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First record of Metapolystoma (Monogenea: Polystomatidae) from Boophis tree frogs in Madagascar, with the description of five new species

Although Madagascar has more than 350 frog species of which all but two are endemic to the island, the known polystome (Monogenea: Polystomatidae) diversity parasitizing Malagasy frogs is low, encompassing five species of Madapolystoma, one species of Kankana and one Metapolystoma. Investigating the parasite diversity of frog parasites at selected Malagasy localities led to the discovery of undescribed polystomes. Five treefrogs, Boophis albilabris, Boophis doulioti, Boophis luteus, Boophis madagascariensis and Boophis occidentalis were found to be infected and are reported here as hosts for new Metapolystoma species. Morphological investigation, combining examination of body length, haptor length, genital bulb width, genital crown diameter, genital spine number, genital spine length, ovary length, egg length, hamulus length, hamulus guard length and hamulus hook length, revealed five distinct morphotypes. Phylogenetic analysis and genetic divergences obtained for three of the five morphotypes, support the distinction of new species. Metapolystoma ansuanum n. sp. is described from B. luteus, Metapolystoma falcatum n. sp. from B. doulioti, Metapolystoma multiova n. sp. from B. occidentalis, Metapolystoma theroni n. sp. from B. madagascareniensis and Metapolystoma vencesi n. sp. from B. albilabris. Finally, although the validity of Metapolystoma as taxon is not fully resolved yet, the phylogenetic position of the described species and their morphology provide clear evidence for new metapolystome taxa.

Metapolystoma brygoonis was initially described as Polystoma and later elevated (Combes, 1976). This separation of Metapolystoma and Polystoma was based on morphological characters, including the large extended uterus containing many eggs that fills the largest part of the body and the posterior position of the large ovary (Combes, 1976). The validity of Metapolystoma as a genus has however been under dispute ever since its nested position within Polystoma was demonstrated at the molecular level (Bentz et al., 2001;Verneau et al., 2002;Olson and Tkach, 2005). Up to the present, the three known Metapolystoma species have been described from grass frogs belonging to Ptychadena: M. brygoonis from P. mascareniensis in Madagascar, M. cachani (Gallien, 1956) from Ptychadena longirostris (Peters, 1870) in Ivory Coast and Metapolystoma porosissimae du Preez and Kok 1992, from Ptychadena porosissima (Steindachner, 1867 in South Africa. It was therefore likely that many Madagascan polystomes awaited discovery (Landman et al., 2018).
Among the 27 anuran genera found in Madagascar (Frost, 2020), Boophis Tschudi, 1838 is endemic to Madagascar and the Mayotte Island in the Comoros (Frost, 2020;Glaw and Vences, 2007), hence totalling 79 currently recognised species (Frost, 2020) representing a diverse and species-rich group within the Mantellidae Günter, 1859. During herpetological surveys conducted in Madagascar in 2005Madagascar in , 2006Madagascar in and 2007, several species of Boophis from different localities were examined for polystome parasites. Boophis albilabris (Boulenger, 1888), Boophis doulioti (Angel, 1934), Boophis luteus (Boulenger, 1882), Boophis madagascariensis (Peters, 1874) and Boophis occidentalis Glaw & Vences, 1994 were found to be infected with five distinct, unknown Metapolystoma species. Since the collection of additional materials was constrained by permit restrictions and administrative difficulties in Madagascar, and because preliminary molecular and morphological data converged towards the same result, our objective was to describe new parasites collected from distinct frog species despite their small sample size.

