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

Abstract

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.

Graphical abstract

1. Introduction

Madagascar is well known for its high species diversity and endemicity, particularly in the cases of plants and vertebrates (Myers et al., 2000). When it comes to amphibian diversity, Madagascar is globally ranked in the top twelve (Andreone et al., 2008). Its unique herpetofauna serve as hosts for an equally unique and diverse assemblage of parasites (see Wohltmann et al., 2007; Junker et al., 2010; Rocha et al., 2012; Kuzmin et al., 2013; Landman et al., 2018). However, little is known about Madagascar's anuran polystome flatworm diversity, since only 86 of the 356 known frog species (Frost, 2020) have been screened for polystomes at a few accessible localities in the past (Verneau et al., 2009). Malagasy polystomes are currently represented by four genera encompassing a single chelonian and seven anuran species, all of these were found in the urinary bladder of their host. Uropolystomoides chabaudi (Euzet and Combes, 1965) from Pelomedusa subrufa (Bonnaterre, 1789) is the only polystome known from turtles. Metapolystoma brygoonis (Euzet and Combes, 1964) was the first polystome found within a Malagasy anuran host, as described from Ptychadena mascareniensis (Duméril and Bibron, 1841). Besides these two species, Kankana manampoka Raharivololoniaina et al. 2011 was described from Cophyla pollicaris (Boulenger, 1888). Five other species of Madapolystoma were reported from mantellids, namely Madapolystoma biritika Du Preez et al. (2010) from Mantella madagascariensis (Grandidier, 1872), M. isaloensis Landman et al. (2018) from Mantella expectata Busse and Böhme, 1922 and M. magnahami Landman et al. (2018) from Blommersia domerguei Guibé, 1974. Madapolystoma cryptica Berthier et al. 2014 and M. ramilijaonae Berthier et al. 2014 were conversely described from the same host species Guibemantis liber (Peracca, 1893).

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 2005, 2006 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.

2. Material and methods

2.1. Host and parasite sampling

Annually during February–March of 2005, 2006 and 2007, several 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. Specimens were rinsed in water, stained with acetocarmine, dehydrated, cleared in xylene and mounted in Canada balsam. Five additional, fixed specimens of B. albilabris collected in 1972 by Mr. Blane at Andringita Massif, Madagascar, and archived at the Muséum National d’Histoire 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, IUCN, 2016b, IUCN, 2016c, IUCN, 2016d, IUCN, 2016e).

Table 1.

Frog species under survey with their site of collection in Madagascar.

Frog species	No. of specimens collected	Locality
B. madagascariensis	30	Andasibe (Indri Reserve)
B. albilabris	6	Ambatolahy and Andringita massif
B. doulioti	22 frogs, 10 tadpoles	Ankarafantsika
B. luteus	9	Isalo (Cascade des Nymphes Special Reserve)
B. occidentalis	5	Isalo (Cascade des Nymphes Special Reserve)

Fig. 1.

Map of Madagascar showing parasite type localities and distribution areas of hosts.

2.2. DNA extraction and amplification

All polystome DNA samples used in this study were sourced from Verneau et al. (2009) and Berthier (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) and Héritier 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.

2.3. 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).

2.4. 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 series (Table 2).

Table 2.

Body measurements in micrometres for all Metapolystoma spp. values.

Measurements	Metapolystoma vencesi n. sp.	Metapolystoma falcatum n. sp.	Metapolystoma ansuanum n. sp.	Metapolystoma theroni n. sp.	Metapolystoma multiova n. sp.
Number of mature specimens	6	1	2	1	2
Total length	9190	7871	2680–3952	9086	4915–5537
Greatest width	3496	3272	1035–1436	3208	2257–2419
Width at vagina	2315	1783	804–1001	2011	1469–2086
Haptor length ‒ Body length ratio	0.16	0.19	0.23–0.25	0.18	0.18
Haptor length	1437	1492	669–912	1634	916
Haptor width	2344	2636	1166–1385	2572	1478
Hamulus length X	269‒332 (299 ± 32; 3)	393–407	205	392–408	196‒248 (215 ± 29; 3)
Hamulus length Y	225‒302 (270 ± 40; 3)	303–314	179	252–257	121‒174 (153 ± 23; 4)
Hamulus hook length Z	40‒52 (47)	66–70	62	50–55	58.2
Oral disk	391	297	307–329	486	248–271
Pharynx length	302–420	327	256–308	401	302–405
Pharynx width	291–390	290	183–222	385	289–320
Genital bulb diameter	115	86	64–73	65	86
Ovary length	861	772	265–338	479	446
Ovary width	194	196	98–144	385	158
Number of genital spines	8	8	10	7	6
Genital spine length	31.5–33.4 (32.5 ± 0.6; 9)	24.3–31.6 (27.9 ± 1.78; 18)	24.5–28.5 (26.19 ± 1.16; 5)	28.9–34.9 (32.9 ± 4; 3)	40.4–43.7 (42 ± 1.4; 5)
Genital crown diameter	31.8	25.7	29.4–32.8	24.2–27.6 (25.8 ± 1; 10)	24.5
Sucker diameter	366‒452(424 ± 37; 12)	435‒498(468 ± 29; 6)	241‒305(273 ± 25; 12)	404‒424 (416 ± 8; 6)	329‒356 (339 ± 10; 9)
Maximum number of eggs in utero	367	11	41	176	499+
Egg length	210‒230 (219 ± 7; 16)	161‒185 (171 ± 8; 11)	196‒217 (205 ± 8; 8)	239‒265(250 ± 7; 12)	198‒228 (212 ± 9; 23)
Egg width	120‒147 (130 ± 8; 16)	108‒123 (117 ± 5; 11)	119‒137 (127 ± 7; 8)	136‒152(143 ± 5; 12)	144‒165 (154 ± 7; 23)
Marginal hooklet 1 length	29.7–34.2 (32.5 ± 1.3; 13)	32.6–39.7 (36.9 ± 2.3; 13)		34.3–39.4 (936.2 ± 1.4; 21)	28.6–38.7 (33.8 ± 3.3; 11)
Marginal hooklet 2–7 length	20.8–25 (23.6 ± 1; 30)	21.8–27 (23.8 ± 1.52; 30)	22.3–24.79 23.5 ± 1.17; 4)	22.6–27.2 (24.8 ± 1.1; 44)	21.3–26.9 (24.7 ± 2; 17)
Marginal hooklet 8 length	27.4–32.4 (30.5 ± 1.6; 12)	28.8–33.5 (31.3 ± 1.5; 14)		30.5–34.1 (32.2 ± 1; 19)	28.4–36.3 (32.4 ± 2.8; 5)
Widest section from front	57%	66%	16–65%	51%	50%–57%
Times longer than wide	2.7	2.4	2.6–2.8	2.6	2.2–2.3

