Revisiting the type material of two African Diplozoinae (Diplozoidae: Monogenea), with remarks on morphology, systematics and diplozoid specificity

Introduction

The Diplozoidae Palombi, 1949 are hermaphroditic parasites which mature and reproduce after two larval diporpa fuse in permanent cross-copula on a suitable host. They attach to the gills of their teleost fish hosts using a haptor armed with clamps and a pair of hooks. The sclerotised structures making up these clamps and hooks are of great taxonomic importance, being crucial for diplozoid species differentiation. The morphology of the anterior end of the median sclerite and the sclerites connecting this structure to the clamp jaws are of particular importance in this regard, as well as the size of the hooks. The size of the clamps has also been used for diplozoid species identification historically, but the size of these structures can be a function of other variables such as host size and environmental factors, making it an unreliable taxonomic feature (Gläser & Gläser, 1964; Matějusová et al., 2002; Milne & Avenant-Oldewage, 2012). Within the Diplozoidae, there are two subfamilies differentiated by the number of clamp pairs in the haptor. Diplozoinae Palombi, 1949 have four pairs of clamps and contain five genera, while Neodiplozoinae Khotenovsky, 1980 have more than eight pairs of clamps and have two monotypic genera. All Diplozoinae species in Africa currently belong to the genus Paradiplozoon Akhmerov, 1974, while the Neodiplozoinae are represented by a single Afrodiplozoon Khotenovsky, 1981 species.

Of the six Paradiplozoon spp. in Africa (Table 1), Paradiplozoon ichthyoxanthon Avenant-Oldewage in Avenant-Oldewage, Le Roux, Mashego & Jansen van Vuuren, 2014, Paradiplozoon vaalense Dos Santos, Jansen van Vuuren & Avenant-Oldewage, 2015, Paradiplozoon krugerense Dos Santos & Avenant-Oldewage, 2016 and Paradiplozoon moroccoense Koubková, Benovics & Šimková in Benovics, Koubková, Civáňová, Rahmouni, Čermáková & Šimková, 2021 were described relatively recently. Adequate taxonomic morphometric data is available for these species either from their original descriptions, or subsequent sclerite studies (Dos Santos & Avenant-Oldewage, 2015; Dos Santos, Dzika & Avenant-Oldewage, 2019), allowing sufficient morphological and morphometric comparison to other diplozoid taxa. Data for the second internal transcribed spacer of ribosomal DNA (ITS2 rDNA) is also available for these four species, enabling effective comparison with other diplozoids. In contrast, the descriptions of the remaining two species, Paradiplozoon ghanense (Thomas, 1957) and Paradiplozoon aegyptense (Fischthal & Kuntz, 1963), lack key taxonomic characteristics for proper taxonomic differentiation. Unidentified diplozoid parasites have also been reported in Africa by Thurston (1970) and Truter et al. (2023), but these species were not fully identified or described.

Both P. ghanense and P. aegyptense were described as Diplozoon von Nordmann, 1832, but subsequently moved to Paradiplozoon when the family was revised by Khotenovsky (1985). As the text is in Russian and not freely accessible, later works still consider these taxa as Diplozoon species (Echi & Ezenwaji, 2010; Dar et al., 2012a, 2012b; Ahmad et al., 2015a, 2015b, 2015c). Paradiplozoon ghanense was described from Brycinus macrolepidotus Valenciennes, 1850 (Alestidae) from the Black Volta near Lawra, Ghana. This was the first diplozoid description from Africa, and one of only two diplozoid species to be described from non-cyprinoid host species. The initial description provides most morphometric data required for diplozoid taxonomy, except for sclerite detail of the attachment clamps and the size and shape of the central hooks. According to Thomas (1957), no hooks were detected, and he noted that “it is probable that they were torn out during removal of the parasite from the gills” (sic.). The description of this species included two diagrams: one illustrating a complete specimen with internal organs and the overall body shape, and the second presenting a clamp. The clamp illustration unfortunately lacked detail, appeared stylised, and is thus not taxonomically informative. Paradiplozoon aegyptense was described from Labeo forskalii Rüppell, 1835 (Cyprinidae) obtained from the Giza fish Market in Egypt (Fischthal & Kuntz, 1963). Like P. ghanense, the description of P. aegyptense provided most morphometric data and included a detailed diagram illustrating a whole parasite with the arrangement of internal organs. Unfortunately, no diagrams of the haptoral sclerite details were provided, although the description of P. aegyptense did include measurements for the central hook. Moreover, the description of the clamp sclerites of P. aegyptense was exceptionally brief, only stating that they are similar to those of P. ghanense.

