General remarks

The taxonomic history of the superfamily Diplostomoidea is highly complex. Numerous publications (e.g., Dubois Reference Dubois1938, Reference Dubois1953, Reference Dubois1968, Reference Dubois1970a, Reference Dubois1970b, Reference Dubois1982, Reference Dubois1987, Reference Dubois1989; Sudarikov, Reference Sudarikov and Skrjabin1959, Reference Sudarikov and Skrjabin1960a,Reference Sudarikov and Skrjabinb, Reference Sudarikov and Skrjabin1961, Reference Sudarikov1997; Yamaguti Reference Yamaguti1958, Reference Yamaguti1971) have introduced and disputed changes to taxonomy, composition, and systematics of diplostomoideans based on morphology and host-associations. We provide an abbreviated history of the superfamily, without focus on generic composition of its constituent families, followed by histories and details for each family.

Diplostomoidea

Originally, Blanchard (Reference Blanchard1847) placed what are currently known as diplostomoideans into the family Holostomidae Blanchard, 1847. Poirier (Reference Poirier1886) examined a series of digeneans from crocodilians to determine their relationship with Diplostomum spp. He used the name Diplostomidae but did not establish the superfamily Diplostomoidea in his work. Nicoll (Reference Nicoll1937) first proposed the name Diplostomatoidea Nicoll 1937, which was not broadly recognized until Sudarikov (Reference Sudarikov and Skrjabin1960a) and Gibson (Reference Gibson1996) reintroduced the taxon as Diplostomoidea Poirier, Reference Poirier1886.

Railliet (Reference Railliet1919) erected the superfamily Strigeoidea Railliet, 1919 and family Strigeidae; the family was further split into 5 subfamilies: the Strigeinae Railliet, 1919; Polyotylinae Monticelli, 1888 (which included diplostomids); Cyathocotylinae Mühling, 1898; Alariinae Hall et Wigdor, 1918; and Braunininae Wolf, 1903. Poche (Reference Poche1925) provided an alternative classification system, which included Tribus Fascioloidae Poche, 1925 that contained 4 families, including the Strigeidae. In the same work, Poche (Reference Poche1925) erected the supersuperfamily Strigeida Poche, 1925 that consisted of the Strigeidae and Cyathocotylidae.

Dubois (Reference Dubois1936b) accepted supersuperfamily Strigeida of Poche (Reference Poche1925) containing superfamilies Strigeides (amended spelling of Strigeoidea) of Railliet (Reference Railliet1919) and Cyathocotylides Dubois, 1936. The Strigeides was further split into subsuperfamilies Strigeines Dubois, 1936; Diplostomines Dubois, 1936; and Bolbocephalodines Dubois, 1936. The Strigeines and Bolbocephalodines only included 1 family each, the family Strigeidae and Bolbocephalodidae, while the Diplostomines included the Diplostomidae and Proterodiplostomidae. It is worth noting that in this classification system, only species with a cup-shaped prosoma and bilobed holdfast organ belonged to the Strigeidae; all diplostomids of reptiles were included in the Proterodiplostomidae based on host association. The Cyatocotylides included the families Cyathocotylidae and Brauninidae. Dubois (Reference Dubois1953) maintained this classification system, although the spelling of the Diplostomines was amended to Diplostomatines.

In contrast, La Rue (Reference La Rue1957) maintained the superfamily Strigeoidea of Railliet (Reference Railliet1919) with families Strigeidae, Diplostomidae, Cyathocotylidae, Proterodiplostomidae, Bolbocephalodidae, and Brauninidae. Shortly after, Sudarikov (Reference Sudarikov and Skrjabin1960a) recognized the superfamily Diplostomatoidea that contained the families Diplostomidae, Alariidae Tubangui, 1922, and Bolbocephalodidae. Further, Sudarikov (Reference Sudarikov and Skrjabin1960b) considered Proterodiplostomatoidea Sudarikov, 1960 as a separate superfamily containing two families, Proterodiplostomatidae and Ophiodiplostomatidae Sudarikov, 1960. The most recent major systematic re-evaluation of the group by Niewiadomska (Reference Niewiadomska, Gibson, Jones and Bray2002a–Reference Niewiadomska, Gibson, Jones and Brayg) in the Keys to the Trematoda recognized 6 families: Bolbocephalodidae, Brauninidae, Cyathocotylidae, Diplostomidae, Proterodiplostomidae, and Strigeidae. However, since its publication, Niewiadomska’s classification system underwent several changes, mostly associated with the introduction of molecular phylogenetics (see overview below). Most notably, the family Brauninidae was synonymized with the Cyathocotylidae by Achatz et al. (Reference Achatz, Pulis, Junker, Binh, Snyder and Tkach2019d).