Measurement	Metapolystoma porosissimaea	Metapolystoma brygoonisb	Metapolystoma cachanic
Number of mature specimens	15	10	10	3	57
Total length	4150–6710 (5870 ± 0.6; 10)	3400–4800 (4200)	4000	3880–5628 (4594)	3.680–7.250 (5.070)
Greatest width	1570–2360 (2040 ± 0.2; 11)	1220–1500 (1340)	1500	1120–2880 (1793)	1.160–2.880 (1.980)
Width at vagina	1.120–1.700 (1.460 ± 0.2; 11)	650‒1030 (800)			880–1.760 (1.220)
Haptor length ‒ Body length ratio	0.26–0.33 (0.29 ± 0.1; 10)	0.22–0.23 (0.22)	0.25	0.17–0.21 (0.19)	0.15–0.32 (0.23)
Haptor length	1.540–1.800 (1.670 ± 01; 12)	760‒1070 (900)	1000	720‒1072 (864)	910–1.500 (1.15)
Haptor width	2.010–2.660 (2.360 ± 0.2; 12)	1220–1760 (1530)	1500	1248–2800 (1858)	1.300–2.350 (1.730)
Hamulus length X	432‒472 (447 ± 14.3; 26)	330‒420 (380)	310	280‒450 (348)	330‒520 (430)
Hamulus length Y	332.4‒385.7 (361.2 ± 18.52; 13)
Hamulus hook length Z	72‒86.6 (78.3 ± 3.97; 15)
Oral disk	461‒566 (521)	220‒340 (270)
Pharynx length	267‒348 (310 ± 31.8; 10)
Pharynx width	234‒298 (262 ± 20.4; 10)
Genital bulb diameter	93‒129.5 (105.7 ± 11.91; 7)	80	25
Ovary length	546‒755 (651 ± 89.1; 11)	410‒460 (440)
Ovary width	266	90‒160 (120)
Number of genital spines	9–10	8	8
Genital spine length	31/21.83‒25.97 (24.144 ± 1.46; 6)	30‒32 (31)
Genital crown diameter	25.18‒26.96 (26.35 ± 0.57; 7)
Sucker diameter	461‒566 (521 ± 30; 65)	260‒420 (340)	350–400	272–580 × 288–544 (386 × 379)	300‒500 (420)
Maximum number of eggs in utero	250	100–140	70–100	4	8‒390 (170)
Egg length	201‒229 (214 ± 7.2; 30) 192‒207 (197 ± 5.1; 10)	160‒200 (180)	190	176‒208 (200)	122‒159 (0.138)
Egg width	113‒134 (128 ± 6.6; 30) 100‒131 (115 ± 9.3; 10)	90‒120 (100)	120	96‒112 (101)
Marginal hooklet 1	35
Marginal hooklet 2–7	22
Marginal hooklet 8	33
Marginal hooklet 1–2		31 ‒ 34 (33)	30
Marginal hooklet 6–8		31

Values: (Min ‒ Max (Mean ±Ơ; N)), numbers in bold indicate additional measurements taken from species described prior to the present study.

^aFrom Du Preez and Kok (1992a).

^bFrom Euzet and Combes (1964).

^cFrom Gallien (1956) (left), Kulo (1981) (middle) and Murith (1981) (right).

3. Results

3.1. Taxonomic summary

Class: Monogenea van Beneden. 1858 Order: Polystomatidea Lebedev. 1988 Family: Polystomatidae Gamble. 1896 Genus: Metapolystoma Combes, 1976.

3.1.1. Metapolystoma vencesi n. sp. (Fig. 2, Fig. 3; Table 2)

Fig. 2.

