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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.

Two frog species, Blommersia domerguei and Mantella expectata, are reported as hosts for new species of Madapolystoma. Phylogenetic analyses and genetic divergences observed in the genus supported the distinction of two morphotypes infesting selectively each host species and morphological investigation combining marginal hooklet morphometrics, genital spine number and measurements further showed that polystomes from the two host species differed from each other and from all other known polystomes. Madapolystoma magnahami n. sp. and Madapolystoma isaloensis n. sp. are therefore described as two new species. Advanced in utero development was illustrated in both polystome species following the observation of well developed hamuli and two pairs of haptoral suckers in developing embryos. Inside some of these in utero embryos a F2 generation embryo was also observed. This is the first report of true viviparity among polystomatid flatworms.

Purpose Despite Madagascar’s high amphibian diversity of more than 400 species, only a few polystome species are known from the island. The dissection of frogs from museum collections, together with amphibian and parasite surveys conducted in Madagascar led to the discovery of an undescribed polystome infecting Aglyptodactylus madagascariensis. The purpose of this study is to formally describe this species. Methods Polystomes recovered from A. madagascariensis were stained (Acetocarmine) and mounted (Canada balsam) to facilitate morphometrics and taxonomic drawings. Some specimens were fixed in absolute alcohol, a Bayesian tree inferred from the analysis of concatenated 18S, 28S and COI gene sequences was constructed and pairwise distances were calculated. Parasites collected from archived hosts in museums were used for histology and scanning electron microscopy (SEM). Results Polystomes recovered from A. madagascariensis display characteristics of the genus Metapolystoma and morphologically differed from all other known metapolystomes. The Bayesian phylogeny shows that Metapolystoma n. sp. ex. A. madagascariensis and M. falcatum are sister species with high Bayesian posterior probability. Histological and SEM investigations contributed to morphological descriptions. Conclusions Morphological examination supported by phylogenetic analysis and genetic divergences revealed distinct differences from all known metapolystome species, supporting the description of a new species. Differences between the life cycles of Metapolystoma and Polystoma provided additional evidence for the validity of that genus as taxon. Whereas Polystoma may display ovoviviparity on rare occasions after incomplete egg expulsion towards the end of the breeding season, Metapolystoma displays true ovoviviparity. We emphasize the need for parasite surveys in Madagascar and recommended for museum material to be examined for polystomes to provide supplementary material and localities for further field investigation. Graphical Abstract

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