it did historically. At the present time, only two
polystome species have been described from the conjunctival sacs of trionychids, namely *A. moleri*
from *A. ferox* in the USA and *Fornixtrema palpebrae* (Strelkov, 1950) from the Chinese softshell
turtle (*Pelodiscus sinensis* Wiegmann) in Lake Hanka, East Asia.

372

373 Acknowledgements

We thank the following people for helping with fieldwork in Florida (USA): Jamie Casteel,
Jeremy Geiger, and Chris Jones. Ewa Hughes and Vertigo Moody facilitated access to laboratory
facilities at Santa Fe College (USA) and provided some necessary laboratory materials. We also thank
Simon Smith (Property Consultant, Durban, South Africa) and Georgia Shemitz (Karst
Environmental Services, High Springs, Florida, USA) for permission to use the photographs in Fig.
2 and Fig. 3, respectively, Richard Tinsley for his valuable comments on an earlier version of this
manuscript, and two anonymous reviewers for helpful remarks and suggestions. Olivier Verneau
381 received financial support from Centre de Formation et de Recherche sur les Environnements

382 Méditerranéens of France and Louis Du Preez from the South African Institute for Aquatic

383 Biodiversity and the National Research Foundation of South Africa. This research did not receive any

384 specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

385
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581
582 **Figures captions**

583

584 **Fig. 1.** Bayesian phylogram inferred from analysis of four concatenated nuclear and mitochondrial genes. Nodes supported by less than 0.9 posterior probabilities were collapsed. Values next to branches indicate, from left to right, Bayesian Posterior Probabilities, parsimony and distance bootstrap proportions. Letters A1, A2, B, C1-C4, and D to F show the six well differentiated polystome lineages and sublineages discussed in the text with host family names indicated on the righthand side. * Species that were not investigated in Héritier et al. (2015). (A.s.), *Apalone spinifera*; (C.s.), *Chelydra serpentina*; (G.p.), *Graptemys pseudogeographica*; (K.b.), *Kinosternon baurii*; (P.c.), *Pelusios castaneus*; (P.n.), *Pseudemys nelsoni*; (P.sp.), *Pelomedusa* sp.; (T.s.), *Trachemys scripta*.

593

594 **Fig. 2.** Turtles (*Pelusios* sp.) basking on the back and head of a hippopotamus. Photograph courtesy of Simon Smith (Property Consultant, Durban, South Africa).

595

596 **Fig. 3.** Florida softshell turtle *Apalone ferox*. Photograph courtesy of Georgia Shemitz (Karst Environmental Services, High Springs, Florida, USA).

599

600
<figure><img src="image_1.png" /></figure>

Polystomoides tunisiensis

Polystomoides asiaticus

Polystomoides euzeti

Polystomoides cayensis*

Polystomoides orbiculare

Polystomoides sp. (A.s.)

Polystomoides soredensis (T.s.)

Polystomoides oris

Uteropolystomoides multifalx

Aussietrema spratti

Pleurodirotrema chelonidae

Fornixtrema palpebrae

Fornixtrema liewi

Fornixtrema fentoni*

Fornixtrema sp. (K.b.)*

Fornixtrema elizabethae*

Fornixtrema sp. (G.p.)

Fornixtrema sp. (C.s.)

Fornixtrema guianensis

Fornixtrema scorpioides*

Fornixtrema sp. (T.s.)*

Fornixtrema sp. (P.n.)*

Polystomoidella whartoni*

Uropolystomoides malayi

Uropolystomoides siebenrockiellae

Uropolystomoides sp. (P.sp.)

Uropolystomoides sp. (P.c.)