Ventral view of Metapolystoma vencesi n. sp. holotype. Abbreviations: eg, egg; gb, genital bulb; gc, genito–intestinal canal; ha, hamuli; hp, haptor; ic, intestinal caecum; mo, mouth; oc, oncomiracidium; od, oviduct; oi, oö–vitelline canal; os, false oral sucker; ov, ovarium; ph, pharynx; su, sucker; sv, semen vesicle; te, testis; ut, uterus; va, vagina; vd, vas deferens; vi, vitelline; vv, vitello–vaginal canal.

Fig. 3.

Metapolystoma vencesi n. sp. from Boophis doulioti.a, marginal hooklets 1 (top), 2–7 (middle) and 8 (bottom) from holotype and paratypes; b, hamuli from holotype; c, genital crown from holotype. Abbreviations: X, outer length; Y, inner length; Z, hook length.

3.1.1.1. Type host

Boophis albilabris (Mantellidae)

3.1.1.2. Type locality

Ambatolahy, Madagascar (Fig. 1), (−21.2438667S; 47.4262167E).

3.1.1.3. Site in host

Urinary bladder.

3.1.1.4. 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.

3.1.1.5. Type material

The morphological description is based on six mature, ten oncomiracidia and four juvenile parasites. Three specimens were sexually mature (Holotype NMBP 578; Paratypes NMBP 579–580) and two immature (Paratypes NMBP 581–582). The holotype and paratypes NMBP 581 and NMBP 582 originated from Ambatolahy, Madagascar, while paratypes NMBP 579 and NMBP 580 originated from Andringita Massif, Madagascar. Juvenile parasites and oncomiracidia were used for marginal hook measurements and drawings. The type material was deposited in the parasitic worm collection, National Museum, Aliwal Street, Bloemfontein 9301, South Africa.

3.1.1.6. Voucher material

Remaining specimens in polystome collection, North-West University, Potchefstroom, South Africa.

3.1.1.7. Zoobank registration

The Life Science Identifier (LSID) of the article is: 59F6A99A-C667-48 EB-9EA4-881D43956065. The life science identifier (LSID) of the new name Metapolystoma vencesi n. sp. Landman et al. is: urn:lsid:zoobank.org:act:680E18A9-64D6-40C9-87D9-1AC5F28A1030.

3.1.1.8. Etymology

In recognition of Professor Miguel Vences, Technical University of Braunschweig, Germany, for his dedication to the study of Madagascan herpetofauna.

3.1.1.9. Description

Measurements reflected in 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).

Ovary elongate, not lobed, positioned posterior to midbody, length 4.4 times greater than width, measuring 9% of body length. Oviduct 596 long, 29–53 (30 ± 7; 1) wide. Uterus massive and wide, occupying most of body proper, tubiform, convolute, containing 367 ovoid, operculate eggs; some with fully developed oncomiracidia. Hatched intrauterine oncomiracidia present. Mehlis’ gland obscure. Two vaginae 180–420 long, 16–31 wide, on lateral margins, bearing multiple marginal openings, vaginal vestibule elongate, positioned at 18% from anterior. Vitellaria extended throughout most of body and haptor, surrounds female reproductive organs. Genito-intestinal canal prominent, 580 long, 40–133 (62 ± 28; 11) wide, situated posterior to ovary.

3.1.2. Metapolystoma falcatum n. sp. (Fig. 4, Fig. 5, Fig. 6, Fig. 7; Table 2)

Fig. 4.

Ventral view of Metapolystoma falcatum n. sp. holotype. Abbreviations: eg, egg; gb, genital bulb; gc, genito–intestinal canal; ha, hamuli; hp, haptor; ic, intestinal caecum; mg, Mehlis gland; mo, mouth; oc, oncomiracidium; 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 canal; vd, vas deferens; vi, vitelline; vl, vitelline duct; vv, vitello–vaginal canal.

Fig. 5.

Metapolystoma falcatum n. sp. from Boophis doulioti. 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. 6.

Ventral view of Metapolystoma falcatum n. sp. from Boophis doulioti, neotenic form. Abbreviations: gb, genital bulb; gc, genito–intestinal canal; hp, haptor; ic, intestinal caecum; mg, Mehlis gland; mo, mouth; od, oviduct; oi, oö–vitelline canal; oö, oötype; os, false oral sucker; ov, ovarium; ph, pharynx; su, sucker; te, testis; ut, uterus; vi, vitelline; vl, vitelline duct; vv.

Fig. 7.

Metapolystoma falcatum n. sp. from Boophis doulioti, neotenic form. a, marginal hooklets 2–7; b, marginal hooklets 8; c, marginal hooklet 1.

3.1.2.1. Type host

Boophis doulioti (Mantellidae).

3.1.2.2. Type locality

Ankarafantsika, Madagascar (Fig. 1), (16.115976S; 47.095631E).

3.1.2.3. Site in host

Gills of tadpoles and urinary bladder of mature frogs.

3.1.2.4. 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.

3.1.2.5. Type material

Morphological descriptions are based on one mature parasite, eight juveniles and seven neotenics. One sexually mature specimen (Holotype NMBP 560), four immature bladder parasites (Paratypes NMBP 561–564) and four neotenics (Paratypes NMBP 565–568), all from the type locality, were deposited in the parasitic worm collection, National Museum, Aliwal Street, Bloemfontein 9301.

3.1.2.6. Voucher material

Remaining specimens in polystome collection, North-West University, Potchefstroom, South Africa.

