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Keywords Aglyptodactylus madagascareniensis · Life cycle · Madagascar · Metapolystoma · Polystome abundant in amphibians, more particularly within anurans. Introduction It was shown that polystomes would have originated during the evolutionary transition between aquatic and terrestrial Although parasites are often perceived in a negative con- vertebrates and subsequently evolved within amphibians text, parasitism is the most successful and widespread life- [4, 5]. They could have secondarily switched to freshwater cycle strategy, to the extent that more animals are parasites turtles and the common hippopotamus from African cae- than not and those that are not parasitic serve as hosts for cilians . Polystomes co-evolved with their amphibian parasites. This is the result of a long evolutionary history hosts for at least 250 million years , and this may have in which hosts and parasites co-evolved and developed led to their high diversity of reproductive strategies. All successive adaptations . According to  parasite diver- polystomes exhibit direct life cycles which involve a larval sity correlates strongly with host diversity, which is espe- oncomiracidium. There are, however, major differences in cially true for amphibian parasites. Since amphibians are polystome infestation strategies. Some of these differences exposed to aquatic and terrestrial environments, they can are exemplified in the genera Protopolystoma infecting a be considered, to some extent, as the perfect host group. primarily aquatic host, Metapolystoma and Polystoma Aquatic environments provide ample opportunities for infecting amphibious hosts that spend a reasonable time infection. Following completion of metamorphosis, most at the water, Eupolystoma that infects terrestrial hosts that amphibians move out on land and serve as staple food spend little time at the water and Pseudodiplorchis that for all kinds of vertebrates, creating new opportunities for infects an arid adapted host (see [6, 7]). parasite transmission. One example is the African clawed Unlike all other polystomes, Metapolystoma and Poly- frog Xenopus laevis (Daudin 1802) that serves as host for stoma species share two different external reproductive 25 different parasite genera and more than 30 parasite spe- strategies. A neotenic cycle is followed when an oncomira- cies , which, in turn, also illustrate a large diversity of cidium attaches to the gills of a tadpole in pre-metamor- life-cycles. phosis. The oncomiracidium then develops into a neotenic Monogenea (Polystomatidae) is one of the parasite parasite and initiates egg production after two to three groups that infects amphibians. Although these parasites weeks until the host reaches metamorphosis. Thereafter were also reported from the Australian lungfish, freshwater the parasite detaches from the gills and dies [8–10]. On chelonians, and the common hippopotamus, they are most 1 3 Acta Parasitologica the contrary, if an oncomiracidium attaches to the gills of a tadpole in pro-metamorphosis, the parasite develops slowly into a urinary bladder destined parasite [11, 12]. This usually takes place at Gosner developmental stage 22 when the hindlegs of the developing tadpole bend at the knee. Upon host metamorphosis, the young parasite leaves the branchial chamber and migrates over the host’s abdomen to the cloaca, where it enters and migrates to the urinary bladder to mature and reproduce in synchrony with the host [11, 12]. However, a third internal reproduc- tive strategy has been documented within Metapolystoma. Metapolystomes are partly ovoviviparous where some eggs develop and hatch in utero to reinfect the same host along- side the parent . Eggs that do not hatch in utero hatch after being expelled from the host. The oncomiracidium then infects a tadpole and partakes in either a neotenic branchial or a bladder-destined branchial cycle . Ovo- viviparity, though uncommon in Polystoma, has been observed in Polystoma integerrimum (Frölich 1791) and Polystoma pelobatis Euzet and Combes, 1966 . This ovoviviparous cycle is, however, not part of these para- site’s normal reproductive cycle. Polystoma integerrimum, similar to P. pelobatis, has a short annual egg produc- tive and deposition cycle of only four days per year . One egg may not get expelled by the end of the parasite’s annual reproductive cycle [6, 8] resulting in the retention of an egg. In such an instance, the egg may develop and hatch in utero, whereafter