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, Alvey, 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.

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.