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International Journal for Parasitology: Parasites and Wildlife logoLink to International Journal for Parasitology: Parasites and Wildlife
. 2022 Sep 16;19:180–186. doi: 10.1016/j.ijppaw.2022.09.004

Classification of pleurodire polystomes (Platyhelminthes, Monogenea, Polystomatidae) revisited with the description of two new genera from the Australian and Neotropical Realms

Louis Heyns du Preez a,b,∗, Marcus Vinícius Domingues c, Olivier Verneau a,d,e
PMCID: PMC9519787  PMID: 36188110

Abstract

Polystomatids are platyhelminth parasites that infect mostly amphibian and chelonian hosts. Polystomatid of testudines were, for more than seven decades, classified in the three genera – Neopolystoma Price, 1939, Polystomoides Ward, 1917 and Polystomoidella Price, 1939. The genus delimitation was primarily based on the absence of hamuli in Neopolystoma, the presence of one pair of hamuli in Polystomoidella, and two pairs in Polystomoides. From 2016 to 2020, five new genera were erected - namely Uropolystomoides Tinsley and Tinsley, 2016, Uteropolystomoides Tinsley, 2017, Apaloneotrema Du Preez and Verneau, 2020, Aussietrema Du Preez and Verneau, 2020 and Fornixtrema Du Preez and Verneau, 2020. The generic diagnosis was based not only on the size and shape of morphological characters such as hamulus 1, uterus and eggs, but also on the site of infestation (i.e. urinary bladder, oral cavity or conjunctival sacs). Despite large advancements in polystome classification over the last decade, Neopolystoma was still polyphyletic with some species nested within Polystomoides and others being closely related to the Australian Aussietrema. Regarding the distribution of freshwater turtles of the two suborders Pleurodira (Southern continents) and Cryptodira (distributed worldwide except in Australia), one may wonder whether Australian chelonian polystomes of the genus Neopolystoma may have diverged from species infecting other pleurodires of South America. In the present study based on the analysis of several species selected among all genera, we reveal striking morphological differences within polystomes infecting pleurodiran turtles, which herein led to the proposal of two new chelonian polystome genera, Pleurodirotrema n. g. and Manotrema n. g. Pleurodirotrema n. g. is characterized by the absence of hamuli, presence of latero-ventral vaginae and includes species that infect either the oral region or the urinary bladder of Australian hosts of the Pleurodira. Manotrema n. g. is characterized by the presence of small hamuli, latero-ventral vaginae, deep incisions between suckers, a low genital spine number (<10) and includes species that infect the oral region of South American Pleurodira.

Keywords: Monogenea, Polystomatidae, Pleurodirotrema, Manotrema, Vaginae, Suckers

Graphical abstract

Image 1

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Highlights

  • •

    Two new polystome genera are described from chelonian hosts.

  • •

    They differ from all other chelonian polystomes in that they only infect pleurodire turtles from the Australian and Neotropical Realms.

  • •

    Describing these two genera resolves a polyphyletic clade as pointed out by Heritier and others.

  • •

    Solid morphological characters are supported by molecular evidence for Pleurodirotrema.

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    Haptor and suckers for Manotrema are unique among chelonian polystomes.

1. Introduction

The Polystomatidae Gamble, 1896 sensu Sinnappah et al. (2001) is the largest family of the Monogenea Van Beneden, 1858 that infects aquatic or semi aquatic vertebrates of the Rhipidistia. Although monogenean parasites are mostly fish ectoparasites, polystomatids infect all three extant orders of Amphibia, namely Anura (frogs), Caudata (salamanders) and Gymnophiona (caecilians) where adult worms are generally found in the urinary bladder. Polystomatids are also found within the urinary bladder, pharyngeal cavities and/or conjunctival sacs of freshwater turtles of the two testudines suborders Pleurodira and Cryptodira, in the mouth and on the gills of the Australian lungfish Neoceratodus forsteri and in the conjunctival sacs of the common hippopotamus Hippopotamus amphibius. Since their discovery in the late 1700s from the common frog Rana temporaria, about 200 species have been reported and described from all over of the world except Antarctica. They are classified in 30 genera, of which 20 are found exclusively within amphibians, eight within freshwater turtles and, one each, within the Australian lungfish and the common hippopotamus, respectively.