3.1.2.7. Zoobank registration.

The Life Science Identifier (LSID) of the article is: 59F6A99A-C667-48 EB-9EA4-881D43956065. The LSID of the new name Metapolystoma falcatum n. sp. Landman et al. is: urn:lsid:zoobank.org:act:FB1716E2-5C61–4C53-9A2F-BB364D6782A8.

3.1.2.8. Etymology

The species epithet refers to the exceptionally long curved tips of marginal hooklets two to seven (falcatum = sickle shaped, curved, hooked, armed with scythes).

3.1.2.9. Description

Mature parasite (Fig. 4‒5).

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).

Ovary elongated, not lobed, positioned posterior to midbody, length four times greater than width,10% of body length. Oviduct 411 long, 22–48 (30 7; 1) wide. Uterus massive and narrow, occupying most of the space between intestinal caeca, tubiform, serpentines between oötype and genital bulb. Eggs operculated. Holotype released 171 eggs with only 11 remaining in utero. Some eggs contain fully developed oncomiracidia. Mehlis’ gland distinct. Two parallel vaginae 262‒235 long, 102 wide, situated on lateral margins, bearing multiple marginal openings. Vaginal vestibule cup-shaped, 21% from anterior. Vitellaria dorsal to intestinal tract, extends throughout most of body and haptor, except areas occupied by female reproductive organs. Genito-intestinal canal prominent, 943 long, 10–46 (23 ± 9; 1) wide, situated directly posterior to ovary.

Neotenic parasite (Fig. 6‒7).

Measurements obtained from seven egg-producing neotenic parasites. Body pyriform (Fig. 6), dorsoventrally flat, ventrally concave, 1.419–3.307 (2.265 ± 667; 7) long. Maximum width 904–1.376 (1121 ± 368; 3). Body length 2–5 (3 ± 1; 6) times greater than width. Mouth 100–148 (124 ± 17; 5) in diameter, sub-ventral, surrounded by false oral sucker. Posterior haptor 313–526 (411 ± 89; 7) long, 541–1.166 (936 ± 305; 7) wide. Haptor length-body length ratio 0.13–0.24 (0.2 ± 0.04; 6), haptor bearing three pairs of haptoral suckers equal in size 103–226 (178 ± 32; 47). Hamuli absent. Marginal hooklets placed as for M. vencesi n. sp., pairs one 28.2 long (Fig. 7a), larger than pairs two to seven 22.6–27.7 (25.5 ± 1.5; 15), (Fig. 7b), pairs eight 27.5–35 (30.5 ± 3.155; 8) long (Fig. 7c). Medial pharynx length 199–312 (244 ± 45; 5) equal to width 207–312 (245 ± 41; 5), positioned immediately posterior or at the margin of false oral sucker. Intestine bifurcates at distance 27–30% (30% ± 2%; 3) from anterior, situated posterior to pharynx, converging posteriorly at 77–81% (80% ± 2.7%; 3) from anterior, stretching into area between haptoral suckers; no prehaptoral anastomosis. Intestine with lateral diverticula, length greater than width. Diverticula in posterior half longer than anterior. Medial diverticula only posterior to ovary, length greater than width, narrower than lateral diverticula.

Testis follicular, though only a small section was visible, positioned directly posterior to vitello-vaginal canal, ventral to intestine. Vas deferens not visible. Genital pore opening mid-ventral, posterior to intestinal ceca bifurcation, at distance 42–51% (46% ± 4%; 3) from anterior. Genital bulb diameter 31–51 (41 ± 9; 4), muscular, surrounded by glandular cells, armed with genital crown bearing eight to eleven genital spines 7.9–11.8 (9.8 ± 1; 10) long.

Ovary elongate, 256–580 (401 ± 120; 7) long, 67–117 (91 ± 19; 7) wide, not lobed, situated in middle of body, length three-six times greater than width, measuring 16–21% (17 ± 2.18%; 6) of body length. Oviduct 15 long, 6–28 (13 ± 6; 1) wide. Oötype 171–173 long, ovoid, containing a maximum of one ovoid, operculate egg 171–173 long, 132–142 wide. Uterus absent, eggs laid immediately after production. Mehlis’ gland distinct. Vaginae absent. Vitellaria dorsal to intestinal tract, extended throughout most of body, except area occupied by female reproductive organs. Genito-intestinal canal prominent, 257 long, 7–47 (18 ± 8; 1) wide, situated posterior to ovary.

3.1.3. Metapolystoma ansuanum n. sp. (Fig. 8‒9; Table 2)

Fig. 8.

Ventral view of Metapolystoma ansuanum n. sp. holotype. Abbreviations: eg, egg; gb, genital bulb; gc, genito–intestinal canal; ha, hamuli; hp, haptor; ic, intestinal caecum; mg, Mehlis gland; mo, mouth; oc, oncomiracidium; 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; vd, vas deferens; vi, vitelline; vv, vitello–vaginal canal.

3.1.3.1. Type host

Boophis luteus (Mantellidae).

3.1.3.2. Type locality

Cascade des Nymphes, Isalo National Park, Madagascar (Fig. 1), (22.46977S; 45.260701E).

3.1.3.3. Site in host

Urinary bladder.

3.1.3.4. Level of infection

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

3.1.3.5. Type material

The morphological descriptions are based on two mature parasites (Holotype NMBP 569; Paratype NMBP 570) collected from the same locality in Cascade des Nymphes. The type material was deposited in the parasitic worm collection, National Museum, Aliwal Street, Bloemfontein 9301.