Host and parasite sampling
Annually during February-March of 2005, 2006 Boophis species were collected in Madagascar (Table 1). Following collection by hand at night, frogs were individually kept overnight in clear plastic bags containing 50 ml of tap water. After 24 h, the water in which the frogs were kept was poured through two plankton sieves with mesh sizes of 500 and 100 μm respectively. The 500 μm sieve retained course debris and most of the faeces, while the 100 μm sieve retained parasite eggs and fine debris. The content of the 100 μm sieve was then washed into a Petri dish and inspected for the presence of polystome eggs using a dissecting microscope. Infected frogs were euthanized with MS222 (Ethyl-3-aminobenzoate methanesulfonate) and dissected for parasite investigation using a Nikon SMZ-645 dissecting microscope. Where no eggs were observed, two representatives per species per locality were dissected to check for the presence of non egg-producing polystomes. The kidneys, urinary bladder and accessory bladders were removed and inspected in a Petri dish containing 0.6% Ringers solution. For the purpose of molecular studies, some juvenile polystomes were preserved in absolute ethanol. The remainder of the juvenile polystomes were mounted in ammonium picrate glycerine or preserved in 10% NBF. Adult parasites were fixed in 10% NBF under coverslip pressure. Naturelle of Paris, were dissected and parasites retrieved and processed as explained above. Host and parasite distribution maps ( Fig. 1) were drawn in ArcMap 10.5.1 (Esri, California), and shape files for frog distributions were provided by the IUCN (IUCN, 2016a-e).

DNA extraction and amplification
All polystome DNA samples used in this study were sourced from Verneau et al. (2009) andBerthier (2011). According to these authors, DNA extractions were conducted in 100-150 μl of 10% Chelex 100 sodium (Sigma-Aldrich, L'Isle d'Abeau Chesnes, France) with the proteinase K 1 mg⋅mL− 1 final concentration at 55 • C for the duration of 1 to 1,5 h. Enzymatic reactions were stopped at 100 • C for 15 min and DNA were stored at − 20 • C until use. Amplifications of 18S, 28S and COI were conducted following the procedure described in Héritier et al. (2015), regardless of the gene of interest or primers used. The complete 18S rRNA gene and the partial 28S rRNA gene were amplified in two rounds with the respective combinations of primers F18/18Rg and 18F3/IR5 for 18S and LSU5'/IR16 and IF15/LSU3 ′ for 28S. Primer sequences are reported in Sinnappah et al. (2001), Verneau et al. (2009) andHéritier et al. (2015). COI was amplified using the combination of primers L-CO1p/H-Cox1p2 (Littlewood et al., 1997). All PCR products were sent to the Genoscreen Company (Lille, France) for purification and sequencing with their respective forward and reverse PCR primers. Sequences were read and edited with the software Geneious (Saint Joseph, Missouri, USA) to check chromatograms before use for phylogenetic and distance analyses.

Phylogenetic and distance analyses
All Metapolystoma sequences were edited and aligned independently using Clustal W implemented in MEGA version 7 (Kumar et al., 2016) under default parameters (Thompson et al., 1994). The alignment included five African Polystoma species and a single European Polystoma species, that is, Polystoma integerrimum (Frölich, 1791), which was used as an outgroup according to Bentz et al. (2001). They were subsequently concatenated in a single alignment for Bayesian analysis. The two ribosomal genes were treated as two separate partitions, and the COI genes as three distinct partitions according to their codon position. A two substitution rates model with a proportion of invariable characters was selected for the 18S partition, whereas a GTR + I model was selected for the 28S partition following the Akaike Information Criterion (AIC) implemented in Modeltest 3.06 (Posada and Crandall, 1998). Concerning COI, six types of substitutions and gamma rates each comprising four gamma rate categories were applied to the first and second positions, whereas six types of substitutions and invariable-gamma rates each comprising four gamma rate categories were applied to the third position. Evolutionary parameters were estimated independently for all five partitions. The Bayesian analysis was run using MrBayes 3.04b (Huelsenbeck and Ronquist, 2001), employing four chains running for ten million generations and sampled every 100 cycles. The Bayesian consensus tree was subsequently drawn after removing the first 10 000 trees (10%) as the burn-in phase, and viewed with TreeView version 1.6 (Page, 1996). Corrected pairwise distances were calculated independently for partial 18S, 28S and COI sequences using the Kimura 2-parameter model and 1000 bootstrap replicates in MEGA version 7 (Kimura, 1980).