The proposal of phylogenetic hypotheses for polystomes based on morphological characters has always been a very difficult, to near impossible, task, due to limited morphological interspecies variation. With the advancement of molecular technique in the late 1900s, particularly with the PCR approach, more and more phylogenetic studies have been focused on taxonomic groups for which morphological characters were inadequate. As an outcome, the first molecular phylogenies on polystomes were published in the early 2000s with Sinnappah et al. (2001), Bentz et al. (2001, 2006) and Verneau et al. (2002, 2009), providing a phylogenetic framework for discussion of both their evolution since their origin in the Paleozoic period and dispersal in more recent times. Though other publications contributed later to a better understanding of their evolution through time and space (Badets et al., 2011; Héritier et al., 2015), molecular phylogenies also proved to be an essential tool to the taxonomy of amphibian polystomes (Berthier et al., 2014; Chaabane et al., 2019; Du Preez et al., 2007, 2010, 2014; Fan et al., 2020; Landman et al., 2018, 2021; Raharivololoniaina et al., 2011; Yildirimhan et al., 2012) as well as chelonian polystomes (Du Preez et al., 2017; Dutton et al., 2021; Héritier et al., 2018).

Whereas phylogenetic and genetic studies provided an essential source of information in species delimitation of polystomes, it also helped in the revision of their systematics, more particularly for polystomes infecting freshwater turtles. Five new polystome genera have been described from freshwater turtles during the past ten years, namely Uropolystomoides Tinsley and Tinsley, 2016, Uteropolystomoides Tinsley, 2017, Apaloneotrema Du Preez and Verneau, 2020, Aussietrema Du Preez and Verneau, 2020 and Fornixtrema Du Preez and Verneau, 2020, however, none of them derived from the discovery of new species. Uropolystomoides was created to accommodate a particular clade of Polystomoides Ward (1917) occurring in the urinary bladder only and differing from all Polystomoides species of the pharyngeal cavity by the size of hamulus 1 - which is greater than the sucker diameter (Tinsley and Tinsley, 2016). Uteropolystomoides was created to accommodate Polystomoides nelsoni Du Preez and Van Rooyen, 2015 which was the single species of Polystomoides with a uterus holding numerous eggs and a massive genital bulb with more than 120 genital spines (Tinsley, 2017). Finally, Apaloneotrema, Aussietrema and Fornixtrema were created to accommodate three distinct lineages of Neopolystoma Price, 1939 species which all infect the conjunctival sacs of their host, but differ from each other mainly by the shape of the egg, i.e. a large fusiform egg with rounded tips for Apaloneotrema, a spherical egg for Aussietrema and a fusiform to diamond-shaped egg with acute tips for Fornixtrema which also has an egg-cell maturation chamber (Du Preez and Verneau, 2020).

