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REMARKS ON ALARIA The members of Alaria in the two phylogenies based on 28S had only slight differences in topology (Figs 1, 2). At the same time, the phylogenies of 28S and cox1 limited to members of Alaria showed more pronounced differences in branch topology (Figs 2, 3). Alaria mustelae was positioned as a sister taxon to the other Alaria spp. in the second 28S analysis (Fig. 2), while in the cox1 phylogeny, A. ovalis and A. procyonis formed an unsupported clade that was placed as a sister group to the other members of Alaria (Fig. 3). The positions of A. alata + Alaria sp. 1 and A. marcianae + Alaria sp. 3 varied between the two analyses as well (Figs 2, 3). Discordance between phylogenies based on ribosomal and mitochondrial data has been well documented among other diplostomoideans (e.g. Brabec et al., 2015; Heneberg et al., 2020; Hoogendoorn et al., 2020; Achatz et al., In press). Faster mutating genes, such as cox1, are more reliable for distinguishing between closely related diplostomoidean species/species-level lineages (Table 2; Supporting Information, Table S1), but slower mutating genes, such as 28S, remain more suitable for phylogenetic inference at taxonomic levels above genus. All Alaria spp. in the present study, except for A. alata, were collected from North America. The nested phylogenetic position of A. alata clearly suggests a geographic expansion from the Nearctic into the Palaearctic (Figs 1–3). It is difficult to address questions related to host switching of Alaria spp., considering that many species have been historically reported in a diversity of mammalian hosts (e.g. see Dubois, 1968 and references therein). The accuracy of Alaria spp. identifications in previous reports is questionable considering that most publications lack DNA sequence data and many Alaria spp. are morphologically similar. Some Alaria spp., such as A. arisaemoides, are also known to have substantial morphological variation (e.g. Hall & Wigdor, 1918; Dubois, 1968). The topology of our molecular phylogeny based on the 28S of Alaria spp. (Fig. 2) is not well enough supported to confidently infer evolutionary patterns of definitive host associations; the discordance between topologies of 28S (Fig. 2) and cox1 (Fig. 3) further complicates the situation. Our specimen of Alaria sp. 3 from the cougar Puma concolor (Linnaeus, 1758) is immature; hence, additional collection of well-fixed, mature specimens of Alaria sp. 3 is crucial for accurate species identification and confirmation of its definitive host. It is worth noting that our specimens of A. arisaemoides (Fig. 4B) conform closely to the original description of the species and subsequent descriptions of the species (e.g. Augustine & Uribe, 1927; Dubois, 1968). However, the cox1 sequences of our specimens are only 1.9–2.6% different from material identified as Alaria americana Hall & Wigdor, 1918 by Locke et al. (2018) (Supporting Information, Table S1). The material described by Locke et al. (2018) is somewhat different to the original description of A. americana described by Hall & Wigdor (1918). For instance, A. americana was originally described with vitellarium that does not extend anteriorly beyond the level of the ventral sucker. The vitellarium of A. americana from Locke et al. (2018) extends anteriorly to the level of the ventral sucker, similar to the condition in A. arisaemoides. In our opinion, the specimens identified as A. americana by Locke et al. (2018) are likely misidentified specimens of A. arisaemoides., Published as part of Achatz, Tyler J, Chermak, Taylor P, Martens, Jakson R, Woodyard, Ethan T, Rosser, Thomas G, Pulis, Eric E, Weinstein, Sara B, Mcallister, Chris T, Kinsella, John M & Tkach, Vasyl V, 2022, Molecular phylogeny supports invalidation of Didelphodiplostomum and Pharyngostomoides (Digenea: Diplostomidae) and reveals a Tylodelphys from mammals, pp. 124-136 in Zoological Journal of the Linnean Society 196 (1) on page 132, DOI: 10.1093/zoolinnean/zlab114, http://zenodo.org/record/7031477, {"references":["Brabec J, Kostadinova A, Scholz T, Littewood TJ. 2015. Complete mitochondrial genomes and nuclear ribosomal RNA operons of two species of Diplostomum (Platyhelminthes: Trematoda): a molecular resource for taxonomy and molecular epidemiology of important fish pathogens. Parasites & Vectors 8: 336.","Heneberg P, Sitko J, Tesinsky M. 2020. Paraphyly of Conodiplostomum Dubois, 1937. Parasitology International 76: 102033.","Hoogendoorn C, Smit NJ, Kudlai O. 2020. Resolution of the identity of three species of Diplostomum (Digenea: Diplostomidae) parasitizing freshwater fishes in South Africa, combining molecular and morphological evidence. International Journal for Parasitology. Parasites and Wildlife 11: 50 - 61.","Hall MC, Wigdor M. 1918. Two new flukes from the dog. Annual Report of the Michigan Academy of Science 20: 139.","Augustine DL, Uribe C. 1927. Alaria arisaemoides n. sp., a trematode from Vulpes fulva. Parasitology 19: 236 - 244."]}