Morphology and morphometry
Parasites were examined and photographed using a Zeiss Imager Axio10 compound microscope (Zeiss, Germany) fitted with a Zeiss Axio cam 305 camera (Zeiss, Germany). Morphological structures and organs were measured in micrometres using the Zeiss Zen Blue elements (Zeiss, Germany) software program. Hamuli were respectively measured from the apex behind the hook to the tip of the guard (Length X) and to the handle (Length Y), and the hook from tip to base (Length Z). Full-body images of type specimens were taken using a Nikon AZ100M microscope (Nikon, Netherlands) fitted with a low powered 1× objective. Illustrations were done in Adobe Illustrator CC (Adobe, California). Additional taxonomic measurements for M. porosissimae were taken from the type   Values: (Min -Max (Mean ±Ơ; N)), numbers in bold indicate additional measurements taken from species described prior to the present study. a From Du Preez and Kok (1992a). b From Euzet and Combes (1964). c From Gallien (1956) (left), Kulo (1981) (middle) and Murith (1981) (right).

Level of infection.
One of six frogs examined from Ambatolahy was infected with one mature and four juvenile parasites (prevalence 16.6%). One of four frogs examined from Ambohitantely was infected with seven mature parasites (prevalence 25%). The prevalence for the combined sample was 20% and the mean intensity 6.5.
3.1.1.6. Voucher material. Remaining specimens in polystome collection, North-West University, Potchefstroom, South Africa.  Table 2. Body pyriform (Fig. 2), dorsoventrally flattened, widest section at 57% of total length from anterior end, body length 2.7 times greater than width. Mouth sub-ventral, surrounded by false oral sucker. Posterior haptor 16% of body length, bearing three pairs of haptoral suckers of equal size. Marginal hooklets placed as for other polystomes: pairs one and two between hamuli, pairs three to five embedded in suckers, pairs six to eight found between anterior suckers, pairs one and eight larger than pairs two to seven (Fig. 3a). Well-developed hamuli between posterior-most haptoral suckers without cut between handle and guard (Fig. 3b). Medial pharynx length greater than width, positioned immediately posterior or at the margin of false oral sucker. Intestine bifurcates immediately posterior to pharynx, 8% of total length from most anterior point, converging posteriorly at position of 80% of total length from most anterior point, extending into haptor; no prehaptoral anastomoses. Lateral diverticula length equal to width, found only in last third of intestine. Medial diverticula only posterior to ovary, length greater than width.
Testis follicular, positioned in a narrow band posterior to the ovary, ventral to intestine. Vas deferens widens anteriorly to form sinuous semen vesicle 20-32 (27 ± 5; 1) wide, 179 long, 2% of body length, narrowing towards genital bulb, opening in common genital opening. Genital pore opening on left ventral half, posterior to intestinal ceca bifurcation, 10% of total length from most anterior point. Genital bulb muscular, surrounded by glandular cells, armed with genital crown bearing eight genital spines (Fig. 3c

Site in host.
Gills of tadpoles and urinary bladder of mature frogs.

Level of infection.
Three of 22 frogs collected were infected. One mature and 13 juvenile parasites were recovered, with a maximum of 12 recovered from a single host (prevalence 13.6%. mean intensity 4.7). Seven of 10 tadpoles collected were infected with a total of 13 neotenic parasites, while as many as three parasites were infecting a single host (prevalence 70%; mean intensity 1.85). Though the name neotenic for some polystomes can be confusing (see Badets and Verneau, 2009), it refers here to egg producing parasites that were recovered from the gills of tadpoles.
Measurements reflected in Table 2. Body pyriform (Fig. 4), dorsoventrally flat, widest section at 66% of total length from anterior end, body length 2.4 times greater than width. Mouth sub-ventral, surrounded by false oral sucker. Posterior haptor 19% of body length bearing three pairs of haptoral suckers equal in size. Marginal hooklets placed as for other polystomes and as described for M. vencesi n. sp., pairs one and eight larger than pairs two to seven (Fig. 5a). Well- developed hamuli between posterior-most haptoral suckers with deep cut between handle and guard (Fig. 5b). Hamuli development presented in Fig. 5c. Medial pharynx length greater than width, positioned immediately posterior to or at the margin of false oral sucker. Intestine bifurcates immediately posterior to pharynx at 11% from anterior, converging posteriorly at 82% from anterior, stretching in between haptoral suckers; no prehaptoral anastomoses. Intestine bears lateral diverticula, length equal to width. Medial diverticula only posterior to ovary, length greater than width.
Testis follicular, only a small section visible, positioned directly posterior to the ovary, ventral to intestine. Vas deferens widens anteriorly to form sinuous semen vesicle 23-40 (30 ± 6; 1) wide, 301 long, measuring 4% of total length, narrowing towards genital bulb, opening     in common genital opening. Genital pore opening mid-ventral, posterior to intestinal ceca bifurcation at 12% from anterior, genital bulb muscular, surrounded by glandular cells, armed with genital crown bearing eight genital spines (Fig. 5d).