3.1.3.6. Zoobank registration

The Life Science Identifier (LSID) of the article is: 59F6A99A-C667-48 EB-9EA4-881D43956065. The LSID of the new name Metapolystoma ansuanum n. sp. Landman et al. is: urn:lsid:zoobank.org:act:4761BF6E-F309-4625-A7D7-CA64F3A3F6F8.

3.1.3.7. 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.

3.1.3.8. Description

Measurements reflected in 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.

Fig. 9.

Metapolystoma ansuanum n. sp. from Boophis luteus. a, hamulus from holotype; b, marginal hooklets 2–7; c, genital crown from holotype.

Testis follicular, large, kidney-shaped, positioned posterior to ovary, ventral to intestine. Vas deferens widens anteriorly to form sinuous semen vesicle 17–31 (26 ± 4; 1) wide, 422 long, measuring 11% of body length, narrowing towards genital bulb, opening in common genital opening. Genital pore opening mid-ventral, posterior to intestinal ceca bifurcation at position 16–18% from anterior, genital bulb muscular, surrounded by glandular cells, armed with genital crown bearing 10 genital spines (Fig. 9c).

Ovary, elongate, not lobed, positioned posterior to midbody, length 2.3–2.7 times greater than width, measuring 9–10% of body length. Oviduct 345 long, 19–39 (28 ± 6; 1) wide. Uterus large, occupying one-third of body proper, tubiform, convolute. Uterus contains 41 ovoid, operculate eggs, some contain fully developed oncomiracidia. Intrauterine oncomiracidia present. Mehlis’ gland distinct. Two parallel vaginae, each 94–132 long (119 ± 18; 4), 66–81 (75 ± 8; 4) wide, found on lateral margins, bearing multiple marginal openings; vaginal vestibule cup-shaped at 21–26% from anterior. Vitellaria dorsal to intestinal tract, extending throughout most of body, except area occupied by female reproductive organs. Genito-intestinal canal prominent, 264 long, 30–40 (37 ± 4; 1) wide, situated directly posterior to ovary.

3.1.4. Metapolystoma theroni n. sp. (Fig. 10‒11; Table 2)

Fig. 10.

Ventral view of Metapolystoma theroni n. sp. holotype. Abbreviations: eg, egg; gb, genital bulb; gc, genito–intestinal canal; ha, hamuli; hp, haptor; ic, intestinal caecum; mg, Mehlis gland; mo, mouth; oc, oncomiracidium; 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 canal; vd, vas deferens; vi, vitelline; vl, vitelline duct; vv, vitello–vaginal canal.

3.1.4.1. Type host

Boophis madagascariensis (Mantellidae).

3.1.4.2. Type locality

Indri Reserve in Andasibe, Madagascar (Fig. 1), (18.930856S; 48.413611E).

3.1.4.3. Site in host

Urinary bladder.

3.1.4.4. 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).

3.1.4.5. Type material

The morphological descriptions are based on one mature and 27 juvenile parasites. One sexually mature specimen (Holotype 573) and four immature ones (Paratypes 574–577), all from the type locality, were deposited in the parasitic worm collection, National Museum, Aliwal Street, Bloemfontein 9301.

3.1.4.6. Voucher material

Remaining specimens in polystome collection, North-West University, Potchefstroom, South Africa.

3.1.4.7. Zoobank registration

The Life Science Identifier (LSID) of the article is: 59F6A99A-C667-48 EB-9EA4-881D43956065. The LSID of the new name Metapolystoma theroni n. sp. Landman et al. is: urn:lsid:zoobank.org:act:55203AE0-217D-45D0-B3C8-07DDE53FA9A3.

3.1.4.8. 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. 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.

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.

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).

Ovary elongate, not lobed, positioned posterior to midbody, length 1.2 times greater than width, measuring 5% of body length. Oviduct 1167 long, 20–51 (33 ± 9; 1) wide. Uterus massive, occupying 50% of body proper, tubiform, serpentines between posterior connection at oötype and anterior connection at genital bulb, containing 176 ovoid, operculate eggs, some contain fully developed oncomiracidia. Hatched intrauterine oncomiracidia present. Mehlis’ gland distinct. Two parallel vaginae 270–304 long, 132–177 wide, on lateral margins, with multiple marginal openings, vaginal vestibule cup-shaped at 18% from anterior. Vitellaria dorsal to intestinal tract, extended throughout most of body and haptor, except areas occupied by female reproductive organs. Genito-intestinal canal prominent 457 long, 30–77 (52 ± 16; 1) wide, situated posterior to ovary.

3.1.5. Metapolystoma multiova n. sp. (Fig. 12‒13; Table 2)

Fig. 12.

Ventral view of Metapolystoma multiova n. sp. holotype. Abbreviations: 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.

3.1.5.1. Type host

Boophis occidentalis (Mantellidae).

3.1.5.2. Type locality

Cascade des Nymphes, Isalo National Park, Madagascar (Fig. 1), (22.46977S; 45.260701E).

3.1.5.3. Site in host

Urinary bladder.

3.1.5.4. 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.