Bolbocephalodidae

Dubois (Reference Dubois1934) erected the family Bolbocephalidae Dubois, 1934 for the lone species Bolbocephalus intestiniforax Dubois, 1934 parasitic in birds; however, since the original genus name was preoccupied, Strand (Reference Strand1935) amended the genus name to Bolbocephalodes and the family name to Bolbocephalodidae. Sudarikov (Reference Sudarikov and Skrjabin1960a) considered it to be one of the families within the Diplostomatoidea, where it has remained since. Currently, the Bolbocephalodidae is the smallest family in the superfamily consisting of a single monotypic genus, Bolbocephalodes Strand, 1935. The morphology of Bolbocephalodes intestiniforax Dubois, 1934 is highly similar to strigeids as recognized by Niewiadomska (Reference Niewiadomska, Gibson, Jones and Bray2002a) (i.e., a cup-shaped portion of prosoma and bilobed holdfast organ). Despite these similarities, it remains in its own family, with sequence data from this genus currently lacking.

Cyathocotylidae

The Cyathocotylidae has proven to be the least controversial family of diplostomoideans, in part because it contains the only diplostomoideans with a cirrus sac. Mühling (Reference Mühling1896) initially established the Cyathocotyleae for the genus Cyathocotyle Mühling, 1896. Later, Poche (Reference Poche1925) amended the name to the Cyathocotylidae. More recently, Achatz et al. (Reference Achatz, Pulis, Junker, Binh, Snyder and Tkach2019d) synonymized the Brauninidae (containing a single genus Braunina Heider, 1900 also characterized by the presence of a cirrus sac) with the Cyathocotylidae based on morphological and molecular analyses. Braunina was transferred into the subfamily Braunininae of the Cyathocotylidae. At present, the Cyathocotylidae includes 6 subfamilies: Cyathocotylinae (3 genera), Muhlinginae Mehra, 1950 (1 genus), Prohemistominae Lutz, 1935 (5 genera), Prosostephaninae Szidat, 1936 (3 genera), Szidatiinae Dubois, Reference Dubois1938 (3 genera), and Suchocyathocotylinae Achatz, Pulis, Junker, Binh, Snyder et Tkach, 2019 (1 genus).

Few molecular phylogenetic studies have focused on cyathocotylids (Achatz et al. Reference Achatz, Pulis, Junker, Binh, Snyder and Tkach2019d, Reference Achatz, Von Holten, Binh and Tkach2024b; Sokolov et al. Reference Sokolov, Vlasenkov, Bugmyrin, Kalmykov and Lebedeva2024). These studies have revealed somewhat contradictory evidence between morphology and molecular phylogenetic results. For instance, Achatz et al. (Reference Achatz, Pulis, Junker, Binh, Snyder and Tkach2019d) transferred Holostephanoides Dubois, 1983 from the Cyathocotylinae into the Szidatiinae based on strongly supported molecular evidence. At the same time, the adult morphology of Holostephanoides is highly similar to other cyathocotylines and quite different from the other 2 szidatiine genera, Gogatea Lutz, 1935 and Neogogatea Chandler et Rausch, 1947 (Achatz et al. Reference Achatz, Pulis, Junker, Binh, Snyder and Tkach2019d, Reference Achatz, Von Holten, Binh and Tkach2024b). Likewise, Paracoenogonimus Katsurada, 1914 (Prohemistominae) was shown to be nested among szidatiines, while Georgduboisia Sokolov, Vlasenkov, Bugmyrin, Kalmykov et Lebedeva, 2024 (Prosostephaninae) formed a strongly supported clade with cyathocotylines (Sokolov et al. Reference Sokolov, Vlasenkov, Bugmyrin, Kalmykov and Lebedeva2024). Molecular phylogenies revealed similar problems with the subfamily classification systems among diplostomids and proterodiplostomids (Achatz et al. Reference Achatz, Chermak, Martens, Pulis, Fecchio, Bell, Greiman, Cromwell, Brant, Kent and Tkach2021c; Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020; see discussion below) as well as in some other digenean families (Tkach et al. Reference Tkach, Kudlai and Kostadinova2016, Reference Tkach, Achatz, Hildebrand and Greiman2018). Hence, subfamily classification systems have been recently abandoned in several digenean families—for example Cryptogonimidae Ward, 1917; Dicrocoeliidae Looss, 1899; and Echinostomatidae Looss, 1899. Some of the recent authors (Achatz et al. Reference Achatz, Chermak, Martens, Pulis, Fecchio, Bell, Greiman, Cromwell, Brant, Kent and Tkach2021c; Tkach et al. Reference Tkach, Achatz, Hildebrand and Greiman2018, Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020) pointed out that members of those subfamilies lack consistent differentiating morphological features. Based on these arguments and for the stability of the classification system, we do not recognize subfamilies of the Cyathocotylidae here and abandon the subfamily-based classification system of the family.