Level of infection.
One of nine frogs collected were infected with two mature parasites (prevalence 11.1%).

Etymology. This species is named for Mrs Anna-Susan van der
Linde, known as Ansu, in acknowledgement of her teaching and inspiration of many secondary school pupils in the field of biology. Table 2. Body pyriform (Fig. 8), dorsoventrally flat, ventrally concave, widest section at position 16-65% from anterior end of body. Body length 2.6-2.8 times greater than width. Mouth sub-ventral, surrounded by false oral sucker. Posterior haptor measures 24% of total length, bearing three pairs of haptoral suckers of equal size. Well-developed hamuli positioned between posterior-most haptoral suckers with deep cut between handle and guard, hook length Z long relative to Length X (Fig. 9a). Marginal hooklets placed as for other polystomes: pairs one and two between hamuli, pairs three to five embedded in haptoral suckers, pairs six to eight positioned between anterior-most haptoral suckers (Fig. 9b). Medial pharynx length greater than width, positioned immediately posterior to or at margin of false oral sucker. Intestine bifurcates immediately posterior to pharynx at 15-18% from anterior, converging posteriorly at 79-81% from anterior; no prehaptoral anastomoses. Lateral intestinal diverticula length equal to width in anterior half, length greater than width in posterior half. Medial diverticula only posterior to ovary.

Level of infection.
Three of 30 frogs collected were infected with a total of one mature and 71 juvenile parasites, while as many as 40 parasites were infecting a single host (prevalence of 10%, mean intensity 24).

Etymology.
This species is named in honour of emeritus Professor Pieter Daniel Theron at the North-West University, South Africa, in recognition of 54 years of inspiring teaching and dedication to the field of zoology.
3.1.4.9. Description. Measurements reflected in Table 2. Body pyriform Fig. 11. Metapolystoma theroni n. sp. from Boophis madagascariensis. a, marginal hooklets 1 (top), 2-7 (middle) and 8 (bottom) from holotype and paratypes; b, hamuli from holotype; c, hamulus development; d, genital crown from holotype. (Fig. 10) dorsoventrally flat, widest section at 51% from anterior end, body length 2.6 times greater than width, mouth sub-ventral, surrounded by false oral sucker. Posterior haptor occupying 18% of total body length, bearing three pairs of haptoral suckers, equal in size. Marginal hooklets placed as for other polystomes, pairs one and two between hamuli, pairs three to five embedded in suckers, pairs six to eight in area between anterior-most suckers, pairs one and eight larger than pairs two to seven (Fig. 11a). Well-developed hamuli positioned between posterior-most haptoral suckers with deep cut between handle and guard (Fig. 11b). Hamuli development presented in Fig. 11c. Medial pharynx length greater than width, positioned immediately posterior to or at margin of false oral sucker. Intestine bifurcates immediately posterior to pharynx at 10% from anterior, converging posteriorly at 80% from anterior; no prehaptoral anastomoses. Lateral intestinal diverticula in first three quarters length equal to width, in last quarter length greater than width. Medial diverticula only posterior to ovary, length greater than width.
Testis follicular, u-shaped, mainly positioned posterior to the ovary with two lateral processes extending forward along the lateral line past the ovary up to one-third of the body proper, ventral to intestine. Vas deferens widens anteriorly to form sinuous semen vesicle 23-65 (46 ± 18; 1) wide, 122 long, measuring 1% of body length, narrowing towards genital bulb, opening in common genital opening. Genital pore opening mid-ventral, posterior to intestinal ceca bifurcation, positioned 13% from anterior, genital bulb muscular, surrounded by glandular cells, armed with genital crown bearing seven genital spines (Fig. 11d).