According to Du Preez and Verneau (2020), Neopolystoma still represents a polyphyletic taxon with some species nested within Polystomoides and others being closely related to Aussietrema. With regard to the distribution of modern freshwater turtles, pleurodires are restricted to southern continents, i.e. across the Australian, Ethiopian and Neotropical Realms, while cryptodires are distributed worldwide with the exception of Australia (Rhodin et al., 2021). According to Pereira et al. (2017), the breakup of Pangaea drove the divergence between cryptodires and pleurodires. Whereas the biogeographic history of cryptodires is assumed to be complicated by the complex paleogeographic history of Laurasia, the biogeographic history of pleurodires was shown to be tightly related to the paleogeographical history of the Gondwana (Pereira et al., 2017). Though Ferreira et al. (2018) concluded that the current distribution of pleurodires could not be fully explained using vicariance or extinctions as sole explanations, they showed that the divergence time between South American and Australian Chelidae should be pushed back to the end of the Early Cretaceous as some kind of barrier prevented any dispersal of chelids. While Australian pleurodires might have been isolated from all other freshwater turtles at least 100 Million years ago (Mya), ancestral trionychids (Cryptodira) would have dispersed to Australia in the Miocene (Pereira et al., 2017). This would have left ample time for polystomes of Australian pleurodires to diverge from all other polystome lineages and accumulate morphological changes. This agrees with the molecular dating proposed by Héritier et al. (2015) for chelonian polystomes that indicated a separation of about 98 Mya with a 95% confidence interval of 66–136 Mya between the Australian polystome lineage, including Aussietrema and species of Australian Neopolystoma infecting specifically pleurodires, and its sister polystome lineage including species of Polystomoides and Neopolystoma, both infecting cryptodires. By reanalyzing morphological types of several species of Aussietrema, Neopolystoma and Uropolystomoides of the Australian Realm on the one hand, followed by morphological comparisons with types of other pleurodiran and cryptodiran polystome species of the genera Polystomoides, Neopolystoma and Uropolystomoides on the other, we might expect to find morphological characters (synapomorphies) that delimitate a new genus within polystomes of pleurodires - at least in Australia.

The objectives of the present study were thus to focus on as many species as possible of the genera Apaloneotrema, Aussietrema, Fornixtrema, Neopolystoma, Polystomoides Polystomoidella, Uropolystomoides and Uteropolystomoides in order to identify these characters if they exist.

2. Material and methods

2.1. Polystome sampling

Observations for the present study were based on: (i) a representative loan of several Australian polystome specimens from the Queensland Museum, Australia, including Aussietrema cribbi (Pichelin, 1995), Aussietrema queenslandensis (Pichelin, 1995), Aussietrema spratti (Pichelin, 1995), Aussietrema tinsleyi (Pichelin, 1995), Neopolystoma chelodinae (MacCallum, 1918), Neopolystoma kreffti Rohde, 1984, Neopolystoma maclayi Rohde, 1984, Neopolystoma novaeguineae Fairfax, 1990, Uropolystomoides australiensis (Rohde and Pearson, 1980) and Uropolystomoides scottae (Pichelin, 1995); (ii) specimens of Polystomoides brasiliensis Vieira et al., 2008 examined at the Oswaldo Cruz Institute in Rio de Janeiro, Brazil and a subsequent loan of material from this museum; (iii) Uteropolystomoides nelsoni (Du Preez and Van Rooyen, 2015) from the Parasitic Worm Collection, National Museum, Bloemfontein, South Africa; (iv) specimens of Neopolystoma cayensis Du Preez et al. (2017), N. chelodinae, Neopolystoma orbiculare (Stunkard, 1916), Polystomoidella whartoni (Wright, 1879), Polystomoides asiaticus Rohde, 1965, Polystomoides multifalx (Stunkard, 1924), Polystomoides scriptanus Héritier et al. (2018), Polystomoides soredensis Héritier et al. (2018), Uropolystomoides chabaudi (Euzet and Combes, 1965), Uropolystomoides malayi (Rohde, 1963) and U. nelsoni from the collection of the authors at the North-West University in Potchefstroom, South Africa.

2.2. Specimen preparation and morphological structures of investigation

Fixed specimens from the authors’ collection were rinsed in water, stained with acetocarmine, dehydrated, cleared in xylene and mounted in Canada balsam. Parasites were examined for morphological features including body size, position and size of the vaginae, genital spine number, shape of haptor and suckers and shape and size of hamuli, when present. Morphological features were photographed using a Nikon AZ100M microscope (Nikon, Netherlands) fitted with a 0.5X, 1X and 4X objectives as well as a Nikon U3 digital camera. Measurements were captured using the Nikon NIS software. Small structures were examined, measured and photographed using a Zeiss Imager Axio10 compound microscope (Zeiss, Germany) fitted with a Zeiss Axio cam 305 camera (Zeiss, Germany) and Zeiss Zen Blue elements (Zeiss, Germany) software program. Finally, type drawings for all recorded chelonian polystome species (see Morrison and Du Preez, 2011) were also investigated for their morphology, genital spine number and intervaginal distances.