Diplostomidae

The history of the Diplostomidae was tumultuous. Poirier (Reference Poirier1886) erected the family for Diplostomum spp. Railliet (Reference Railliet1919) did not recognize the family and included diplostomids within the subfamily Polyotylinae in family Strigeidae. In Railliet’s classification system, the subfamily Alariinae established by Hall and Wigdor (Reference Hall and Wigdor1918) for diplostomids of mammals (i.e., Alaria spp.) was maintained as a separate subfamily. Tubangui (Reference Tubangui1922) elevated its status to the family Alariidae.

Dubois (Reference Dubois1936b, Reference Dubois1951) considered the Diplostomidae to be a separate family from the Strigeidae; in his classification system, the Diplostomidae included subfamilies Diplostominae (subsubfamilies Diplostomini Dubois, 1936 and Crassiphialini Dubois, 1936) and Alariinae; these subfamilies were almost entirely separated based on parasitism in avian or mammalian definitive hosts, respectively. Sudarikov (Reference Sudarikov and Skrjabin1960a,Reference Sudarikov and Skrjabinb) did not accept this arrangement and considered the family to include the subfamilies Diplostominae, Codonocephalinae Sudarikov, 1959, and Crassiphialinae Sudarikov, 1960. Later, Dubois (Reference Dubois1970a,Reference Duboisb) considered the Diplostomini and Crassiphialini, along with the Codonocephalini Sudarikov, 1959, to represent tribes of the Diplostomidae rather than subfamilies.

Shoop (Reference Shoop1989) provided a substantial revision to the classification system of the Diplostomidae by breaking it up into 3 families: Bolbophoridae Shoop, 1989; Diplostomidae (subfamilies Alariinae and Diplostominae); and Neodiplostomidae Shoop, 1989 (Crassiphialinae and Neodiplostominae Shoop, 1989). Nieiwadomska (Reference Niewiadomska, Gibson, Jones and Bray2002d) did not accept the classification system of Shoop (Reference Shoop1989) and considered Diplostomidae comprising Alariinae, Codonocephalinae, Crassiphialinae, and Diplostominae. However, molecular phylogenetic studies (e.g., Achatz et al. Reference Achatz, Pulis, Fecchio, Schlosser and Tkach2019c, Reference Achatz, Bell, Melo, Fecchio and Tkach2021a,Reference Achatz, Chermak, Martens, Pulis, Fecchio, Bell, Greiman, Cromwell, Brant, Kent and Tkachc, Reference Achatz, Martens, Kostadinova, Pulis, Orlofske, Bell, Fecchio, Oyarzún-Ruiz, Syrota and Tkach2022c–Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkache, Reference Achatz, Von Holten, Kipp, Fecchio, LaFond, Martens and Tkach2023b, Reference Achatz, Chacko, Prasadan and Tkach2024a; Blasco-Costa and Locke Reference Blasco-Costa and Locke2017; Hernández-Mena et al. Reference Hernández-Mena, García-Varela and Pérez-Ponce de León2017; Locke et al. Reference Locke, Van Dam, Caffara, Pinto, López-Hernández and Blanar2018) revealed these subfamilies to be extensively non-monophyletic. This non-monophyly along with extensive morphological review led Achatz et al. (Reference Achatz, Chermak, Martens, Pulis, Fecchio, Bell, Greiman, Cromwell, Brant, Kent and Tkach2021c) to reject the use of subfamilies within the Diplostomidae. Importantly, several prior studies (e.g., Achatz et al. Reference Achatz, Chermak, Martens, Pulis, Fecchio, Bell, Greiman, Cromwell, Brant, Kent and Tkach2021c, Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022e; Blasco-Costa and Locke Reference Blasco-Costa and Locke2017 and references therein) demonstrated that molecular phylogenies did not support host-based classification systems of diplostomoideans and emphasized the extensive amount of evolutionary host switching events between various host groups in this diverse, cosmopolitan digenean lineage.