3.1.5.6. Zoobank registration

The Life Science Identifier (LSID) of the article is: 59F6A99A-C667-48 EB-9EA4-881D43956065. The LSID of the new name Metapolystoma multiova n. sp. Landman et al. is: urn:lsid:zoobank.org:act:6854087D-44B5–492B-9750-3647188F0F26.

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).

3.1.5.8. Description

Measurements reflected in 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 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.

Fig. 13.

Metapolystoma multiova n. sp. from Boophis occidentalis. a, marginal hooklets 1 (top), 2–7 (middle) and 8 (bottom) from holotype and paratypes; b, hamuli from holotype (left) and from paratype (right); c, genital crown from holotype.

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).

Ovary elongate, not lobed, positioned posterior to midbody, length 2.8 times greater than width, measuring 8% of body length. Oviduct 988 long, 20–48 (31 ± 9; 1) wide. Uterus contains 336–499 ovoid, operculate eggs, some contain fully developed oncomiracidia, some hatched intrauterine oncomiracidia present. Mehlis’ gland obscured. Two parallel vaginae, each 266–311 long, 58–62 wide, situated on lateral margins bearing multiple marginal openings. Vaginal vestibule cup-shaped, situated at 18% from anterior. Vitellaria dorsal to intestinal tract, extended throughout most of body and haptor, except areas occupied by female reproductive organs. Genito-intestinal cannel prominent 633 long, 7–103 (24 ± 24; 1) wide, situated at level of ovary.

3.2. 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 characters, contained 19 taxa, among which 13 specimens of Metapolystoma and six specimens characterizing distinct species of Polystoma.

Table 3.

Polystome species investigated, host species, locality and GenBank accession numbers for 18S, 28S and COI.

Polystome species	Host species	Locality	18S Accession number	28S Accession number	COI Accession number
Metapolystoma brygoonis	Ptychadena mascareniensis	Madagascar: Ambatolampy	FM897287a	FM897270a	FM897300a
Metapolystoma brygoonis	Ptychadena mascareniensis	Madagascar: Ambatolampy	MW054243g	JN800281b	MW053457g
Metapolystoma brygoonis	Ptychadena mascareniensis	Madagascar: Ankarafantsika	MW054242g	MW054236g	JN800285b
Metapolystoma brygoonis	Ptychadena mascareniensis	Madagascar: Makira	MW054246g	MW054239g	JN800288b
Metapolystoma brygoonis	Ptychadena mascareniensis	Madagascar: Makira	MW054247g	MW054240g	JN800289b
Metapolystoma brygoonis	Ptychadena mascareniensis	Madagascar: Ranomafana	MW054245g	MW054238g	JN800286b
Metapolystoma brygoonis	Ptychadena mascareniensis	Madagascar: Vohiparara	MW054244g	MW054237g	JN800287b
Metapolystoma cachani	Ptychadena longirostris	Africa: Nigeria	FM897280a	FM897262a	JN800294b
Metapolystoma falcatum n. sp.	Boophis doulioti	Madagascar: Ankarafantsika	MW054248g	JN800283b	JN800291b
Metapolystoma falcatum n. sp.	Boophis doulioti	Madagascar: Ankarafantsika	FM897286a	FM897269a	MW053458g
Metapolystoma multiova n. sp.	Boophis occidentalis	Madagascar: Isalo	FM897285a	FM897268a	FM897301a
Metapolystoma theroni n. sp.	Boophis madagascareniensis	Madagascar: Andasibe	FM897284a	FM897267a	FM897298a
Metapolystoma theroni n. sp.	Boophis madagascareniensis	Madagascar: Andasibe	MW054249g	MW054241g	JN800293b
Polystoma claudecombesi	Amietia delalandii	South Africa	FM897281a	FM897263a	(−)
Polystoma dawiekoki	Ptychadena anchietae	South Africa	AM051069c	AM157204d	AM913856e
Polystoma integerrimum	Rana temporaria	France	AM051071c	AM157206d	JF699306f
Polystoma marmorati	Hyperolius marmoratus	South Africa	AM051073c	AM157208d	AM913859e
Polystoma occipitalis	Hemisus marmoratus	Ivory Coast	AM051075c	FM897264a	(−)
Polystoma testimagna	Strongylopus fasciatus	South Africa	AM157194c	AM157217d	AM913860e

^aFrom Verneau et al. (2009).

^bFrom Berthier and Verneau, unpublished.

^cFrom Bentz et al. (2006).

^dFrom Badets et al. (2011).

^eFrom du Preez et al. (2007).

^fFrom Raharivololoniaina et al. (2011).

^gSequences reported in the present study; (−) Not available.

Minimum and maximum distances with standard deviation as estimated among Metapolystoma species were as follows. M. falcatum n. sp. (18S: 0.051% ± 0.0003–0.051 ± 0.0005%; 28S: 0.42% ± 0.001–1.42% ± 0.002; COI: 8.6% ± 0.016–15.68% ± 0.023); M. multiova n. sp. (18S: 0–0.1% ± 0.0007); 28S: 0.28% ± 0.001–1.64% ± 0.003; COI: 12.54% ± 0.020–13.78% ± 0.022); M. theroni n. sp. (18S: 0–0.1% ± 0.0007; 28S: 0.28% ± 0.001–1.93% ± 0.003; COI: 11.4% ± 0.019–15.68% ± 0.023). With regard to 18S, total differences between species showed no character difference between M. multiova n. sp. and M. theroni n. sp. while the total 18S differences among all other Metapolystoma spp. ranged from 1 to 2 (Table 4). Regarding total 28S differences (Table 5), values ranged from 4 to 27 among all Metapolystoma spp., while the lowest value occurred between M. multiova n. sp. and M. theroni n. sp. Finally, regarding total COI differences (Table 6), values ranged from 25 to 45 between all Metapolystoma spp. Though no 18S character difference was detected between M. multiova n. sp. and M. theroni n. sp., the high levels of genetic divergence between M. falcatum n. sp., M. multiova n. sp., M. theroni n. sp., M. brygoonis and M. cachani (as estimated on the basis of 28S and COI sequences) led us to consider three new Metapolystoma species.