Level of infection.
One of five frogs collected were infected with two adult parasites (prevalence 20%).
3.1.5.5. Type material. The morphological descriptions are based on two mature parasites. Two sexually mature specimens (Holotype 571, Paratype 572), both from the type locality, were deposited in the parasitic worm collection, National Museum, Aliwal Street, Bloemfontein 9301. 3.1.5.7. Etymology. The species epithet is derived from Latin and related to the vast number of eggs (±500) carried by this species in contrast with other currently known species in the genus (multi = many + ova = eggs). Table 2. Body pyriform (Fig. 12), dorsoventrally flat, ventrally concave, widest section at 50-57% from anterior end of body. Body length 2.2-2.3 times greater than width. Mouth directed sub-ventrally, surrounded by false oral sucker. Posterior haptor measures 18% of total length bearing three pairs of haptoral suckers of equal size. Marginal hooklet pairs six to eight situated between anterior-most haptoral suckers, pairs one and eight larger than pairs two to seven, pairs one and two between hamuli, pairs three to five embedded in suckers (Fig. 13a). Well-developed hamuli situated between posterior-most haptoral suckers with deep cut between handle and guard (Fig. 13b). Medial pharynx length greater than width, positioned immediately posterior to or at the margin of false oral sucker. Intestine bifurcates immediately posterior to pharynx at 13% from anterior, converging posteriorly at 79-85% from anterior; no  eg, egg; gb, genital bulb; gc, genito-intestinal canal; ha, hamuli; hp, haptor; ic, intestinal caecum; mo, mouth; oc, oncomiracidium; od, oviduct; os, false oral sucker; ov, ovarium; ph, pharynx; su, sucker; te, testis; va, vagina; vi, vitelline; vv, vitello-vaginal canal. prehaptoral anastomoses. Lateral intestinal diverticula situated in first third of intestine, where length is equal to width, absent in the second third while length is greater than width in the posterior third. Medial diverticula only posterior to ovary, length greater than width.

Description. Measurements reflected in
Testis follicular, sickle-shaped, posterior to ovary, ventral to intestine. Vas deferens obscured. Genital pore opening mid-ventrally situated on anterior margin of intestinal ceca bifurcation at 4% from anterior, genital bulb muscular, armed with genital crown bearing six genital spines (Fig. 13c).

Genetic divergences between Metapolystoma species and parasite phylogeny
Some of the 18S, 28S and COI sequences used in this study were retrieved from GenBank, while the others were obtained and submitted under accession numbers MW053457, MW053458 and MW054236 to MW054249 (Table 3). The final alignment, which had resulted in 3.977 Minimum and maximum distances with standard deviation as estimated among Metapolystoma species were as follows. M. falcatum n. sp.
When 18S, 28S and COI sequences were combined for Bayesian analysis (Fig. 14), M. cachani appears as the most basal species within Metapolystoma and Malagasy Metapolystoma form a clade. Within this clade, M. multiova n. sp. and M. theroni n. sp. are sister species; two sublineages are well differentiated within M. brygoonis. Sublineage A comprises polystomes found in localities at Makira, Ranomafana and Vohiparara (Fig. 1), while sublineage B comprises polystomes of the Ambatolampy and Ankarafantsika areas (Fig. 1). Because high levels of 28S and COI genetic divergence (Tables 5 and 6) were also observed these two sublineages (28S: 0.14% ± 0.0009; COI: 5.71% ± 0.011), it suggests that there could be two genetic entities within M. brygoonis infecting the same host species in Madagascar, that is, Ptychadena mascareniensis.