Proterodiplostomidae

Dubois (Reference Dubois1936a) erected the Proterodiplostomidae for diplostomids collected from reptiles, predominantly crocodilians. Most proterodiplostomids are characterized by the presence of a paraprostate that is associated with the male reproductive system. However, members of Mesodiplostomum and Proalarioides Yamaguti, 1933 lack this organ. The monophyly of this lineage, including Mesodiplostomum, has been demonstrated for most proterodiplostomids, with the exception of Proalarioides (Achatz et al. Reference Achatz, Chermak, Junker and Tkach2022a, Reference Achatz, Chacko, Prasadan and Tkach2024a; Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020; Figures 1 and S1). Achatz et al. (Reference Achatz, Chacko, Prasadan and Tkach2024a) transferred Proalarioides to the Diplostomidae. A brief systematic history of this family was published by Tkach et al. (Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020); therefore, we are not presenting it in detail here. Among other systematic changes, Tkach et al. (Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020) demonstrated the non-monophyly of the proterodiplostomid subfamilies and proposed to abandon their use. These findings were further supported by subsequent works on proterodiplostomids (Achatz et al. Reference Achatz, Brito, Fecchio and Tkach2021b, Reference Achatz, Chermak, Junker and Tkach2022a, Reference Achatz, Chacko, Prasadan and Tkach2024a).

Recently, Achatz et al. (Reference Achatz, Martens, Kudlai, Junker, Boe and Tkach2022d) described the diplostomid Neofibricola Achatz, Martens, Kudlai, Junker, Boe et Tkach, 2022 from Nile crocodile Crocodylus niloticus Laurenti. Despite the host overlap, these new digeneans were demonstrated to be well separated from proterodiplostomids and lacked a paraprostate. This finding further demonstrates that host-based classification systems are not suitable for diplostomoidean systematics.

Strigeidae

The Strigeidae as originally recognized by Railliet (Reference Railliet1919) was a mixture of contemporary strigeids, diplostomids, and cyathocotylids. Dubois (Reference Dubois1936b) proposed the Strigeidae as it is currently accepted and split the family into the subfamilies Strigeinae with subsubfamilies Strigeini Dubois, 1936 and Cotylurini Dubois, 1936. Baer (Reference Baer1938) erected the subfamily Duboisiellinae Baer, 1938 for Duboisiella proloba Baer, 1938 collected from mammals, while all members of the Strigeinae only parasitize avian definitive hosts. Dubois (Reference Dubois1953) accepted Duboisiellinae as a subfamily of the Strigeidae. Bisseru (Reference Bisseru1956) described a few species of Neostrigea Bisseru, 1956 and Prostrigea Bisseru, 1956 from crocodilians and placed them into a new family Neostrigeidae Bisseru, 1956 based primarily on parasitism in a specific group of hosts. Dubois (Reference Dubois1968) considered the infection in crocodilians to be accidental and synonymized the family with the Strigeidae.

Sudarikov (Reference Sudarikov and Skrjabin1959) considered the Strigeini and Cotylurini to be subfamilies and separated Pseudapatemon Dubois, 1936 into a new subfamiliy, Pseudapatemoninae Sudarikov, 1959, based on its massive ‘cork plug-like’ holdfast organ. Dubois (Reference Dubois1968) considered the Strigeini and Cotylurini to be tribes within the subfamily Strigeinae and rejected the Pseudapatemoninae. In turn, Sudarikov (Reference Sudarikov1984) disagreed with Dubois (Reference Dubois1968) and maintained the Pseudapatemoninae. Dubois (Reference Dubois1968) separated his tribes based on the presence of vitellarium in prosoma and opisthosoma (Strigeini) or vitellarium entirely or tending to be confined to opisthosoma (Cotylurini). In Keys to the Trematoda, Niewiadomska (Reference Niewiadomska, Gibson, Jones and Bray2002f) did not include the tribes into her key, but the first step of the key aligns with the separation of the former tribes.