Apaloneotrema moleri*

Oculotrema hippopotami

Nanopolystoma tinsleyi

Polystoma gallieni

Concinnocotyla australensis
<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>
<table><caption>Table 1. Parasite species investigated for their phylogenetic relationships</caption><thead><tr><th rowspan="2">Parasite species</th><th rowspan="2">Infection site</th><th rowspan="2">Host species</th><th rowspan="2">Locality</th><th rowspan="2">Host family</th><th colspan="4">Genbank Accession numbers</th></tr><tr><th>18S</th><th>28S</th><th>12S</th><th>COI</th></tr></thead><tbody><tr><td>Apaloneotrema moleri</td><td>Conjunctival sacs</td><td>Apalone ferox</td><td>USA</td><td>Trionychidae</td><td>MW029406</td><td>MW029412</td><td>MW029418</td><td>MW029424</td></tr><tr><td>Aussietrema spratti</td><td>Conjunctival sacs</td><td>Chelodina longicollis</td><td>Australia</td><td>Chelidae</td><td>AJ228788</td><td>FM992702</td><td>KR856105</td><td>Z83007</td></tr><tr><td>Fornixtrema elizabethae</td><td>Conjunctival sacs</td><td>Trachemys scripta</td><td>USA</td><td>Emydidae</td><td>MW029402</td><td>MW029408</td><td>MW029414</td><td>MW029420</td></tr><tr><td>Fornixtrema fentoni</td><td>Conjunctival sacs</td><td>Rhinoclemmys pulcherrima</td><td>Costa Rica</td><td>Geoemydidae</td><td>KR856134</td><td>KR856153</td><td>KR856110</td><td>FR822555</td></tr><tr><td>Fornixtrema guianensis</td><td>Conjunctival sacs</td><td>Rhinoclemmys punctularia</td><td>French Guiana</td><td>Geoemydidae</td><td>KY200987</td><td>KY200989</td><td>KY200992</td><td>KY200995</td></tr><tr><td>Fornixtrema liewi</td><td>Conjunctival sacs</td><td>Cuora amboinensis</td><td>Malaysia</td><td>Geoemydidae</td><td>KR856128</td><td>KR856147</td><td>KR856102</td><td>FR822530</td></tr><tr><td>Fornixtrema palpebrae</td><td>Conjunctival sacs</td><td>Pelodiscus sinensis</td><td>Vietnam</td><td>Trionychidae</td><td>FM992696</td><td>AF382065</td><td>KR856104</td><td>FR822601</td></tr><tr><td>Fornixtrema scorpioides</td><td>Conjunctival sacs</td><td>Kinosternon scorpioides</td><td>French Guiana</td><td>Kinosternidae</td><td>Unavailable</td><td>KY200990</td><td>KY200993</td><td>KY200996</td></tr><tr><td>Fornixtrema sp.</td><td>Conjunctival sacs</td><td>Kinosternon bauri</td><td>USA</td><td>Kinosternidae</td><td>MW029401</td><td>MW029407</td><td>MW029413</td><td>MW029419</td></tr><tr><td>Fornixtrema sp.</td><td>Conjunctival sacs</td><td>Chelydra serpentina</td><td>USA</td><td>Chelydidae</td><td>KR856131</td><td>KR856150</td><td>KR856107</td><td>FR822529</td></tr><tr><td>Fornixtrema sp.</td><td>Conjunctival sacs</td><td>Graptemys pseudogeographica</td><td>USA</td><td>Emydidae</td><td>KR856132</td><td>KR856151</td><td>KR856108</td><td>FR822553</td></tr><tr><td>Fornixtrema sp.</td><td>Conjunctival sacs</td><td>Trachemys scripta</td><td>USA</td><td>Emydidae</td><td>MW029403</td><td>MW029409</td><td>MW029415</td><td>MW029421</td></tr><tr><td>Fornixtrema sp.</td><td>Conjunctival sacs</td><td>Pseudemys peninsularis</td><td>USA</td><td>Emydidae</td><td>MW029404</td><td>MW029410</td><td>MW029416</td><td>MW029422</td></tr><tr><td>Pleurodirotrema chelodinae</td><td>Urinary bladder</td><td>Chelodina longicollis</td><td>Australia</td><td>Chelidae</td><td>KR856126</td><td>KR856145</td><td>KR856100</td><td>Z83005</td></tr><tr><td>Polystomoidella whartoni</td><td>Urinary bladder</td><td>Kinosternon bauri</td><td>USA</td><td>Kinosternidae</td><td>MW029405</td><td>MW029411</td><td>MW029417</td><td>MW029423</td></tr></tbody></table>
<table>
  <tr>
    <td>Polystomoides asiaticus</td>
    <td>Pharyngeal cavity</td>
    <td>Cuora amboinensis</td>
    <td>Malaysia Geoemydidae</td>
    <td>FM992697</td>
    <td>FM992703</td>
    <td>KR856113</td>
    <td>Z83009</td>
  </tr>
  <tr>
    <td>Polystomoides cayensis</td>
    <td>Urinary bladder</td>
    <td>Rhinoclemmys punctularia</td>