Various attempts to obtain fresh material for both P. ghanense and P. aegyptense were unsuccessful and thus only museum deposited material could be studied. Most of the type material (holotype and seven paratypes) for P. aegyptense was deposited to the U.S. National Museum Helminthological Collection (later the U.S. National Parasite Collection) and is now housed at the Smithsonian National Museum of Natural History (SNMNH), Washington. Material for P. aegyptense (one paratype and two vouchers) was also found in the collection of the Royal Museum for Central Africa (RMCA), Tervuren, Belgium. The type material of P. ghanense was not designated nor the collection indicated by Thomas (1957). However, two voucher specimens deposited by the species authority are housed at the SNMNH. With the exception of the data generated by Sicard et al. (2003) for an unidentified diplozoid from a recorded host of P. aegyptense in the Ivory Coast, and unsubstantiated and unlikely data for P. aegyptense by Ahmad et al. (2015a, 2015b) from India (see Dos Santos & Avenant-Oldewage (2020) for details), molecular data for both species remain elusive.

Diplozoid species were generally considered to be strictly host specific, with only selected species like Paradiplozoon homoion (Bychowsky & Nagibina, 1959), Paradiplozoon megan (Bychowsky & Nagibina, 1959) and Paradiplozoon bingolense Civáňová, Koyun & Koubková, 2013 infecting several hosts (Khotenovsky, 1985; Matějusová et al., 2002; Benovics et al., 2021; Nejat et al., 2023). However, specificity of diplozoid species appear highly variable, being impacted by limited data, singular reports/collections for several species, taxonomic uncertainty, and unsupported identifications. The infection of more than one host family or order by a diplozoid species is still exceedingly rare, with only a few Diplozoinae species suspected of doing so. It has been noted that diporpa are less selective than adult diplozoids, with Paperna (1963) infecting Oreochromis niloticus (Linnaeus, 1758) with juvenile Paradiplozoon minutum (Paperna, 1964), but they were presumably not able to fuse and mature on incompatible hosts. Paperna (1979) furthermore, collected adult diplozoids from B. macrolepidotus (the type host of P. ghanense) and identified them as P. aegyptense even though this diplozoid was described from a cyprinid host and all other records of this taxon are from cyprinoids. Most collections of diplozoids from non-cyprinoids, including this collection of P. aegyptense from B. macrolepidotus by Paperna (1979), lack detail substantiated by morphological or taxonomic reports.

To improve understanding and differentiate diplozoid taxa in Africa, the morphology of both P. ghanense and P. aegyptense are redescribed here. Some of the specimens studied here likely represent the singular collection of P. aegyptense from B. macrolepidotus by Paperna (1979), enabling re-evaluation of this record. Additionally, a summary of all records for diplozoids occurring on non-cyprinoid hosts is presented, scrutinized and discussed here.

Materials and Methods

Slide mounted material of P. aegyptense and P. ghanense from both the SNMNH and the RMCA were studied using standard compound microscopy techniques, including phase contrast and differential interference contrast (DIC) microscopy. Details for the studied specimens are given in Table 2. The specimens from the RMCA collection were studied at the University of Johannesburg using a Zeiss Axioplan 2 Imaging Light Microscope with Axiovision 4.7.2. The specimens from the SNMNH collection were studied on site in Washington using an Olympus BX51 with Leica Application Suite v4.4.0, or an Olympus BX6F with Olympus CellSens Dimensions 1.13. Morphometric analyses were carried out using either AxioVs40 V 4.8.2.0 or LCmicro 2.4 (Build 29191) software. Measurements were taken following Khotenovsky (1985) and Pugachev et al. (2010). Measurements of structures not in the correct orientation (for example clamps in lateral view), or those that were damaged, were excluded. All measurements are in μm unless otherwise noted, providing three significant figures only. Measurements are presented as a mean with range in parentheses. Illustrations of the haptoral sclerites were produced using both camera lucida illustrations and from digital photomicrographs, using Corel DRAW® Graphics Suite X6 software to digitally create them. Sclerite terminology (Fig. S1) was adapted from Nejat et al. (2023). The obtained data were compared to the descriptions of P. ghanense and P. aegyptense by Thomas (1957) and Fischthal & Kuntz (1963) respectively, as well as the summaries by Khotenovsky (1985). Morphometric data of P. aegyptense were also compared to the data for this species by Ahmad et al. (2015a) and that of P. homoion by Bychowsky & Nagibina (1959), Khotenovsky (1985) and Matějusová et al. (2002). All literature and collection records of Diplozoinae from Africa were summarized and tabulated, as well as all collection records of diplozoids from hosts other than Cypriniformes.