More recently, molecular phylogenetic studies have demonstrated the Strigeidae to be clearly non-monophyletic (Achatz et al. Reference Achatz, Pulis, Junker, Binh, Snyder and Tkach2019d, Reference Achatz, Chermak, Martens, Pulis, Fecchio, Bell, Greiman, Cromwell, Brant, Kent and Tkach2021c, Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022e, Reference Achatz, Von Holten, Kipp, Fecchio, LaFond, Martens and Tkach2023b; Blasco-Costa and Locke Reference Blasco-Costa and Locke2017; Gudla et al. Reference Gudla, Orlofske, Brant, Tkach, Dubay, Holtz and Achatz2023; Hernández-Mena et al. Reference Hernández-Mena, García-Varela and Pérez-Ponce de León2017; Locke et al. Reference Locke, Van Dam, Caffara, Pinto, López-Hernández and Blanar2018; Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020). However, as subsequent studies have added a greater diversity of strigeid taxa, the family has broken up into multiple non-monophyletic clades/branches (Achatz et al. Reference Achatz, Von Holten, Kipp, Fecchio, LaFond, Martens and Tkach2023b; Achatz et al. Reference Achatz, Chacko, Prasadan and Tkach2024a; Gudla et al. Reference Gudla, Orlofske, Brant, Tkach, Dubay, Holtz and Achatz2023; Figures 1 and S1). In some analyses, the diplostomid Codonocephalus urniger and Proalarioides sp. have clustered with strigeids (Figures 1 and S1). Furthermore, there are additional questions that may require attention at the genus level, as demonstrated by the apparent non-monophyly of Apharyngostrigea and weakly supported monophyly of Strigea in our analysis.

Proposal of a new classification system

The molecular phylogenies including the phylogeny obtained in the present work (Figures 1 and S1) consistently showed a clear separation between the Cyathocotylidae and Diplostomidae + Proterodiplostomidae + Strigeidae. This has been demonstrated in several recent molecular phylogenetic studies (Achatz et al. Reference Achatz, Pulis, Junker, Binh, Snyder and Tkach2019d, Reference Achatz, Chermak, Martens, Pulis, Fecchio, Bell, Greiman, Cromwell, Brant, Kent and Tkach2021c, Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022e, Reference Achatz, Von Holten, Kipp, Fecchio, LaFond, Martens and Tkach2023b; Blasco-Costa and Locke Reference Blasco-Costa and Locke2017; Gudla et al. Reference Gudla, Orlofske, Brant, Tkach, Dubay, Holtz and Achatz2023; Hernández-Mena et al. Reference Hernández-Mena, García-Varela and Pérez-Ponce de León2017; Locke et al. Reference Locke, Van Dam, Caffara, Pinto, López-Hernández and Blanar2018; Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020), and it is not surprising as cyathocotylids possess a cirrus sac that is absent in members of the other 3 groups. There is no doubt that the Cyathocotylidae represents a distinct family. However, the highly non-monophyletic Diplostomidae and Strigeidae have not been supported in any of the larger studies. It is also clear that none of the historical classification systems of the Diplostomoidea is supported by either molecular data or morphology.

Strigeids, as commonly recognized, have a tubular, cup-shaped or bulbiform prosoma and a bilobed holdfast organ; diplostomids have a flattened, foliate, caliciform or bulbous prosoma with a sucker-like holdfast organ. However, for example, Pseudapatemon aldousi McIntosh, 1940 (a strigeid) has a concave, but not cup-shaped, prosoma. On the other hand, several diplostomids (e.g., Codonocephalus urniger, Pseudocrassiphiala tulipifera Achatz, Von Holten, Fecchio et Tkach, 2023, Uvulifer elongatus Dubois, 1988) have a cup-shaped prosoma, and numerous species have ventral concavities expressed to varying extents (e.g., Alaria Schrank, 1788, Crassiphiala, Diplostomum, Neodiplostomum Railliet, 1919, Posthodiplostomum Dubois, 1936) (see illustrations and photographs of Dubois (Reference Dubois1968, Reference Dubois1970b, Reference Dubois1985, Reference Dubois1988), Niewiadomska (Reference Niewiadomska, Gibson, Jones and Bray2002d) and Achatz et al. (Reference Achatz, Curran, Patitucci, Fecchio and Tkach2019a,Reference Achatz, Dmytrieva, Kuzmin and Tkachb, Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022e, Reference Achatz, Burkman, Fecchio, Pulis and Tkach2023a,Reference Achatz, Von Holten, Kipp, Fecchio, LaFond, Martens and Tkachb)).