the oncomiracidium will exit the genital atrium and attach alongside the parent . Until the end of the 2020s, Metapolystoma (Monoge- nea: Polystomatidae) was only known from Ptychadena Boulenger, 1917 found in Madagascar and the African continent. This genus included Metapolystoma brygoonis (Euzet and Combes 1964) from Ptychadena mascareniens (Duméril and Bibron 1841) in Madagascar, Metapolys- toma cachani (Gallien 1956) from Ptychadena longiro- stris (Peters, 1870) in West-Africa and Metapolystoma porosissimae Du Preez and Kok 1992 from Ptychadena porosissima (Steindachner 1867) in South Africa. It is only recently that Landman et al.  described, from Madagascar, Metapolystoma ansuanum Landman, Verneau, Raharivololoniainan and Du Preez 2021 from Boophis luteus (Boulenger 1882), Metapolystoma fal- catum Landman, Verneau, Raharivololoniainan and Du Preez 2021 from Boophis doulioti (Angel 1934), Metapol- ystoma multiova Landman, Verneau, Raharivololoniainan and Du Preez 2021 from Boophis occidentalis Glaw and Vences, 1994, Metapolystoma theroni Landman, Verneau, Raharivololoniainan and Du Preez 2021 from Boophis Fig. 1 A Distribution of Aglyptodactylus madagascareniensis in madagascariensis (Peters 1874) and Metapolystoma venc- Madagascar with sites where parasites were found. B Aglyptodacty- esi Landman, Verneau, Raharivololoniainan and Du Preez lus madagascariensis. ArcMap 10.5.1 (Esri, California) was used to 2021 from Boophis albilabris (Boulenger 1888). After the compile a host distribution map, based on genetically verified records examination of archived collections of Malagasy frogs of Köhler et al.  1 3 Acta Parasitologica in Antanarivo (Madagascar) and Paris (France) together weak acetocarmine solution, they were dehydrated and with field surveys in Madagascar, Aglyptodactylus mada- mounted in Canada balsam. Juvenile specimens were gascariensis (Duméril 1853) was found to be infected mounted as temporary preparations in ammonium picrate with a polystomatid flatworm. Based on molecular evi- glycerine for examination of sclerites. One specimen for dence , this parasite represents a yet undescribed molecular analysis was preserved in 100% ethanol and Metapolystoma species. Our objectives were therefore processed in Verneau et al. . to formally describe this new species recovered from A. madagascariensis and to indicate the value of museum Phylogenetic and Distance Analyses collections in the assessment of parasite diversity, espe- cially in protected areas where the collection of potential All 18S, 28S and COI sequences used in this study, except hosts is restricted. for Metapolystoma sp. ex. A. madagascariensis, were extracted from Genbank and listed in Landman et al.  under Accession numbers AM051069, AM051071, AM051073, AM051075, AM157194, AM157204, Material and Methods AM157206, AM157208, AM157217, AM913856, AM913859, AM913860, JF699306, JN800281, JN800283, Host and Parasite Sampling JN800285–JN800289, JN800291, JN800293, JN800294, FM897262–FM897264, FM897267–FM897270, In January 2005, two specimens of A. madagascariensis, FM897280, FM897281, FM897284–FM897287, FM897298, collected at Andasibe, were examined and found to be FM897300, FM897301, MW054236–MW054249, uninfected with polystomes. In February 2006, a variety MW053457 and MW053458 for the description of Boophis of formalin preserved specimens were examined at the metapolystomes. Sequences for Metapolystoma sp. were also Tsimbazaza Zoological Garden, Antananarivo, Mada- extracted from Genbank, however listed in Verneau et al. gascar for polystomes. Among them, 12 specimens of  under Accession numbers FM897266, FM897283 and A. madagascariensis collected in November 2005 from FM897299 for a global phylogenetic analysis of Madagas- two distinct localities of Madagascar, namely Besariaka can polystomes. Clustal W, which is implemented in Mega and Mangabe-Ranomena-Sahasarotra (Fig. 1A) were 7 , was selected to edit and align 18S, 28S and COI examined. In May 2006, a variety of Malagasy frog spe- sequences independently using default parameters . cies were examined at the Muséum National d’Histoire Resulting 18S and 28S alignments comprised 20 taxa, con- Naturelle of Paris. A small cut was made across the lower taining 14 Metapolystoma and six distinct Polystoma spe- abdomen to expose the urinary bladder which was pulled cies, with Polystoma integerrimum (Frölich 1791) used as out for inspection. Thirty-three specimens of A. madagas- an outgroup, while the resulting COI alignment comprised