Results

Both voucher specimens of P. ghanense from the SNMNH had highly similar morphometric and haptoral sclerite details, supporting their conspecificity. However, one of the specimens in lot USNM 1367213 bears a striking resemblance to the illustration by Thomas (1957) and thus the specimens in this lot are likely the specimens used to describe the taxon. This is supported by the fact that the collection dates and localities of the specimens correspond to the description. The specimens were also deposited by J.D. Thomas, even though no designation of types was made in Thomas (1957). As such, we consider the specimens in lot USNM 1367213 as the syntypes of P. ghanense following Articles 72.1.1 and 72.4.1.1 and Recommendation 73F of The International Code of Zoological Nomenclature (ICZN, 1999). Due to the similarity of the illustration in Thomas (1957) to one of the specimens in USNM 1367213, this specimen is designated here as the lectotype for the taxon (Article 74.1, Recommendation 73F and 74B (ICZN, 1999)), with the second specimen becoming the paralectotype (Article 73.2.2, 74.1.3 and 74.4 (ICZN, 1999)), separated into a new lot - USNM 1548457.

Of the 12 specimens designated as P. aegyptense (11 adults and one diporpa), most had similar morphometrics and haptoral sclerite characteristics. However, the hook sizes, clamp sclerites and general morphology of one slide (RMCA_VERMES_35512; one adult and one diporpa) resembled the details for P. ghanense instead. This specimen is thus re-designated as a voucher of P. ghanense, leaving 10 P. aegyptense specimens. USNM 1355449 was designated as the “type” of P. aegyptense in the original description and is catalogued in the SNMNH as such. However, paratypes were also designated by Fischthal & Kuntz (1963), and thus USNM 1355449 is actually the holotype (Article 73.1.1 (ICZN, 1999)). Furthermore, only USNM 1355450 is designated as paratype material in the original description, but USNM 1363700 is designated as paratype material in the SNMNH catalogue, while RMCA_VERMES_35196 shares the same collection and collector details as the type material. Additionally, even though only three specimens (one “type” and two paratypes) are catalogued by Fischthal & Kuntz (1963), the authors state that the “species is represented in the collection by nine pairs of adults in permanent copula” (sic.) suggesting that USNM 1363700 and RMCA_VERMES_35196 are the remaining six specimens of the type series. As such, all material deposited by the species authors are considered part of the type series for P. aegyptense, supporting the designation of USNM 1363700 as paratypes and indicting that RMCA_VERMES_35196 should be designated as a paratype as well. RMCA_VERMES_35580 is now the only voucher specimen of P. aegyptense.

The morphometric data presented here for both P. ghanense and P. aegyptense are based only on the type material of each species, while the voucher material is discussed in the remarks section and their data presented in Tables 3 and 4 respectively. The voucher diporpa of P. ghanense is discussed separately in the morphometric section of this species.

Family: Diplozoidae Palombi, 1949

Subfamily: Diplozoinae Palombi, 1949

Genus: Paradiplozoon Akhmerov, 1974

Paradiplozoon ghanense (Thomas, 1957)

Synonym: Diplozoon ghanense Thomas, 1957

Type host: Brycinus macrolepidotus Valenciennes, 1850

Other hosts: Alestes baremoze (Joannis, 1835)

Type locality: Black Volta, Lawra, Ghana

Other localities: Lake Volta, Ghana; Lake Albert, Uganda; Anambra River, Nigeria (See Table 1 for detail).