The holdfast organ of strigeids is bilobed with ventral and dorsal lobes, while diplostomids and proterodiplostomids possess a sucker-like holdfast organ (Nieiwadomska Reference Niewiadomska, Gibson, Jones and Bray2002d–Reference Niewiadomska, Gibson, Jones and Brayf; Figure 2). However, the holdfast organ of Co. urniger (a diplostomid) is weakly developed and appears almost bilobed. The holdfast organ of Pseudapatemon spp. (strigeids) is described as ‘massive’ (Dubois Reference Dubois1970b; Niewiadomska Reference Niewiadomska, Gibson, Jones and Bray2002d) or ‘plug like’ (Dubois Reference Dubois1970b). Diplostomids of the genera Allodiplostomum Yamaguti, 1935, Parallelorchis Harkema et Miller, 1961 and Pseudoscolopacitrema Palmieri, Krishnasamy et Sullivan, 1979 have holdfast organs that are trilobed or with 2 ventrolateral projections (Harkema and Miller Reference Harkema and Miller1961; Palmieri et al. Reference Palmieri, Krishnasamy and Sullivan1979).

Figure 2. Some of the major morphological features of diplostomids. (a) Alaria marcianae, anterior part of prosoma with horn-like pseudosuckers, ventral view (after Young et al. Reference Young, Orlofske, Jadin, Tkach, Greiman, Locke, Fernandes, Brant, Bates, Michalski and Achatzin press); (b) Alaria trashpandae, anterior part of prosoma with invaginated pseudosuckers, ventral view (after Young et al. Reference Young, Orlofske, Jadin, Tkach, Greiman, Locke, Fernandes, Brant, Bates, Michalski and Achatzin press); (c) Schwartzitrema sp., anterior part of prosoma with pseudosuckers with auricular expansions, ventral view; (d) Neodiplostomum nephrocystis, anterior part of prosoma with apical organ, ventral view (after Achatz et al. in press); (e) Herpetodiplostomum vogti, sucker-like holdfast organ, ventral view (after Achatz et al. Reference Achatz, Brito, Fecchio and Tkach2021b); (f) Cotylurus cornutus, bilobed holdfast organ, lateral view (after Dubois Reference Dubois1968); (g) Nematostrigea annulata, bilobed holdfast organ with thin, crimped lobes, ventral view (after Gudla et al. Reference Gudla, Orlofske, Brant, Tkach, Dubay, Holtz and Achatz2023); (h) Allodiplostomum scolopacis, holdfast organ with 3 massive lobes (from Dubois Reference Dubois1970b); (i, j) Posthodiplostomum dawnsherryae, posterior part of opisthosoma with genital cone and preputial fold in genital atrium (i) and everted (j), ventral view (after Achatz et al. Reference Achatz, Von Holten, Pritchard and Keel2025); (k, l) Pseudocrassiphiala tulipifera, posterior part of opisthosoma with genital cone and preputial fold in genital atrium, ventral (k) and lateral (l) views (after Achatz et al. Reference Achatz, Von Holten, Kipp, Fecchio, LaFond, Martens and Tkach2023b); (m) Bolbophorus indianus, posterior part of opisthosoma with genital cone and bulb, lateral view (after Dubois Reference Dubois1970b); (n) Neofibricola smiti, genital atrium with atrial sphincter (after Achatz et al. Reference Achatz, Martens, Kudlai, Junker, Boe and Tkach2022d); (o) Australapatemon intermedius, posterior part of opisthosoma with well-developed genital cone with internal rugae, lateral view (after Dubois Reference Dubois1968); (p) A. marcianae, seminal vesicle and ejaculatory duct with muscular pouch-like part, ventral view (after Young et al. Reference Young, Orlofske, Jadin, Tkach, Greiman, Locke, Fernandes, Brant, Bates, Michalski and Achatzin press); (q) Crassiphiala jeffreybelli, seminal vesicle with dilated part, ventral view (after Achatz et al. Reference Achatz, Von Holten, Kipp, Fecchio, LaFond, Martens and Tkach2023b); (r) Dungalabatrema kostadinovae, posterior part of opisthosoma with pouch surrounding paraprostate, lateral view (after Achatz et al. Reference Achatz, Chermak, Junker and Tkach2022a); (s) Pseudocrocodilicola americaniense, posterior part of opisthosoma with muscular pouch surrounding hermaphroditic duct, ventral view (after Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020); (t) Sphincterodiplostomum joaopinhoi, posterior part of opisthosoma with muscular sphincter and genital pore, dorsal view (after Achatz et al. Reference Achatz, Bell, Melo, Fecchio and Tkach2021a); (u) Polycotyle ornata, posterior part of opisthosoma with sucker-like structures on body wall, lateral view (after Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020); (v) Proterodiplostomum longum, posterior part of opisthosoma with well-developed sucker-like structure in genital atrium, lateral view (after Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020); (w) Afroproterodiplostomum ingwenyae, posterior part of opisthosoma with massive genital cone with sucker-like structure, lateral view (after Achatz et al. Reference Achatz, Chermak, Junker and Tkach2022a). Abbreviations for structures: AO, apical organ; AS, atrial sphincter; D, dilated part of seminal vesicle; E, egg; EP, pouch like part of ejaculatory duct; GA, genital atrium; GB, genital bulb; GC, genital cone; GP, genital pore; HD, hermaphroditic duct; H, holdfast organ; P, paraprostate; Po, pouch; PF, preputial fold; PG, proteolytic gland; Ps, pseudosucker; R, genital cone with rugae; SV, seminal vesicle; S, sphincter; SS, sucker-like structure; PT, posterior testis; U, uterus; VS, ventral sucker.