cariensis were examined. These specimens were collected only 18 taxa. The 18S, 28S and COI sequences were finally in November 1971 at Chaines Anosyennes, Madagascar concatenated in a single alignment for Bayesian analysis. (Fig. 1A) Host specimens from Chaines Anosyennes were 18S and 28S sequences were treated as two separate par- collected outside the currently known distribution of A. titions while COI sequences were treated as three distinct madagascariensis (Fig. 1A) and may be Aglyptodactylus partitions according to their codon position. A two substi- australis Köhler, Glaw, Pabijan, and Vences, 2015. For tution rates model was selected for the 18S partition and a this reason, specimens from this locality were not included GTR + I model for the 28S partition following the Akaike in the morphometric description. In February 2008 fol- Information Criterion (AIC) implemented in Modeltest 3.06 lowing a rainstorm at Andasibe (Fig. 1A), Madagascar, . Six types of substitutions and four gamma rates cat- two specimens of A. madagascariensis (Fig. 1B) were egories were applied for COI partitions one and two, while collected dead on the road and 32 frogs were collected six types of substitutions with invariable-gamma rates were by hand around various forest pools in the area. Frogs applied for COI partition three. Evolutionary parameters for were euthanized using Ethyl-3-aminobenzoate methane- the five partitions were estimated separately with MrBayes sulfonate (MS222) and dissected to inspect for polystomes. 3.04b . Four chains were run for ten million generations, The accessory bladders, Wolffian ducts and kidneys were sampling every 100 cycles. The first 10 000 trees (10%) were also examined. Parasites that were retrieved were trans- removed at the burn-in phase. The Bayesian consensus tree ferred to glass cavity blocks containing 0.6% amphibian was drawn (Fig. 2) and inspected with Tree View version 1.6 saline solution. . Corrected pairwise distances used for species delimi- Adult specimens used for morphological examination tation were calculated independently for the three genes in were fixed and preserved in 10% neutral buffered formalin MEGA version 7 with the Kimura 2-parameter model . (NBF) under coverslip pressure. Following staining in a 1 3 Acta Parasitologica Fig. 2 Bayesian tree inferred from the analysis of concatenated 18S, 28S and COI gene sequences. Node values indicate Bayesian posterior probabilities Total character differences between species were also esti- parasites. Taxonomic measurements in micrometres mated for all three genes. were captured using the Zeiss Zen Blue elements (Zeiss, Germany) software program. A Nikon AZ100M Morphology and Morphometry stereomicroscope (Nikon, Netherlands) was used to capture full-body micrographs of type specimens. Adobe A Zeiss Imager Axio10 compound microscope (Zeiss, Illustrator CC (Adobe, California) was used to prepare Germany), fitted with a Zeiss Axio cam 305 camera illustrations from micrographs. (Zeiss, Germany) was used to investigate and photograph 1 3 Acta Parasitologica Eggs were measured in length (x) and width (y), plotted for histological examination. Specimens were washed on a scatterplot, and encircled by a 95% confidence ellipse and dehydrated in an ethanol series, impregnated with with the mean x;y as pivot in order to discriminate between paraffin wax and embedded for sectioning using a Slee different species groups. Measurements came from the type MPS histocene embedding machine (Slee, London, UK). series of M. ansuanum, M. falcatum, M. multiova, M. poro- Specimens were sectioned at 6 μm using a Reichert Jung sissimae, M. theroni and M. vencesi and from Kulo  for motorised microtome (Reichert-Jung, Nosloch, Germany). M. cachani. Concerning M. brygoonis, measurements were Sections were stained with routine eosin and Harris’ from parasites collected in September 1988 at Ranomafana, hematoxylin and mounted using Entellan mounting medium Madagascar that were stored in the polystome collection of . the North-West University, Potchefstroom, South Africa. Marginal hooklets were measured according to the protocol Sclerite Isolation and Scanning Electron Microscopy of Du Preez and Maritz  in order to discriminate spe- cies groups. Sclerotized hooks were isolated from adult parasites through enzyme digestion of soft body parts using Proteinase K Histology [200 µg/ml] (see ). Haptors were separated and cut into smaller fragments, rehydrated in distilled water, and placed Specimens