Infection site: Gills

Lectotype: USNM 1367213 (one slide, USNPC 071636)

Paralectotype: USNM 1548457 (one slide, USNPC 071636)

Voucher: RMCA_VERMES_35512 (one slide, M.T. 35.512)

Morphology

Adult (n = 2; Figs. 1, S2 and S3; Tables 3 and S1): Specimens permanently fused in cross-copula. Body 3,780 (3,240–4,250). Broad, dorsoventrally flattened anterior, 2,300 (1,850–2,810) long, 694 (636–754) wide, tapered toward oral and fusion areas. Sub-cylindrical fusion area, 563 (508–625) long, 951 (925–977) wide. Posterior subcylindrical, 664 (596–755) long, 136 (93–226) wide. Widened haptor at posterior end, 467 (325–661) long, 494 (362–602) wide, without any conspicuous features or protrusions. Clear constriction between posterior and fusion area. Tegument with small, delicate plicae, slightly larger in posterior between fusion area and haptor, not pronounced. No dilations in posterior nor any diagnostically pronounced plicae. Oral opening “U”-shaped, sub-terminal, ventral. Buccal cavity with two suckers, 62.5 (56.3–68.5) long, 57.6 (52.9–67.9) wide, close to each other, 21.4 (17.2–29.3) between them. Suckers 24.9 (14.6–32.8) from anterior. Prepharynx short, 22.9 (15.8–31.8). Pharynx, 54.7 (45.4–65.2) long, 43.1 (38–45.4) wide, posterior to suckers. Intestine single caecum from pharynx to posterior, approaching haptor, diverticula in anterior only. Vitellaria densely packed from just posterior of pharynx to mostly anterior of fusion area.

Gonads entirely in fusion area, single testes of one specimen close to anterior body. Ovary anterior to testes, long, folded, 281 (193–395) long, 233 (178–327) wide. Testes post-ovarian (but in contact or slightly overlapping ovary), conspicuous, round to oval, single, smooth, 134 (123–152) long, 118 (115–123) wide. Egg large, spindle shaped (Fig. 1A), 258 long, 114 wide. Long, coiled filament attached to operculum, anoperculum 167 long.

Haptor with two rows of ventrally directed clamps, four in a row on opposite ventrolateral margins. Clamps 95.8 (80.4–106) long, 140 (105–162) wide on average. First clamp 94.4 (90.5–102) long, 125 (105–138) wide, second clamp 100 (93.3–104) long, 144 (123–162) wide, third clamp 97.7 (87.6–106) long, 147 (126–158) wide, fourth clamp 92.2 (80.4–98) long, 142 (122–162) wide. Clamps consist of a pair of “J”-shaped anterior clamp jaws, a pair of posterior clamp jaws and a ‘‘U’’-shaped median plate (Fig. 1B). Clamp jaws (Figs. 1C and 1D) slender but not delicate. Lateral sclerite absent. Anterior of median sclerite rounded, with elongate obovate to spoon-shaped anterior spur extending towards anterior clamp jaws (Fig. 1E). No anterior joining sclerite between anterior clamp jaws and anterior end of median sclerite. Board trapezoid dorsal joining sclerite above anterior spur (Fig. 1B and S3A, S3B). Tendon guiding termination at posterior end of median sclerite claw-shaped with large central opening (Fig. 1F). Single posterior joining sclerite with broad base and rounded anterior sometimes visible between posterior clamp jaws and posterior of median sclerite.

Central hooks present (Fig. 1G), single pair, each roughly at level of first clamp towards medial aspect of haptor. Hook 21.5 (21–21.9), handle 40.3 (39.1–42.6). Wing mostly overlaying blade of hook.

Diporpa (n = 1; Figs. S4A, S4B and S5A; Tables 3 and S1): Body length 1,000. Broad, dorsoventrally flattened anterior, 537 long, 288 wide, tapered towards anterior. Fusion area absent. Slight constriction between anterior and posterior. Posterior subcylindrical, 464 long, 201 wide, no pronounced dilations. Widened haptor at posterior, 271 long, 285 wide, without any conspicuous features or protrusions. Tegument with small, fine plicae, slightly more noticeable in anterior of posterior, not pronounced. Oral opening “U”-shaped, sub-terminal, ventral. Buccal cavity with two suckers, 55 (55–56) long, 55 (52–59) wide, close to each other, 11.5 between them. Suckers 28 from anterior. Suckers 28.4 from anterior. Prepharynx short, 20.6 long. Pharynx, 59.9 long, 48.2 wide, posterior to suckers. Intestine single caecum from pharynx to middle of haptor. Vitellaria and gonads absent, only primordial mass present.