3. Results

3.1. Position and size of the vaginae

All polystomes infecting the oral region, urinary bladder or the conjunctival sacs of pleurodires have latero-ventral vaginae. These include species of the Australian Realm belonging to the genera Aussietrema, Neopolystoma (A in Fig. 1) and Uropolystomoides (B in Fig. 1), species of the Neotropical Realm of the genus Polystomoides infecting pleurodires (C in Fig. 1) and species of the Ethiopian Realm of the genus Uropolystomoides infecting pleurodires. On the contrary, with a few exceptions - Apaloneotrema moleri (Du Preez and Morrison, 2012) and Uropolystomoides spp. - all other polystomes infecting the oral cavity, the urinary bladder or the conjunctival sacs of cryptodires, show peripheral vaginae. These include species of the Nearctic, Neotropical, Oriental and Palearctic Realms belonging to the genera Fornixtrema, Neopolystoma and Polystomoides, but also U. nelsoni from the Nearctic Realm. Apaloneotrema moleri and all species of the genus Uropolystomoides show opposite, near-peripheral vaginae. The vaginae for A. moleri, which were reported as latero-ventral in Du Preez and Verneau (2020), are actually near peripheral and lateral to the intestinal caeca, unlike all polystomes from pleurodires where the vaginae are more medial. Furthermore, the vaginae for A. moleri are pre-ovarian and located at a position about one third from the anterior tip (Du Preez and Verneau, 2020), whereas they are post-ovarian at the level of the anterior margin of the testis for all other chelonian polystomes.

Fig. 1.

Fig. 1

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Lateroventral vaginae as observed for Neopolystomakreffti (A), Uropolystomoides scottae (B) and Polystomoides brasiliensis (C). Annotations: gb, genital bulb; ov, ovary; te, testis; vg, vaginae. Scale bar = 200 μm.

When the inter-vaginal distance is expressed as a percentage of the body width at the level of the vagina, it was determined as 85.5 (76.4–96.6) for Aussietrema infecting pleurodires (A in Fig. 2); 62.1 (57.0–64.8) for Neopolystoma infecting pleurodires (B in Fig. 2); 70.8 (61.3–75.9) for Polystomoides infecting pleurodires (C in Fig. 2); 67.2 (61.5–72.9) for Uropolystomoides infecting pleurodires of the Australian Realm (D in Fig. 2); 73.3 (68.9–79.7) for Uropolystomoides infecting pleurodires of the Ethiopian Realm (E in Fig. 2); 98.3 (96.7–100) for Fornixtrema infecting cryptodires (F in Fig. 2); 97.8 (93.3–99.3) for Neopolystoma infecting cryptodires (G in Fig. 2); 94.7 (84.2–99.4) for Polystomoides infecting cryptodires (H in Fig. 2); 87.8 (75.0–98.4) for Uropolystomoides infecting cryptodires (I in Fig. 2); 96.0 for A. moleri (J in Figs. 2) and 99 for U. nelsoni (K in Fig. 2), both infecting crytodire hosts.

Fig. 2.

Fig. 2

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Violin plot of inter-vaginal distance as % of the body width at the level of the vagina. The plot visualises the probability density of the data at different values. Within each violin is a marker for the median of the data as well as marker indicating the interquartile range. A = Aussietrema infecting pleurodires; B = Neopolystoma infecting pleurodires; C = Polystomoides infecting pleurodires; D = Uropolystomoides infecting pleurodires of the Australian Realm; E = Uropolystomoides infecting pleurodires of the Ethiopian Realm; F = Fornixtrema infecting cryptodires; G = Neopolystoma infecting cryptodires; G = Polystomoides infecting cryptodires; I = Uropolystomoides infecting cryptodires; J = Apalonotrema moleri infecting a cryptodire host. K = Uteropolystomoides nelsoni infecting a cryptodire host.