retrieved from archived host material were on a Millipore filter (pore size 0.5 μm). Specimens were not fixed flat and thus not suitable as whole mounts. They then covered with the enzyme solution and incubated at were preserved in 10% neutral buffered formalin (NBF) 50 ℃ for 10 to 15 min. Subsequently, distilled water was Fig. 3 Ventral view of Metapolystoma ohlerianum n. sp. holotype. eg egg, gb genital bulb, gc genito-intestinal canal, ha hamuli, hp haptor, ic intestinal caecum, mo mouth, mg Mehlis gland, od oviduct, oi oö-vitelline canal, oö oötype, os false oral sucker, ov ovarium, ph pharynx, su sucker, sv semen vesicle, te testis, ut uterus, va vagina, vc vaginal cannel, vd vas deferens, vi general position of vitelline (not visible on type), vl vitelline duct, vv vitello- vaginal cannel 1 3 Acta Parasitologica forced through the filter with a syringe in order to rehydrate Family Polystomatidae Gamble, 1896 specimens and to remove excess salts and debris. Digestion Genus Metapolystoma Combes, 1976 and rehydration steps were repeated seven to eight times until all soft tissue was removed from the hooks. Filter papers with sclerite hooks were dried overnight in a desiccator Type Host prior to the SEM analysis. Dried material was mounted on aluminium SEM stubs with carbon tape and sputter-coated Aglyptodactylus madagascariensis (Duméril 1853), for 90 s with gold palladium using a SPI-Module Sputter Mantellidae (Fig. 1B). Coater (Spi Supplies: Westchester, PA, USA). SEM images were captured using a Phenom Pro Desktop SEM (Phenom- Site in Host World BV., Eindhoven, Netherlands). Adults in urinary bladder, some juveniles in the Muller ducts and urinary bladder. Results Type Material Levels of Infection Morphological descriptions based on 13 mature and 2 Two of the seven frogs examined from Mangabe-Ranomena- juvenile parasites. Holotype NMBP 583 (Fig. 3) and Sahasarotra were infected. One specimen was infected with Paratypes NMBP 584–586 deposited in the Parasitic Worm one mature and eight juvenile parasites and the second collection, National Museum, Aliwal Street, Bloemfontein with 15 mature parasites. All five frogs from Besariaka 9301, South Africa. were uninfected. Of the 33 frogs from Chaines Anosyennes that were examined at the Muséum National d’Histoire Voucher Material Naturelle of Paris, a single frog was infected with nine mature parasites. Finally, four of the 34 live frogs from the Remaining specimens in parasite collection at North-West Andasibe region were infected with a total of four mature University, Potchefstroom, South Africa. and 17 juvenile parasites (prevalence 11.8%, mean intensity 5.3). All four frogs were infected with small, subadult and Type Locality mature parasites. Holotype NMBP 583 and Paratypes NMBP 585–586 from Phylogenetic Relationships and Genetic Andasibe, Madagascar (− 18.938753S; 48.414471E). Divergences Within Metapolystomes Paratype NMBP 584 from Mangabe-Ranomena-Sahasarotra, Madagascar. The Bayesian phylogeny shows that Metapolystoma n. sp. ex. A. madagascariensis and M. falcatum are sister species Zoobank Registration with high Bayesian posterior probability (Fig. 2). The divergence estimates with standard deviations and total The Life Science Identifier (LSID) of the article differences between Metapolystoma n. sp. and M. falcatum is: urn:lsid:zoobank.org:pub:564F7CC4-4EA4- are 0.052% ± 0.00 and 1 for the 18S, 0.141% ± 0.001 and 2 45BD-A480-9CB98304D0F2. The life science for the 28S and 9.372% ± 0,018 and 28 for the COI. These identifier (LSID) of the new name Metapolystoma values exceed the species level threshold of 0.07% for ohlerianum n. sp. Landman et al. is: urn:lsid:zoobank. 28S and 2% for COI as proposed by du Preez et al. , org:act:9B1ECEB9-F5B6-4C46-9199-30035F5F6DE8. suggesting that it is a new species. Etymology Metapolystoma ohlerianum n. sp. (Fig. 3) In recognition of Dr. Annemarie Ohler, Muséum National d’Histoire Naturelle of Paris, for her support over years and for allowing the examination of museum materials. Taxonomic Summary Description Class Monogenea van Beneden, 1858 Measurements were obtained from 13 mature and two Order Polystomatidea Lebedev, 1988 juvenile bladder parasites and are given in micrometres. 