Table 4.

Mean genetic distances (below diagonal) and total character differences (above diagonal) between species groups as inferred from comparisons of 18S rDNA sequences (1931 characters).

		1	2	3	4	5	6	7	8	9	10	11
1	M. brygoonis		1	2	2	2	11	10	13	10	9	25
2	M. falcatum n. sp. (B. d.)	0,001		1	1	1	10	9	12	9	8	24
3	M. multiova n. sp. (B. o.)	0,001	0001		0	2	11	10	13	10	9	25
4	M. theroni n. sp. (B. m)	0,001	0001	0,000		2	11	10	13	10	9	25
5	M. cachani	0,001	0001	0,001	0001		9	8	11	10	9	23
6	P. occipitalis	0,006	0005	0,006	0006	0,005		6	16	15	14	28
7	P. dawiekoki	0,005	0005	0,005	0005	0,004	0003		16	15	14	28
8	P. claudecombesi	0,007	0006	0,007	0007	0,006	0008	0,008		11	10	24
9	P. marmorati	0,005	0005	0,005	0005	0,005	0008	0,008	0006		1	25
10	P. testimagna	0,005	0004	0,005	0005	0,005	0007	0,007	0005	0,001		24
11	P. integerrimum	0,013	0013	0,013	0013	0,012	0015	0,015	0013	0,013	0,0126

Abbreviations: B. d. = Boophis doulioti; B. o. = Boophis occidentalis; B. m. = Boophis madagascariensis

Table 5.

Mean genetic distances (below diagonal) and total character differences (above diagonal) between species groups as inferred from comparisons of 28S rDNA sequences (1418 characters).

		1	2	3	4	5	6	7	8	9	10	11	12
1	M. brygoonis (SlA)	–	2	8	13	15	20	29	29	37	38	37	58
2	M. brygoonis (SlB)	0,001	–	6	11	13	18	27	27	35	36	35	59
3	M. falcatum n. sp. (B. d.)	0,006	0004	–	11	13	20	26	26	34	35	34	56
4	M. multiova n. sp. (B. o.)	0,009	0008	0,008	–	4	23	32	32	40	39	38	62
5	M. theroni n. sp. (B. m.)	0,011	0009	0,009	0003	–	27	34	34	40	39	38	62
6	M. cachani	0,014	0013	0,014	0016	0,019	–	32	33	41	42	41	68
7	P. occipitalis	0,020	0019	0,019	0023	0,024	0023	–	7	36	38	35	59
8	P. dawiekoki	0,021	0019	0,019	0023	0,024	0024	0,005	–	35	35	32	59
9	P. claudecombesi	0,027	0025	0,024	0,09	0,029	0030	0,026	0025	–	22	23	53
10	P. marmorati	0,027	0026	0,025	0028	0,028	0030	0,027	0025	0,016	–	5	53
11	P. testimagna	0,027	0025	0,024	0027	0,027	0030	0,025	0022	0,016	0004	–	52
12	P. integerrimum	0,042	0043	0,041	0045	0,045	0050	0,043	0043	0,039	0039	0,038	–

Abbreviations: SlA = sublineage A; SlB = sublineage B; B. d. = Boophis doulioti; B. o. = Boophis occidentalis; B. m. = Boophis madagascariensis.

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).

		1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17
1	M. brygoonis (Ankarafantsika)	–	1	2	21	21	13	17	27	27	40	39	41	26	36	35	38	55
2	M. brygoonis (Ambatolampy)	0,003	–	3	20	20	12	16	26	26	39	38	40	25	35	34	37	55
3	M. brygoonis (Ambatolampy)	0,006	0009	–	21	21	13	17	26	26	39	39	41	28	36	35	38	56
4	M. brygoonis (Vohiparara)}	0,068	0065	0,068	–	1	13	11	30	30	38	37	39	30	43	40	41	58
5	M. brygoonis (Ranomafana)	0,068	0065	0,068	0003	–	13	12	30	30	38	36	39	29	43	40	41	57
6	M. brygoonis (Makira)	0,041	0038	0,041	0042	0,042	–	8	25	25	37	34	36	22	35	32	36	54
7	M. brygoonis (Makira)	0,055	0051	0,055	0035	0,038	0025	–	30	30	38	36	38	29	40	37	40	55
8	M. falcatum n. sp. (B. d.)	0,089	0086	0,086	0100	0,099	0082	0,100	–	0	38	43	45	32	38	33	37	60
9	M. falcatum n. sp. (B. d.)	0,089	0086	0,086	0100	0,099	0082	0,100	0000	–	38	43	45	32	38	33	37	60
10	M. multiova n. sp. (B. o.)	0,137	0133	0,133	0129	0,129	0125	0,129	0129	0,129	–	40	38	40	49	37	42	61
11	M. theroni n. sp. (B. m.)	0,133	0129	0,133	0125	0,121	0114	0,121	0149	0,149	0138	–	4	36	48	41	45	64
12	M. theroni n. sp. (B. m.)	0,141	0137	0,141	0132	0,133	0121	0,129	0157	0,157	0130	0,013	–	40	50	41	46	65
13	M. cachani	0,085	0082	0,092	0099	0,096	0071	0,096	0106	0,106	0136	0,121	0136	–	35	31	34	51
14	P. dawiekoki	0,121	0117	0,121	0147	0,147	0117	0,135	0128	0,128	0170	0,166	0174	0,117	–	38	42	57
15	P. marmorati	0,117	0114	0,117	0136	0,135	0106	0,124	0110	0,110	0125	0,139	0139	0,103	0129	–	25	54
16	P. testimagna	0,128	0124	0,128	0139	0,139	0121	0,135	0124	0,124	0143	0,155	0158	0,113	0143	0,082	–	53
17	P. integerrimum	0,193	0193	0,198	0205	0,201	0189	0,193	0214	0,214	0218	0,231	0235	0,177	0202	0,189	0185	–