3.2. Genital spine number

The genital spine number is 30 (23–50) for Aussietrema infecting pleurodires (A in Fig. 3); 21 (12–33) for Neopolystoma infecting pleurodires (B in Fig. 3); 6 (2–8) for Polystomoides infecting pleurodires (C in Fig. 3); 75 (73–78) for Uropolystomoides infecting pleurodires of the Australian Realm (D in Fig. 3); 33 (27–37) for Uropolystomoides infecting pleurodires of the Ethiopian Realm (E in Fig. 3); 9 (7–16) for Fornixtrema infecting cryptodires (F in Fig. 3); 19 (14–34) for Neopolystoma infecting cryptodires (G in Fig. 3); 33 (24–44) for Polystomoides infecting cryptodires (H in Fig. 3); 49 (13–77) for Uropolystomoides infecting cryptodires (I in Fig. 3); 12–13 for A. moleri (J in Figs. 3) and 130 (123–136) for U. nelsoni (K in Fig. 3), both infecting cryptodire hosts.

Fig. 3.

Fig. 3

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Violin plot of the genital spines number.

A = Aussietrema infecting pleurodires; B = Neopolystoma infecting pleurodires; C = Polystomoides infecting pleurodires; D = Uropolystomoides infecting pleurodires of the Australian Realm; E = Uropolystomoides infecting pleurodires of the Ethiopian Realm; F = Fornixtrema infecting cryptodires; G = Neopolystoma infecting cryptodires; G = Polystomoides infecting cryptodires; I = Uropolystomoides infecting cryptodires; J = Apalonotrema moleri infecting a cryptodire host. K = Uteropolystomoides nelsoni infecting a cryptodire host.

3.3. Shape of haptor and suckers

The three Polystomoides species known to infect South American pleurodires are the sole polystomes with deep incisions between suckers, giving the impression of a hand with fingers or that of a colony of polyps on narrow stalks. The incisions stretch about halfway to the centre of the hamulus in P. brasiliensis with suckers that are directed ventro-laterally to laterally (Fig. 4). Within the suckers of P. brasiliensis are some skeletal elements which are different from all other known Polystomoides species and polystomes in general. The ring of skeletal blocks, present in Polystomoides, appears to be absent and the skeletal funnel is small (Fig. 5). The walls of the suckers appear to have elongated spatulate sclerites (Fig. 5).

Fig. 4.

Fig. 4

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Haptor of Polystomoides brasiliensis showing the deep incisions between suckers. Scale bar = 200 μm.

Fig. 5.

Fig. 5

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Skeletal elements inside the sucker of Polystomoides brasiliensis. Annotations: sf, skeletal funnel; ss, spatulate sclerites. Scale bar = 100 μm.

3.4. Shape and size of hamuli

Whereas the majority of Polystomoides spp. infecting cryptodires have hamuli in excess of 100 μm long (Fig. 6A–F), those infecting pleurodires have small hamuli ranging from 52 μm in Polystomoides uruguayensis Mané-Garzon and Gil, 1961 to 72 μm in P. brasiliensis (Fig. 6G–I). Furthermore, Polystomoides spp. of cryptodires mostly have broad hamuli with either no cut or a very small cut, between handle and blade, while Polystomoides spp. of pleurodires have a very deep cut leaving both the handle and the guard as long thin structures (Fig. 6G–I).

Fig. 6.