1 3 Acta Parasitologica Fig. 4 Compound microscope micrograph of histology sections bulb surrounded with glandular cells (arrows). E Section through through Metapolystoma ohlerianum n. sp. A Section through false vagina with the vaginal canal branching out (arrow) to form multiple oral sucker interspersed with mucous gland cells (arrow). B Cross- vaginal pores. F Section through testis showing testis follicles (arrow section through pharynx imbedded with glandular cells (arrow). C b) surrounded with connective tissue (arrow a) Section through Mehlis gland (arrow). D Section through genital Body pyriform (Fig. 3), dorsoventrally flat, 3508–5042 measured from anterior end, body length 2.2–4.2 (3.1 ± 0.9; (4517 ± 700; 4) long, 1209–1758 (1515 ± 228; 4) wide, 4) times greater than width. Width at vagina 1041–1340 with widest section 53–69% (58% ± 7.2; 4) of total length (1210 ± 149; 4). Mouth 228–284 (250 ± 26; 4) in diameter, 1 3 Acta Parasitologica Fig. 5 Metapolystoma ohleri- anum n. sp. A Genital crown from holotype B Hamuli from holotype, X - hamulus handle length, Y - hamulus guard length, Z - hamulus hook length C Marginal hooklets 1 (top), 2–7 (middle) and 8 (bottom) from holotype and paratypes sub-ventral surrounded by false oral sucker. False oral quarter of body proper, sack like, surrounded by accessory sucker interspersed with mucous gland cells (Fig. 4A). glands at genital bulb connection (Fig. 4D), containing Medial pharynx length 251–297 (282 ± 21; 4) greater than up to 61 ovoid, operculate eggs 187–242 (211 ± 12.6; width 226–284 (257 ± 27; 4), imbedded with glandular 43) long, 104–182 (127 ± 21.3; 49) wide, some contain cells throughout its length (Fig. 4B). Intestine bifurcates fully developed oncomiracidia; some hatched intrauterine at 14–17% (16% ± 1; 4) of total length from most anterior oncomiracidia present. Two parallel vaginae 195–270 point, converging posteriorly at 58–74% (69% ± 8; 4) of total (229 ± 27; 7) long, 93–177 (137 ± 33; 7) wide, on lateral length, from most anterior point, extending into haptor; no margins, bearing multiple marginal openings formed by prehaptoral anastomoses. Lateral diverticula length equal to branching vaginal canal (Fig. 4E), vaginal vestibule cup width. Medial diverticula posterior to ovary, length greater shaped, 24–27% (25% ± 1.2; 4) from anterior. Vitellaria than with. extended throughout most of body, stretching in between Ovary 349–427 (378 ± 43; 3) long, 136–190 (169 ± 28; 3) haptoral suckers, surrounding the female reproductive wide, elongate, not lobed, positioned posterior to midbody, organs. Genito-intestinal cannel prominent 469–706 long, 7–9% (8% ± 1; 3) of body length, 2–3 (2 ± 0.3; 3) times 22–51 (33 ± 9; 2) wide, situated posterior to ovary. longer than wide. Oviduct 422–491 (455 ± 35; 3) long, Testis follicular, encapsulated in connective tissue that 18–49 (34 ± 9; 3) wide. Mehlis’ glands large, surrounding forms a tube (Fig. 4F), U-shaped, mainly positioned pos- the base of the oötype (Fig. 4C). Uterus large, occupying one terior to the ovary with two lateral processes extending 1 3 Acta Parasitologica Fig. 6 Scanning electron micrographs of Metapolystoma ohlerianum n. sp. showing A Hamuli. B Deep grooves in hamulus guard for muscle attachment. C Deep grooves in hamulus handle for muscle attachment. D Deep grooves in hamulus head for muscle attachment forward along the lateral line past the ovary to a position Posterior haptor 1208–1280 (1239 ± 32; 4) long, about 25% from anterior end of body proper, ventral to intes- 1647–2055 (1852 ± 168; 4) wide, 24–34% (28% ± 4.6; 4) tine. Lateral fields of testis pushed into posterior position of body length, bearing three pairs of cup-shaped haptoral when the uterus is filled with eggs, sometimes causing the suckers equal in diameter 296–526 (387 ± 51; 27). Well- testis to be asymmetrical. Vas deferens widens anteriorly to developed hamuli between posterior–most haptoral suckers form sinuous semen vesicle 14–33 (22 ± 2; 3) wide, 38–88 with deep cut between handle and guard (Fig. 5B), handle (62 ± 25; 3) long, narrowing towards genital bulb, opening length X 340–475 (391 ± 42; 14), guard length Y 263–392 in common genital opening. Genital pore opening ventral, (315 ± 27; 15), and hook length Z 57–69 (63 ± 3.6; 21), directly posterior to intestinal ceca bifurcation, 5–15% some hamuli with posterior crest (Fig. 5B right). Marginal (12% ± 5; 4) of total length from most anterior point, geni- hooklets placed as for other polystomes: pairs one and tal bulb muscular 59–72 (66 ± 4; 7) in diameter, surrounded two between hamuli, marginal hooklet pairs three to five by glandular cells, armed with a genital crown 21.6–27.4 embedded in suckers, pairs six to eight between anterior (23.5 ± 2; 6) in diameter, with seven genital spines 26.7–32.0 suckers. Marginal hooklet pairs one 29.6–33.6 (31.7 ± 1.3; (29.7 ± 1.7; 17) long (Fig. 5A). 