Remarks
The phylogenetic position of M. falcatum n. sp., M. multiova n. sp. and M. theroni n. sp. within the Polystomatidae (Fig. 14) confirms that they are members of Metapolystoma (Polystomatidae). Genetic divergence estimates among Metapolystoma taxa (Tables 4-6  , while the latter two also overlap. Hamulus hook length Z (Fig. 3b) Table 6 Mean genetic distances (below diagonal) and total character differences (above diagonal) between specimens of Metapolystoma species as inferred from comparisons of COI sequences (323 characters). To conclude, due to the small number of parasite specimens that were investigated, we can not exclude that the morphometric variability may represent intra specific variation. However, worms under investigation show a combination of morphological characters, namely shape of the hamulus handle and guard, shape of the hamulus hook, shape of the marginal hooklet tip on hooks and genital spine number that allow

Discussion
Metapolystoma was until now reported only from the Afrotropical realm anuran hosts belonging to Ptychadena (Ptychadenidae). Ptychadena is a successful and widespread genus in Africa being a well-suited host for polystomes (Du Preez and Kok, 1992a). Of the 56 known Ptychadena species (Frost, 2020), 11 are known to host polystomes, including 14 Polystoma and three Metapolystoma species (see Du Preez and Kok, 1992a). According to Verneau et al. (2009), Metapolystoma may have originated in Africa within the time window 19.8-4.3 million years ago (Mya) from ancestors close to Polystoma and further dispersed to Madagascar following natural transoceanic dispersal of the ancestor of Pt. mascareniensis at about 14.2-2.3 Mya . Because M. brygoonis forms a sister group to all other members of Malagasy Metapolystoma, M. cachani being basal within Metapolystoma, a host switch was suggested from the ancestor of Pt. mascareniensis to ancestral Boophis (Mantellidae) , which is confirmed in the present phylogenetic study. Even though Ptychadena and Boophis are not phylogenetically closely related, they both display pleisiomorphic reproductive modes, favouring the possibility of an ancestral host switch in Madagascar (see Verneau et al., 2009), and ultimately the diversification of Metapolystoma within Boophis. While Boophis is not the single representative of the family Mantellidae infected by polystomes in Madagascar, mantellid frogs of the two genera Mantella and Blommersia which both exhibit a derived mode of reproduction, are infected by polystomes of another genus, i.e. Madapolystoma (see Verneau et al., 2009;Du Preez et al., 2010;Landman et al., 2018). In addition, Verneau et al. (2009) reported another undescribed species of Metapolystoma from Aglyptodactylus madagascariensis (Duméril, 1853). Therefore, the five new Metapolystoma species described along this study clearly indicate that a larger diversity of metapolystomes can be expected. This is especially true since Madagascar is inhabited by 79 Boophis species (Frost, 2020) that may serve as hosts for Metapolystoma species. Furthermore, M. brygoonis can be divided in two separate lineages according to genetic differentiation (Fig. 14). These results strengthen the fact that Metapolystoma in Madagascar still continue to diversify and call for further sampling and investigation.
The validity of Metapolystoma has long been disputed. It was suggested that the uterine structure was achieved convergently within Metapolystoma (Tinsley, 1974), through the ability of the parasite to adapt to the ecology of its host (Kok and Seaman, 1987;Murith, 1981;Tinsley, 1983). Bentz et al., (2001) claimed it to be invalid and ascribed its morphological differences from Polystoma in terms of homoplastic characters. Nevertheless our phylogeny supports the monophyly of Metapolystoma, which is however nested within the paraphyletic Polystoma. If the morphological differences between Metapolystoma and Polystoma were the product of reproductive plasticity, Metapolystoma spp. would not have clustered together on a molecular level (see Fig. 14). The fact that they do, therefore, suggests that the long uterus may have been inherited by descent. It is however intriguing to note that two polystome species M. porosissimae and P. sodwanensis, which display completely different life-history strategies, can occur simultaneously in the same host species, i.e. P. porosissima in Africa (Du Preez and Kok, 1992b). Metapolystoma porosissimae displays a strategy where many eggs are stored in a large uterus, which is typical of polystomes that infest hosts within arid environments (Du Preez, 2015;Du Preez and Kok, 1992b). Conversely, P. sodwanensis has a small uterus containing only a few eggs, which is in line with a water-dependent host (Du Preez and Kok, 1992b). Even though morphological differences between the two species are distinct, the simultaneous occurrence of these two species in the same individuals of Pt. porosissima have reinforced the dispute (Du Preez and Kok, 1992b). Though the validity of Metapolystoma at this stage cannot be ruled out, a more in depth genetic investigation of the two African polystome species M. porosissimae and P. sodwanensis should help to conclude.

Declaration of competing interest
The authors declare that there is no conflict of interest.