Fig. 6

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Hamuli of Polystomoides spp. A = Polystomoides japonicum Ozaki, 1935; B = Polystomoides magdalenensis Lenis and Garcia-Prieto, 2009; C = Polystomoides ocellatum (Rudolphi, 1819); D = Polystomoides pauli (Timmers and Lewis, 1979); E = Polystomoides platynotae Combes and Rohde, 1979; F = Polystomoides rohdei; G = Polystomoides brasiliensis; H = Polystomoides fuquesiMané-Garzon and Gil, 1962; I = Polystomoides uruguayensis Mané-Garzon and Gil, 1961. Scale bar = 50 μm.

4. Discussion

4.1. Value of the morphological characters under investigation for the systematic revision of chelonian polystomes

All polystomes infecting pleurodires show latero-ventral vaginae whereas polystomes infecting cryptodires, have peripheral or near-peripheral vaginae. Apaloneotrema moleri is unique in turtle polystomes in that its vaginae are in pre-ovarian positions. Concerning the inter-vaginal distance, when expressed as a percentage of the body width at the level of the vagina, there is no overlap between polystome species infecting pleurodires and those from cryptodires (Fig. 2), except for Aussietrema, but, also for species of Uropolystomoides from the Ethiopian Realm whose inter-vaginal distance overlaps with that of Uropolystomoides spp. infecting pleurodires of the Australian Realm on the one hand and with that of Uropolystomoides spp. infecting cryptodires on the other. Though the position of the vaginae is of limited interest for species delimitation, it provides a valuable and consistent character to separate species of Neopolystoma and Polystomoides infecting pleurodires of the Australian and Neotropical Realms, respectively, from all others infecting cryptodires.

Although the number of genital spines may be a useful character for distinguishing distinct species, it is not often of much use for genus delimitation (Fig. 3) except for Uteropolystomoides that shows 100 or more genital spines (Tinsley, 2017). However, the fewer than 10 genital spines reported for these species, separates this group of species from all other chelonian polystome genera. The reporting of only two genital spines for Polystomoides fuquesi Mané-Garzon and Gil (1962) was surprising, but has been noticed from a sample of 28 specimens (Mané-Garzon and Gil, 1962). However, the fact that both P. fuquesi and P. uruguayensis were described from the same host species in the same geographical area requires further investigation to validate the syntopic occurrence of P. fuquesi and P. uruguayensis. As a consequence, the genital spine number appears to be a valuable taxonomic character to separate Polystomoides spp. infecting South American pleurodires from all other polystomes.

In mature polystomes, the haptor is, as a rule, a rigid discoid structure which is usually dorso-ventrally flattened with a greater width than length. The deep incisions between the suckers of Polystomoides spp. infecting South American pleurodires thus provides a reliable character for these species, distinguishing them from all other polystomes. However, Polystomoides digitatum (MacCallum, 1918), described at the New York Aquarium from the softshell turtle Apalone spinifera (Lesueur, 1827) is currently considered as a junior synonym of Polystomoides coronatum (Leydi, 1888) (Price, 1939). This species has a similar haptor with deep cuts between the suckers judging from the species drawing of the syntype. However, two syntypes examined and photographed in 2015 by one of us at the US Parasite Collection in Baltimore, did not show deep cuts between suckers. The fact that this species has been described from an aquarium where turtles of different species and origins are often kept together in confined spaces, suggests a possibility of a lateral transfer from another host (see Verneau et al., 2011). However, this species shows peripheral vaginae in line with polystomes of cryptodires. Therefore, polystome specimens of the oral and nasal cavities of A. spinifera need to be located in the wild to resolve the systematic status of P. digitatum.

The shape and morphology of polystome suckers vary from soft, simple cups to complex structures with elaborate skeletal elements. Du Preez and Theunissen (2021) studied sucker morphology and proposed a classification system with four types of suckers. All polystomes of chelonian hosts have Type III suckers sensu Du Preez and Theunissen (2021) which entail an elaborate system of skeletal elements aiding in securing a firm grip on the host tissue. While Neopolystoma spp. of the Australian Realm have typical Type III suckers with a skeletal ring and a prominent skeletal funnel, the suckers of South American Polystomoides spp. infecting pleurodires are more delicate and with their spatulate sclerites resembling Type IV suckers sensu Du Preez and Theunissen (2021) reported from Concinnocotyla Pichelin, Whittington and Pearson, 1991 (see Du Preez and Theunissen, 2021).