8) long (Fig. 5C top) and eight 25.9–30.0 (27.5 ± 1.6; 8) 1 3 Acta Parasitologica Fig. 7 Scanning electron micrographs of Metapolystoma ohlerianum ▸ n. sp. showing A Lateral grooves on marginal hooklet one for muscle attachment. B Lateral grooves on marginal hooklet eight for muscle attachment. C The absence of lateral grooves for muscle attachment on marginal hooklets two to seven long (Fig. 5C bottom), larger than pairs two to seven 17.8–22.2 (20.6 ± 1.2; 12) long (Fig. 5C middle). Hamulus guard, handle and head deeply grooved for muscle attach- ment (Fig. 6A–D). Marginal hooklet pairs one (Fig. 7A) and eight (Fig. 7B) laterally grooved between hook tip and guard for muscle attachment, hooks two to seven not grooved (Fig. 7C). Egg and Marginal Hooklet Morphometrics Egg morphometric measurements separated M. ohlerianum n. sp. from M. brygoonis, M. falcatum, M. multiova and M. theroni with no overlap in the scatterplot (Fig. 8). However, they did not separate M. ohlerianum n. sp. from M. ansua- num, M. cachani, M. porosissimae and M. vencesi. Marginal hooklet morphometric measurements sep- arated M. ohlerianum n. sp. from M. falcatum with no overlap in the scatterplot (Fig. 9). Marginal hooklet mor- phometric measurements, however, did not separate M. ohlerianum n. sp. from M. multiova, M. porosissimae, M. theroni and M. vencesi. Remarks Whereas all metapolystomes described to date have tubiform uteri that convolute between the oötype and the genital pore, Metapolystoma ohlerianum n. sp. differs from all Metapolystoma species in that it has a sack-like uterus. Furthermore, M. ohlerianum n. sp., which has seven genital spines, differs from all other metapolystomes except M. theroni. However, these two species differ from each other from egg morphometrics and hamulus hook length. Discussion Wit h M. ohlerianum n. sp., there are now seven Metapolystoma species known from Madagascar. It can, however, be expected that more Metapolystoma species await discovery from Aglyptodactylus species. Aglyptodactylus madagascariensis is a predominant terrestrial species that occurs in the eastern forests of Madagascar and spends most of its time between leaf litter during the daytime, and in and around temporary pools at night [28, 29]. These frogs are explosive breeders that breed following heavy rains [29, 1 3 Acta Parasitologica Fig. 8 Scatter diagram of egg length plotted against egg width for all known Metapolystoma spp. including Metapolystoma ohlerianum n. sp. The ellipses represent 95% of the confidence interval about the mean Fig. 9 Scatter diagram of a × c plotted against b × c for Metapolystoma spp., for which measurements could be obtained. The ellipses represent 95% of the confidence interval about the mean 30]. Since these frogs gather in great numbers to breed, both phylogenetic and morphological results suggest that many frog and parasite eggs are deposited in relatively Metapolystoma is a distinct clade that we recommend to small, temporary pools. These conditions are ideal for be kept as a separate taxon for the time being (see also parasite reinfection since many tadpoles are concentrated ). Metapolystoma, as with Polystoma, has both mature in small waterbodies, which increases the probability of a branchial and bladder adult forms. However, metapolystomes single oncomiracidium to find a host. Although the nested present true ovoviviparity where eggs develop and hatch phylogenetic position of Metapolystoma within Polystoma prior to egg deposition, to reinfect the same host . This raises questions about its validity as a genus (see ), is also evident in re-examined material of Landman et al. 1 3 Acta Parasitologica , within Boophis madagascariensis, i.e., the host of M. which suggests that many more Malagasy polystome species theroni, which was infected with 23 recently established are still undiscovered and undescribed. We therefore empha- oncomiracidia, 19 subadult (non-reproductive) and one size the need for further parasite surveys and recommend mature (reproductive) parasite. The same phenomenon has that, as with this study, museum collections should be exam- been observed for B. doulioti, i.e., the host of M. falcatum, ined for parasites. The investigation of museum specimens which was infected with five small parasites in the Müller will thus provide localities for further field studies, which ducts and respectively six subadult and one mature parasite will help in finding supplementary material for molecular in the urinary bladder (re-examined material of Landman analyses and morphological descriptions. et al. ), suggesting multiple reinfection events. Some Supplementary Information The online version contains supplemen- Polystoma species present ovoviviparity, however, this tary material available at https://doi. or g/10. 