Abbreviations: B. d. = Boophis doulioti; B. o. = Boophis occidentalis; B. m. = Boophis madagascariensis

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 (Table 5, Table 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.

Fig. 14.

Bayesian tree inferred from the analysis of concatenated 18S, 28S and COI gene sequences. Node values indicate Bayesian posterior probabilities.

3.3. 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 (Table 4, Table 5, Table 6) sustain morphological descriptions, for these three species.

The lengths of M. vencesi n. sp. (9190) and M. theroni n. sp. (9086) differ from all other Malagasy metapolystomes, which vary in length from 2680 to 6710. M. theroni n. sp. has a haptor length of 1,634, separating it from all other known Malagasy metapolystomes. Haptor length of M.falcatum n. sp. (1492) and M. vencesi n. sp. (1437) overlap but differ substantially from M. ansuanum n. sp. (669–912), M. brygoonis (760–1070) and M. multiova n. sp. (916). Haptor length-body length ratio of M. vencesi n. sp. (0.16) separates it from all other species while, in the overlapping cases of M. falcatum n. sp. (0.19), M. multiova n. sp. (0.18) and M. theroni n. sp. (0.18), there is a marked difference from M. ansuanum n. sp. (0.23–0.25) and M. brygoonis (0.22–0.23), the latter two of which overlap in turn.

With a hamulus length X (Fig. 3b) of 269–332, M. vencesi n. sp. differs from all other known Malagasy metapolystomes, which range in length between 196 and 420. It is also the single species that has no separation between the hamulus handle and guard. Metapolystoma brygoonis (330–420), M. falcatum n. sp. (393–407) and M. theroni n. sp. (392–408) overlap but differ from M. ansuanum n. sp. (205) and M. multiova n. sp. (196–248), while the latter two also overlap. Hamulus hook length Z (Fig. 3b) separates all newly described metapolystomes from one another, ranging from M. vencesi n. sp. (40–52) with the smallest hooks to M. falcatum n. sp. (66–70) with the largest. Besides M. ansuanum n. sp. differs from all other metapolystomes in that it has an exceptional long hamulus hook relative to the rest of the hamulus and M. falcatum n. sp. differs from all others in that it has an exceptional long curved marginal hooklet tip on hooks two to seven.

With a genital bulb diameter of 115, M. vencesi n. sp. has the largest diameter ranging between 64 and 86. Metapolystoma. brygoonis (80), M. falcatum n. sp. (86), and M. multiova n. sp. (86) overlap but are separated from M. ansuanum n. sp. (64–73) and M. theroni n. sp. (65), while the latter two overlap. Metapolystoma falcatum n. sp. (26), M. multiova n. sp. (25), and M. theroni n. sp. (24–28) overlap in genital crown diameter but are separated from M. ansuanum n. sp. (29–33) and M. vencesi n. sp. (32), while the latter two overlap. Metapolystoma brygoonis, M. falcatum n. sp. and M. vencesi n. sp. all bear eight genital spines and differ from M. ansuanum n. sp., M. multiova n. sp. and M. theroni n. sp. that respectively bear ten, six and seven genital spines. With the greatest genital spine length of 40.4–43.7 M. multiova n. sp. is separated from all other known Malagasy metapolystomes, except M. brygoonis, which ranges between 24.3 and 34.9.

Metapolystoma brygoonis (410–460), M. multiova n. sp. (446) and M. theroni n. sp. (479) overlap in ovary length but differ from M. ansuanum n. sp. (265–338), M. falcatum n. sp. (772) and M. vencesi n. sp. (861), the latter which also differ from one another. With the greatest egg length of 239–265, M. theroni n. sp. differs from all other Malagasy metapolystomes, which range from 160 to 230. Metapolystoma ansuanum (196–217), M. multiova n. sp. (198–228), and M. vencesi n. sp. (210–230) overlap but differ from M. brygoonis (160–200) and M. falcatum n. sp. (161–185), the latter two which overlap.

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 the differentiation of five distinct metapolystome species.

4. 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 (Verneau et al., 2009). 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) (Verneau et al., 2009), 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.