The delicate hamuli, with very deep cuts between the handle and the guard, characterize only Polystomoides spp. of South American pleurodires (Fig. 6G–I). Although, a small cut or incision exists between the handle and the guard within hamuli of Polystomoides rohdei Mané-Garzon and Holcman-Spector, 1968, which infects a cryptodire host from the Neotropical Realm, its hamuli are clearly distinct from polystomes infecting pleurodires (Fig. 6F). In the remaining Polystomoides spp. infecting cryptodires (Fig. 6A–E), the hamulus blade is a solid structure without any incision, enabling this character to also be used to separate Polystomoides spp. of pleurodires from all other polystomes.

4.2. Taxonomy

In the light of the morphological evidence presented above, we propose two new genera for the polystomes infecting pleurodires of the Australian and Neotropical Realms, respectively.

4.2.1. Genus: Pleurodirotrema n. g

4.2.1.1. Generic characteristics

Polystomatidae. Polystomoidinae. Mouth with false oral sucker subterminal. Muscular pharynx. Intestinal caeca extending full length of body proper, not confluent posteriorly. Testis single, compact equatorial. Ovary pretesticular, small. Vitellaria throughout most of body proper, not extending into haptor. Vaginae latero-ventral in line with anterior margin of testis. Uterus absent. Haptor without hamuli. Skeletal elements in suckers arranged as ring of blocks. Sucker type III. Parasitic in urinary bladder and oral region of pleurodires of Australian Realm.

4.2.1.2. Taxonomic summary

Ethymology: Refers to the turtle sub-order Pleurodira.

Type species: Pleurodirotrema chelodinae (MacCallum, 1918) n. comb.

Synonyms: Polystoma chelodinae MacCallum, 1918; Neopolystoma chelodinae Price, 1939.

Site of infection: urinary bladder.

Other species: Pleurodirotrema kreffti (Rohde, 1984) n. comb. Synonym: Neopolystoma kreffti Rohde, 1984. Site of infection: oral cavity; Pleurodirotrema macleayi (Rohde, 1984) n. comb. Synonym: Neopolystoma macleayi Rohde, 1984. Site of infection: urinary bladder; Pleurodirotrema novaeguineae (Fairfax, 1990) n. comb. Synonym: Neopolystoma novaeguineae Fairfax, 1990. Site of infection: oral cavity.

4.2.2. Genus: Manotrema n. g

4.2.2.1. Generic characteristics

Polystomatidae. Polystomoidinae. Mouth with false oral sucker subterminal. Muscular pharynx. Intestinal caeca extending full length of body proper, not confluent posteriorly. Testis single, compact equatorial. Ovary pretesticular, small. Vitellaria throughout most of body proper, not extending into haptor. Vaginae latero-ventral in line with anterior margin of testis. Uterus absent. Haptor with deep incisions between suckers, which appear fingerlike. Skeletal elements in suckers not arranged as a ring of blocks but rather thin spatulate outward bending needles. Sucker type IV. Two small pairs of hamuli present with very deep cuts between handle and guard. Parasitic in urinary bladder of pleurodires of the Neotropical Realm.

4.2.2.2. Taxonomic summary

Etymology: The haptor with deep cuts between the suckers resembles a hand with fingers.

Type species: Manotrema uruguayensis (Mané-Garzón and Gil, 1961) n. comb. Synonym: Polystomoides uruguayensis Mané-Garzón and Gil, 1961. Site of infection: urinary bladder.