1007/ s11686- 023- 00668-z . cannot be considered as part of the parasite’s natural reproductive cycle. This difference between the life-cycles Acknowledgements We thank the Malagasy authorities for the provi- sion of necessary research and export permits, the Volkswagen foun- of the two genera thus provides further evidence supporting dation (Grant no. I/82.201), the CNRS and the Deutsche Forschun- Metapolystoma as a distinct group. gsgemeinschaft (Grant no. VE247/2-1) for funding. Furthermore, we Although great emphasis has been placed on Madagascar are thankful for assistance from colleagues and students, in particular Liliane Raharivololoniaina. Research was conducted in collaboration as a biodiversity hotspot , its parasite diversity remains between the North-West University, the Département de Biologie Ani- underexplored, including that of amphibian polystomes. This male de l’Université d’ Antananarivo and the Association Nationale is especially true when taking into consideration Madagas- pour la Gestion des Aires Protégées. We are also grateful to Jasmin E. car’s rich amphibian diversity of over 400 species  of Randrianirina, Madagascar and to Anne-Marie Ohler from the Muséum National d’Histoire Naturelle of Paris, who gave us access to preserved which 83 species were investigated for polystomes at a few frogs for polystome investigations. selected localities . These studies led to the description of two new polystome genera within amphibians [35, 36] Funding Open access funding provided by North-West University. and 11 new polystome species [15, 35–38], knowing that Data availability Raw data generated from this study is available upon undescribed Madapolystoma species still await description personal request from the corresponding author. . Furthermore, Metapolystoma brygoonis, which could be a complex of species , given the high genetic varia- Declarations tion observed in its host in Madagascar , and therefore requires further investigation. At the current rate of species Conflict of interest The authors declare that there is no conflict of in- extinction many species are becoming extinct before they are terest. formally described [40–42]. The biodiversity of Madagascar Open Access This article is licensed under a Creative Commons is particularly threatened as a result of forest fragmentation Attribution 4.0 International License, which permits use, sharing, and habitat loss [43, 44]. The hosts of some undescribed adaptation, distribution and reproduction in any medium or format, Malagasy polystome species that were discovered during as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate our studies are now Endangered. In fact, 80 (26%) of the 311 if changes were made. The images or other third party material in this anurans assessed in Madagascar are listed as being Endan- article are included in the article's Creative Commons licence, unless gered and 21 (7%) as Critically Endangered . Conse- indicated otherwise in a credit line to the material. If material is not quently, due to collecting permit restrictions, the parasite included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted diversity of these frogs will probably never be assessed. use, you will need to obtain permission directly from the copyright Extinction of any species has a ripple effect as each species holder. To view a copy of this licence, visit http:// creat iveco mmons. is a package of the host with its species-specific parasites. org/ licen ses/ by/4. 0/. With amphibians being the most threatened vertebrates and their numbers continuously declining , this statement is particularly true for their polystomatid parasites as well. Especially since amphibian polystomatids are generally con- References sidered to be host specific [31, 37, 47–50]. With parasites forming such an intricate part of biodiversity and ecology, 1. 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Acta Parasitologica – Springer Journals
Published: Mar 17, 2023
Keywords: Aglyptodactylus madagascareniensis; Life cycle; Madagascar; Metapolystoma; Polystome
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