    <td>French Geoemydidae<br/>Guiana</td>
    <td>KY200986</td>
    <td>KY200988</td>
    <td>KY200991</td>
    <td>KY200994</td>
  </tr>
  <tr>
    <td>Polystomoides euzeti</td>
    <td>Urinary bladder</td>
    <td>Mauremys leprosa</td>
    <td>Algeria Geoemydidae</td>
    <td>KR856127</td>
    <td>KR856146</td>
    <td>KR856101</td>
    <td>FR822587</td>
  </tr>
  <tr>
    <td>Polystomoides orbiculare</td>
    <td>Urinary bladder</td>
    <td>Chrysemys picta</td>
    <td>USA Emydidae</td>
    <td>KR856129</td>
    <td>KR856148</td>
    <td>KR856103</td>
    <td>FR822531</td>
  </tr>
  <tr>
    <td>Polystomoides oris</td>
    <td>Pharyngeal cavity</td>
    <td>Chrysemys picta</td>
    <td>USA Emydidae</td>
    <td>FM992698</td>
    <td>FM992705</td>
    <td>KR856115</td>
    <td>FR822534</td>
  </tr>
  <tr>
    <td>Polystomoides soredensis</td>
    <td>Pharyngeal cavity</td>
    <td>Trachemys scripta</td>
    <td>USA Emydidae</td>
    <td>KR856135</td>
    <td>KR856154</td>
    <td>KR856111</td>
    <td>FR828360</td>
  </tr>
  <tr>
    <td>Polystomoides tunisiensis</td>
    <td>Pharyngeal cavity</td>
    <td>Mauremys leprosa</td>
    <td>Algeria Geoemydidae</td>
    <td>KR856136</td>
    <td>KR856155</td>
    <td>KR856116</td>
    <td>FR822570</td>
  </tr>
  <tr>
    <td>Polystomoides sp.</td>
    <td>Pharyngeal cavity</td>
    <td>Apalone spinifera</td>
    <td>USA Trionychidae</td>
    <td>KR856130</td>
    <td>KR856149</td>
    <td>KR856106</td>
    <td>FR822527</td>
  </tr>
  <tr>
    <td>Uropolystomoides malayi</td>
    <td>Urinary bladder</td>
    <td>Cuora amboinensis</td>
    <td>Malaysia Geoemydidae</td>
    <td>AJ228792</td>
    <td>FM992704</td>
    <td>KR856112</td>
    <td>Z83011</td>
  </tr>
  <tr>
    <td>Uropolystomoides siebenrockiellae</td>
    <td>Urinary bladder</td>
    <td>Siebenrockiella crassicollis</td>
    <td>Malaysia Geoemydidae</td>
    <td>FM992699</td>
    <td>FM992706</td>
    <td>KR856114</td>
    <td>FR822604</td>
  </tr>
  <tr>
    <td>Uropolystomoides sp.</td>
    <td>Urinary bladder</td>
    <td>Pelomedusa sp.</td>
    <td>Nigeria Pelomedusidae</td>
    <td>KR856138</td>
    <td>KR856157</td>
    <td>KR856118</td>
    <td>KR856176</td>
  </tr>
  <tr>
    <td>Uropolystomoides sp.</td>
    <td>Urinary bladder</td>
    <td>Pelusios castaneus</td>
    <td>Nigeria Pelomedusidae</td>
    <td>KR856139</td>
    <td>KR856158</td>
    <td>KR856119</td>
    <td>KR856177</td>
  </tr>
  <tr>
    <td>Uteropolystomoides multifalx</td>
    <td>Pharyngeal cavity</td>
    <td>Pseudemys nelsoni</td>
    <td>USA Emydidae</td>
    <td>KR856137</td>
    <td>KR856156</td>
    <td>KR856117</td>
    <td>FR822603</td>
  </tr>
  <tr>
    <td>Oculotrema hippopotami</td>
    <td>Conjunctival sacs</td>
    <td>Hippopotamus amphibius</td>
    <td>South Africa<br/>Hippopotamidae</td>
    <td>KR856140</td>
    <td>KR856159</td>
    <td>KR856120</td>
    <td>KR856178</td>
  </tr>
  <tr>
    <td>Nanopolystoma tinsleyi</td>
    <td>Urinary bladder</td>
    <td>Typhlonectes compressicauda</td>
    <td>French Typhlonectidae</td>
    <td>KR856124</td>
    <td>KR856142</td>
    <td>KR856077</td>
    <td>KR856164</td>
  </tr>
  <tr>
    <td>Polystoma gallieni</td>
    <td>Urinary bladder</td>
    <td>Hyla meridionalis</td>
    <td>France Hylidae</td>
    <td>AM051070</td>
    <td>AM157205</td>
    <td>KR856084</td>
    <td>JF699305</td>
  </tr>
  <tr>
    <td>Concinnocotyla australensis</td>
    <td>Skin and gills</td>
    <td>Neoceratodus forsteri</td>
    <td>Australia Neoceratodontidae</td>
    <td>AM157183</td>
    <td>AM157197</td>
    <td>Unavailable</td>
    <td>Unavailable</td>
  </tr>
</table>
<figure><img src="image_7.png" /></figure>

Photographs courtesy of (lower left) Simon Smith (Property Consultant, Durban, South Africa) and (upper right) Georgia Shemitz (Karst Environmental Services, Florida, USA).