Bolbocephalodes intestiniforax (Bolbocephalodidae) adds further complexity. The prosoma of this species is bulbous with cup-shaped thickening at its base, which is not all too different as seen in the numerous diplostomids with a concave, but not cup-shaped, prosoma. The holdfast organ of Bolbocephalodes intestiniforax consists of ‘two transverse lips’ (Niewiadomska Reference Niewiadomska, Gibson, Jones and Bray2002a), which clearly appears to be a normal bilobed holdfast similar to that in most strigeids. Thus, Bolbocephalodes intestiniforax anatomy is consistent with both strigeids and diplostomids. The paraprostate organ of proterodiplostomids is not even consistently present in all members of the group; of known species, Mesodiplostomum gladiolum lacks this structure, while molecular phylogeny unequivocally supports its position among other proterodiplostomids (Figure 1; Achatz et al. Reference Achatz, Chermak, Junker and Tkach2022a; Tkach et al. Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020).

Host association-based differentiation was historically used by some authors to separate diplostomoidean families and genera (see Dubois (Reference Dubois1936a,Reference Duboisb, Reference Dubois1968, Reference Dubois1970a,Reference Duboisb) and Niewiadomska (Reference Niewiadomska, Gibson, Jones and Bray2002c–Reference Niewiadomska, Gibson, Jones and Brayg)). However, molecular phylogenetic studies have consistently rejected these classification systems. For instance, Achatz et al. (Reference Achatz, Pulis, Woodyard, Rosser, Martens, Weinstein, Fecchio, McAllister, Bonilla and Tkach2022e) demonstrated that Neodiplostomum (historically viewed as parasites of birds with a single exception of a species parasitic in bat host) and Fibricola Dubois, 1932 (historically viewed as parasites of mammals) represented a single genus, Neodiplostomum. In the case of reptiles, Dubois (Reference Dubois1936a) placed all diplostomids described from reptiles (primarily crocodilians) into the Proterodiplostomidae. Achatz et al. (Reference Achatz, Martens, Kudlai, Junker, Boe and Tkach2022d) erected the genus Neofibricola, a diplostomid, from crocodile. Their molecular phylogenetic analysis, along with the present study, clearly placed that genus in the phylogeny separately from proterodiplostomids. The former Didelphodiplostomum Dubois, 1944, a parasite of mammals, was revealed to be synonymous with Tylodelphys (Achatz et al. Reference Achatz, Chermak, Martens, Woodyard, Rosser, Pulis, Weinstein, McAllister, Kinsella and Tkach2022b). Obviously, host associations do not withstand criticism as the basis for the classification system of diplostomoideans.