Haptor with two rows of ventrally directed clamps, four in a row on opposite ventrolateral margin. Clamps 45.2 (39.7–51.4) long, 66.5 (61.6–73.8) wide on average. First clamp 51.3 (51.2–51.4) long, 64.2 (63.5–65) wide, second clamp 45.6 (43.6–47.7) long, 71.8 (69.8–73.8) wide, third clamp 42.4 (42.3–42.5) long, 67.6 (65.7–69.6) wide, fourth clamp 42.2 (39.7–44.8) long, 62.8 (61.6–64.1) wide. Clamp sclerites highly similar to type material (Figs. S4A and S4B). However, anterior clamp jaws extend anteriorly at their medial connection, forming broad connection dorsal to anterior protrusion of medial sclerite.

Central hooks present, single pair, each roughly at level or anterior to first clamp towards medial aspect of haptor. Hook 20.8 (20.6–21.1), handles 41.7.

Remarks: The morphometric data obtained from the newly identified type series of P. ghanense were similar to that in the original description (Table 3), with only slightly wider ranges recorded for some features. However, even though specimen USNM 1367213 (now only one specimen and designated as lectotype) bore a striking resemblance to the illustration in the description, damage at the extremity of one haptor and the pre-haptoral region of the other were observed (Fig. S2), which was not captured in the illustration by Thomas (1957). Similarly, some of the clamps on both haptors were damaged or missing, with the illustration by Thomas (1957) displaying all clamps intact. Whether damage to the specimen occurred after the description, possibly due to remounting, or whether this damage was edited out during illustration is not known. Unfortunately, the clamps and hooks of the lectotype were not ideally orientated for illustration, but the same morphology was observed in both type series specimens. As such, the third clamps and hook of the paralectotype are illustrated in Fig. 1. The hooks of the taxon could be observed and are described here for the first time. Thomas (1957) does not state how many parasites were collected or studied from the two infected fish, thus the two specimens studied here may be the only material in the type series.

Specimen RMCA_VERMES_35512, which is designated as a voucher of P. ghanense here, is morphologically nearly identical to that of the type series material (Figs. S5B–S5D). This includes the details of the clamp sclerites and the size of the hooks (Figs. S4C–S4E), supporting the re-designation of this material as P. ghanense. However, large deviations in the sizes of the body (total, anterior length and width), fusion area size, gonad size, and especially the sizes of the clamps were observed. Nevertheless, the similarity of the more taxonomically informative clamp sclerites and hook sizes validate the re-identification of the material. The hooks of the diporpa could not be studied in an ideal orientation and thus only their size could be recorded.

Using the keys for Paradiplozoon taxa in South-East Asia and Africa by Khotenovsky (1985), the supplemented data for P. ghanense does not match any of the criteria as there is a gap where taxa with hooks between 20 and 25 μm should fall. However, if the assumption is made that this gap is unintentional and that the 11^th line should refer to taxa with hooks larger than 20 μm instead of larger than 25 μm, then the specimens studied here conform to the identification as P. ghanense. The clamp morphology of P. ghanense differs from all other diplozoid taxa. The most striking difference is the absence of a lateral sclerite. The division of the posterior clamp jaw to form a lateral sclerite is present in most diplozoid taxa, except for P. bingolense from Turkey and Paradiplozoon iraqense Al-Nasiri & Balbuena, 2016 from Iraq. The gross clamp morphology of both P. bingolense and P. iraqense are similar to that of P. ghanense but are more robust. Among African Paradiplozoon, the clamp sclerites of P. ghanense are most similar to those of P. moroccoense, but the latter species has lateral sclerites. Paradiplozoon ghanense differs from all species, as well as all other African Paradiplozoon spp., in terms of general body shape, all lacking the clear constriction between the fusion area and the posterior, as well as restriction of the gonads to the fusion region. Most also have smaller hooks than P. ghanense. The confinement of the reproductive organs to the fusion area in P. ghanense is only shared by Sindiplozoon Khotenovsky, 1981 species.

Paradiplozoon aegyptense (Fischthal & Kuntz, 1963)

Synonym: Diplozoon aegyptensis Fischthal & Kuntz, 1963

Type host: Labeo forskalii Rüppell, 1835

Other hosts: Enteromius paludinosus (Peters, 1852), Labeo coubie Rüppell, 1832, Labeo cylindricus Peters, 1852, Labeo victorianus Boulenger, 1901, Raiamas senegalensis (Steindachner, 1870), Labeo sp.