Other species: Manotrema fuquesi (Mané-Garzón and Gil, 1962) n. comb. Synonym: Polystomoides fuquesi Mané-Garzón and Gil, 1962. Site of infection: urinary bladder; Manotrema brasiliensis (Viera, Novelli, Sousa and SouzaLima, 2008) n. comb. Synonym: Polystomoides brasiliensis Viera, Novelli, Sousa and SouzaLima, 2008. Site of infection: urinary bladder.

4.3. What does sucker morphology within Manotrema n. g. suggest about functionality?

The deep cuts and the outwards directed suckers resemble the branchial generation of two polystome genera infecting anuran hosts, namely Polystoma Zeder, 1800 and Metapolystoma Combes, 1976. When an oncomiracidium of these polystomes establishes on the gills of a young tadpole, well before metamorphosis, it develops rapidly and produces eggs in a matter of 16 days (Kok and Du Preez, 1989; Du Preez and Kok, 1998). This parasite, which is usually considered as a neotenic form, attaches to the branchial filaments inside the branchial chamber of the tadpole. Its haptor shows deep incisions between suckers, increasing flexibility of the haptor. Furthermore, suckers are not directed ventrally like in mature parasites found in the bladder of their host, but ventro-laterally, making attaching to gill filaments within a tightly packed gill chamber challenging. Therefore, one could argue that the deep cuts between the suckers in Manotrema n. g. may also provide additional flexibility to the suckers. However, their functional adaptations require further investigation.

4.4. The impact of chelonian evolution on polystome systematics

Following revision of the chelonian polystome classification, we can now consider 10 genera, among which Apaloneotrema, Fornixtrema, Neopolystoma, Polystomoidella, Polystomoides and Uteropolystomoides that infect cryptodires, Aussietrema, Manotrema n. g. and Pleurodirotrema n. g. that specifically infect pleurodires and Uropolystomoides that infects both cryptodires and pleurodires. With Aussietrema and Pleurodirotrema n. g. - both infecting chelids of the Australian Realm - and Manotrema n. g. infecting chelids of the Neotropical Realm, one may wonder about the origin of these genera. Is there any relationship between parasite speciation and plate tectonics, as was shown for their hosts following biogeographic vicariance (Pereira et al., 2017)? Though it was previously documented that Aussietrema and Pleurodirotrema n. g. (initially considered as Neopolystoma) form a solid monophyletic group (See Du Preez and Verneau, 2020), a hypothesis that results from biogeographic vicariance holds true only if that clade is a sister group of Manotrema n. g., which at this stage cannot be formally demonstrated.

Uropolystomoides currently includes polystomes of both cryptodire and pleurodire turtles. Those infecting pleurodires are found in the Pelomedusidae of the Ethiopian Realm as well as the Chelidae of the Australian Realm. Although the size of hamulus 1 for all species of Uropolystomoides is greater than the sucker diameter, i.e. a unique characteristic for this genus (Tinsley and Tinsley, 2016), all Uropolystomoides species infecting pleurodires differ from Uropolystomoides spp. infecting cryptodires in shape and size of their vaginae. Moreover, Uropolystomoides spp. of the Australian Realm also differ from their congeners of the Ethiopian Realm by several characteristics such as the body size, the sucker diameter and the genital spine number (see Morrison and Du Preez, 2011). As a result, a more thorough analysis of the phylogenetic relationships of the Polystomatidae, including several representatives of the genera Aussietrema, Pleurodirotrema n. g. and Uropolystomoides from the Australian Realm as well as species of Manotrema n. g., will assist in establishing the origins of polystomes infecting pleurodires of the Australian realm.

Declaration of interest

None.

Acknowledgements

We are thankful to Mal Bryant of Queensland Museum for a loan of representatives of Australian polystomes and to Marcello Knoff from the Oswaldo Cruz Institute for assistance during a visit and for a loan of P. brasiliensis specimens. We are also thankfull to Dr Gerhard du Preez for preparing Fig. 2, Fig. 3 and to the reviewers for their valuable comments on the manuscript. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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