As seen in several recent studies, our molecular phylogeny clearly demonstrates the extensive non-monophyly of the Diplostomidae and Strigeidae (Figure 1; see discussion above). The Proterodiplostomidae was nested as a part of the polytomy consisting of many clades of diplostomids and strigeids. Unfortunately, no DNA sequences of Bolbocephalodes intestiniforax (the only member of the Bolbocephalodidae) are currently available. The fact that the major features used to discern between these families are not consistent among their members, coupled with the extensive non-monophyly of families, clearly suggests that the current classification system is not supported and renders it unstable as exemplified by the taxonomic history of family-group diplostomoidean level taxa above. This leaves two possibilities: (i) each independent lineage/branch has to be considered a separate family that will require the erection of more than 10 new families, or (ii) the Proterodiplostomidae and Strigeidae should be synonymized with the Diplostomidae. This will resolve the non-monophyly, finally provide stability for the classification system of the Diplostomoidea at the family level, and allow focus on the enhancement of the classification system of the group at the level of genera. Even if we decide to establish multiple new families, a number of monophyletic groups do not possess morphological or biological characteristics that may clearly separate them from all other monophyletic groups. Therefore, we strongly favor the latter solution and synonymize the Strigeidae, Proterodiplostomidae, and Bolbocephalodidae (based on morphology) with the Diplostomidae. Upon synonymization, the Diplostomidae includes 77 genera. This action leaves only the families Cyathocotylidae and Diplostomidae within the Diplostomoidea. We provide amended diagnoses of the Diplostomidae and Cyathocotylidae below.

Family Diplostomidae Poirier, 1886

Diagnosis: Body composed of prosoma and opisthosoma, separation of body parts may be indistinct. Prosoma flattened, concave or cup-shaped, bulbous, caliciform, foliate, or spatulate; opisthosoma claviform, coniform, cylindrical, or subovate, rarely with thick-walled capsule or series of suckers. Prosoma with or without pseudosuckers. Oral sucker typically present. Ventral sucker present or absent. Holdfast organ variable in size and shape, sucker-like or bilobed, rarely trilobed, with or without papillae, compact gland at base present or absent. Compact proteolytic gland often present, near prosoma-opisthosoma junction. Pharynx typically present. Esophagus short, ceca usually reaching close to posterior end of body. Testes two, variable in shape and arrangement, typically in opisthosoma. Ovary pretesticular or opposite to anterior testis. Seminal vesicle post-testicular; paraprostate present or absent. Cirrus-sac and cirrus absent. Genital cone or bulb often present, variable in level of development. Genital atrium often well developed, muscular, rarely with sucker or sucker-like structures. Genital pore terminal or dorso-subterminal. Vitellarium follicular, variable in extent. Uterus typically in opisthosoma, sometimes extends into prosoma. Parasites of reptiles, birds, and mammals. Afrotropics, Australasia, Indomalaya, Nearctic, Neotropics, and Palearctic. Type-genus Diplostomum von Nordmann, 1832.

Cyathocotylidae Mühling, 1896

Diagnosis: Body composed of unipartite or prosoma and opisthosoma, oval, cordiform, linguiform, or pyriform. Oral and ventral suckers typically present; pseudosuckers absent. Holdfast organ round or oval, may be massive. Pharynx typically present. Esophagus short; ceca usually reaching close to posterior end of body. Position and shape of ovary and testes variable. Cirrus-sac present, occasionally rudimentary, enclosing seminal vesicle and pars prostatica. Cirrus typically present. Genital pore terminal or dorso-subterminal. Vitellarium follicular, variable in extent. Uterus variable in extent. Parasites of fishes, reptiles, birds, and mammals. Afrotropics, Australasia, Indomalaya, Nearctic, Neotropics, and Palearctic. Type-genus Cyathocotyle Mühling, 1896.

Key to diplostomoidean genera

We provide a new key to genera of the Diplostomoidea to follow the new classification system. The removal of the inconsistent features separating the former family-group taxa, including host associations, allows this key to be more suitable for separation among genera. The key provided is based, in part, on the keys of Niewiadomska (Reference Niewiadomska, Gibson, Jones and Bray2002a–Reference Niewiadomska, Gibson, Jones and Brayg), Tkach et al. (Reference Tkach, Achatz, Pulis, Junker, Snyder, Bell, Halajian and Melo2020), and Achatz et al. (Reference Achatz, Chermak, Junker and Tkach2022a,Reference Achatz, Martens, Kudlai, Junker, Boe and Tkachd). The terminology for the structures aligns with the definitions provided in Achatz et al. (Reference Achatz, Martens, Kudlai, Junker, Boe and Tkach2022d). Some of the structures found at the anterior and posterior ends of diplostomids as well as various modifications of the terminal genitalia organization are illustrated in Figure 2.