Unverified hosts: Cyprinus carpio Linnaeus, 1758, Schizopyge niger (Heckel, 1838), Schizothorax progastus (McClelland, 1839), Carassius carassius (Linnaeus, 1758)

Type locality: Giza Fish Market, Cairo, Egypt

Other localities: Lake Volta, Ghana; Lake Albert and Aswa River, Uganda; Nzoia River, Kenya; Ruaha River, Tanzania.

Unverified localities: Several localities in India (See Table 1 for detail).

Infection site: Gills

Holotype: USNM 1355449 (one slide, USNPC 059653)

Paratypes: USNM 1355450 (two slides, USNPC 059654); USNM 1363700 (five slides, USNPC 068090); RMCA_VERMES_35196 (one slide, M.T. 35.196)

Voucher: RMCA_VERMES_35580 (one slide, M.T. 35.580)

Morphology

Adult (n = 9; Fig. 2; Tables 4 and S2): All specimens permanently fused in cross-copula. Body 4,370 (3,640–6,150) long. Broad, dorsoventrally flattened anterior, 2,630 (1,920–3,830) long, 572 (305–925) wide, tapered toward oral and fusion areas. Sub-cylindrical fusion area, 406 (320–459) long, 876 (733–1098) wide. Posterior subcylindrical, 1,080 (839–1840) long, 183 (120–257) wide. Simple, disk-like haptor at posterior, 330 (281–412) long, 270 (189–399) wide, without conspicuous features or protrusions. No constriction between posterior and fusion area, no dilation of posterior. Tegument with small, delicate plicae, slightly more conspicuous in posterior between fusion area and haptor, not pronounced. Oral opening “U”-shaped, sub-terminal, ventral. Buccal cavity with two suckers, 106 (82.9–128) long, 95.1 (75.5–122) wide, close to each other, often touching, 8.93 (4.45–12.6) apart on occasion. Suckers 37.5 (27.1–49) from anterior margin. Prepharynx short, 25.2 (13.8–32.8). Pharynx, 63.2 (51.8–83.2) long, 43.3 (39.1–50.3) wide, posterior to suckers. Intestine single caecum from pharynx to anterior, occasionally reaching slightly into haptor, diverticula in anterior but not in posterior. Vitellaria densely packed from just posterior to pharynx to anterior of gonads in fusion area.

Gonads mainly in fusion area, testes partly or entirely in posterior. Ovary anterior to testis, long, folded, 339 (257–470) long, 166 (121–225) wide. Testes post-ovarian (but in contact or slightly overlapping ovary), conspicuous, round to oval, single, smooth, 127 (100–187) long, 81.8 (63.4–94.7) wide. Eggs large, spindle shaped (Fig. 2A), 280 (239–308) long, 104 (84.6–132) wide. Long, coiled filament attached to operculum, anoperculum 170 (149–191).

Haptor with two rows of ventrally directed clamps, four in a row on opposite ventrolateral margins. Clamps 72.5 (55.7–85.4) long, 97.9 (73.2–120) wide on average. First clamp 68.9 (61.6–84.8) long, 88.7 (73.2–104) wide, second clamp 73.6 (63.2–81.1) long, 101 (82.4–116) wide, third clamp 74.1 (58.3–82.5) long, 103 (88–120) wide, fourth clamp 73.7 (55.7–85.4) long, 101 (87.1–117) wide. Clamps consist of a pair of “J”-shaped anterior clamp jaws, a pair of posterior clamp jaws and a ‘‘U’’-shaped median sclerite (Fig. 2B). Clamp jaws thick and robust (Figs. 2C and 2D), lateral sclerite usually visible. Median sclerite rounded anteriorly with rectangular to truncate anterior spur (Fig. 2E). Trapezoid to rounded anterior joining sclerite between anterior projection of median sclerite and anterior clamps jaws, with a pair of small rectangular anterior distal joining sclerite sometimes visible between anterior joining sclerite and anterior clamp jaws (Fig. 2F). Board trapezoid dorsal joining sclerite observed above anterior of median sclerite (Figs. 2B and S3C–S3F). Tendon guiding termination at posterior end of median sclerite claw-shaped, with large central opening (Fig. 2G). Single, narrow posterior joining sclerite between anterior clamp jaw and posterior end of median sclerites, wider at base with rounded anterior end.

Central hooks present (Fig. 2H), single pair, roughly at level of first clamp towards medial aspect of haptor, ventral. Hook 18.6 (17.6–19.7), handle 44.9 (41.1–47.9). Wing sometimes extending over blade of hook.

Remarks: The morphometric data generated here based on the type series were highly similar to that in the original description of P. aegyptense (Table 4), with only slightly wider or narrower ranges recorded for some features. The only notable difference between the measurements generated here and the original description was the larger size of the hooks, 16.5 (16–17) in the original description and 18.7 (17.7–19.1) here. The similarity in the data generated for all material considered part of the type series here support the designation of USNM 1363700 and RMCA_VERMES_35196 as paratype material even though this material is not specifically designated as such in the original description. Thus, the entire type series of nine adults indicated by Fischthal & Kuntz (1963) was studied here. Some of the clamps on one haptor of the holotype were not fully formed, or malformed, unlike the illustration in the description (and Khotenovsky (1985)), suggesting these anomalies were edited out of the illustrations.

Voucher specimen RMCA_VERMES_35580 was morphologically highly similar to the data for the type series. The only differences were smaller or larger ranges for some structures (e.g., clamps and suckers), but ranges still overlapped with the type series. Some of the internal organs could not be clearly observed as the voucher material was overstained. Unfortunately, the mounting medium of the voucher had badly deteriorated, necessitating remounting. The cracked medium also caused the specimen to fragment, but all pieces of the specimen were retained, remounted, and imaged fragments digitally recombined (Fig. S6). Destaining was attempted before remounting, but to no avail.

Using the keys for Paradiplozoon taxa in South-East Asia and Africa by Khotenovsky (1985), all specimens studied here conform to the identification as P. aegyptense. The morphology of P. aegyptense differs from other African Paradiplozoon spp. mainly based on the clamp sclerite detail, with P. krugerense most similar. However, P. aegyptense has a single posterior joining sclerite while P. krugerense has a proximal and distal posterior joining sclerite. Additionally, P. krugerense does not display the dorsal joining sclerite or the small rectangular distal joining sclerite sometimes visible between the anterior joining sclerite and anterior clamp jaws of P. aegyptense. The lateral sclerite also appears much smaller in relation to the posterior clamp jaws in P. aegyptense than in P. krugerense. Interestingly, a posterior clamp jaw of one of the third clamps of the holotype was not divided to display a lateral sclerite (Figs. 2B and 2D), a characteristic shared by very few diplozoid taxa (see P. ghanense remarks section). This appears to be an anomaly as it was not seen in the other clamps of the holotype or paratypes, indicating that the clamp did not develop normally and suggesting that the clamp sclerites of the holotype may not be the ideal representative for the taxon. The hook length of P. krugerense (18 (17–19)), and to a lesser extent P. vaalense (19 (18–20)), is similar to the updated data for P. aegyptense (18.7 (17.7–19.1)) presented here. Outside of Africa, the clamp sclerites of P. aegyptense are dissimilar to all diplozoid taxa. This includes P. homoion, in which the trapezoid anterior spur of the median sclerite attaches to the anterior clamp jaws. The testes of P. homoion are also lobed, unlike the smooth testes of P. aegyptense. The hook sizes of P. homoion (19–22.4 in Matějusová et al. (2002)) are generally larger than the data for P. aegyptense, supporting the distinctness of the two taxa. However, the hooks were recorded as 17 in the original description of P. homoion, smaller than those of P. aegyptense recorded here, but similar the those in the description of P. aegyptense (16.5 (16–17) in Fischthal & Kuntz (1963)).

Discussion

Conclusion

The present study not only completes the morphometric data for P. ghanense and P. aegyptense to allow comprehensive taxonomic comparison with other diplozoids, but also designated the type series for P. ghanense and confirms the paratype status of six P. aegyptense specimens. Additionally, the re-identification of a P. ghanense specimen has allowed for the partial resolution of discrepancies in the host specificity of this genus in Africa. The next step would be to obtain genetic material for these species to allow for molecular analyses to confirm their taxonomic distinctness and their phylogenetic relations. The more diligent study of diplozoids from non-cyprinoid hosts also appears to be a promising topic for future studies, likely resulting in the description of several diplozoid species.

Supplemental Information