STATUS OF PHARYNGOSTOMOIDES The morphological characteristics of Pharyngostomoides spp. in our material conform to the original descriptions of Ph. procyonis and Ph. ovalis (Fig. 4E, F). Beckerdite et al. (1971) considered Ph. ovalis to be a junior synonym of Ph. procyonis. In addition, Beckerdite et al. (1971) redescribed Ph. procyonis and provided an illustration that appears remarkably similar to Ph. ovalis. Our material of Ph. procyonis and Ph. ovalis differ by 0.4% and 10% in partial sequences of 28S and cox1, respectively (Table 2; Supporting Information, Table S1). The morphology of Ph. procyonis and Ph. ovalis most obviously differs in general body shape (spatulate in Ph. procyonis vs. oval in Ph. ovalis), shape of prosoma (anterior end rounded in Ph. procyonis vs. anterior end square shaped in Ph. ovalis), relative sucker sizes (oral sucker similar in size or smaller than ventral sucker in Ph. procyonis vs. oral sucker usually larger than ventral sucker in Ph. ovalis) and egg size (egg length 82–93 µm in Ph. procyonis vs. egg length 100–115 µm in Ph. ovalis). Considering the genetic and morphological differences listed above, we restore Ph. ovalis. Pharyngostomoides spp. are readily distinguished from Alaria spp. based on the position of the testes (opposite in Pharyngostomoides vs. tandem in Alaria) (Niewiadomska, 2002; Fig. 4E, F vs. Fig. 4 B-D, G, H). However, our molecular phylogeny based on 28S (Fig. 1) positioned Ph. procyonis (type species) and Ph. ovalis among Alaria spp., including the type species A. alata., Published as part of Achatz, Tyler J, Chermak, Taylor P, Martens, Jakson R, Woodyard, Ethan T, Rosser, Thomas G, Pulis, Eric E, Weinstein, Sara B, Mcallister, Chris T, Kinsella, John M & Tkach, Vasyl V, 2022, Molecular phylogeny supports invalidation of Didelphodiplostomum and Pharyngostomoides (Digenea: Diplostomidae) and reveals a Tylodelphys from mammals, pp. 124-136 in Zoological Journal of the Linnean Society 196 (1) on page 129, DOI: 10.1093/zoolinnean/zlab114, http://zenodo.org/record/7031477, {"references":["Beckerdite FW, Miller GC, Harkema R. 1971. Observations of the life cycle of Pharyngostomodies spp. and the description of P. adenocephala sp. n. (Strigeoidea: Diplostomatidae) from the raccoon, Procyon lotor (L.). Helminthological Society of Washington 38: 149 - 156.","Niewiadomska K. 2002. Family Diplostomidae Poirier, 1886. In: Gibson DI, Jones A, Bray RA, eds. Keys to the Trematoda, Vol. 1. London: CAB International and the Natural History Museum, 167 - 196."]}

Achatz, Tyler J, Chermak, Taylor P, Martens, Jakson R, Woodyard, Ethan T, Rosser, Thomas G, Pulis, Eric E, Weinstein, Sara B, Mcallister, Chris T, Kinsella, John M, Tkach, Vasyl V (2022): Molecular phylogeny supports invalidation of Didelphodiplostomum and Pharyngostomoides (Digenea: Diplostomidae) and reveals a Tylodelphys from mammals. Zoological Journal of the Linnean Society 196 (1): 124-136, DOI: 10.1093/zoolinnean/zlab114, URL: https://academic.oup.com/zoolinnean/article/196/1/124/6517659

STATUS OF DIDELPHODIPLOSTOMUM The analysis of 28S (Fig. 1) places Did. variabile (shown as Tylodelphis variabilis comb. nov. in the figure) in the cluster of Tylodelphys and Austrodiplostomum species. The morphology of adult Didelphodiplostomum and Tylodelphys spp. is remarkably similar (Fig. 4A; Dubois, 1968). Furthermore, Didelphodiplostomum and Tylodelphys have identical flame-cell formulas, 2 [(2+2)+(2+[2])] = 16 (Harris et al., 1967; Dubois, 1968, 1970; Niewiadomska, 2002). Dubois (1968) emphasized the remarkable morphological similarity between Didelphodiplostomum and Tylodelphys species. However, the members of the two genera differ in the shape of anterior testis (asymmetrical in Didelphodiplostomum spp. vs. symmetrical in Tylodelphys spp.) and the lack of a genital cone in Didelphodiplostomum spp. (present in Tylodelphys spp., albeit weakly developed in some species). Our molecular phylogeny (Fig. 1) clearly demonstrates that Did. variabile belongs to one of the two major clades of Tylodelphys. Taking into account the results of our phylogenetic analysis (Fig. 1) and minor morphological differences between Didelphodiplostomum and Tylodelphys, we consider Didelphodiplostomum to be a junior synonym of Tylodelphys. As such, we transfer Did. variabile and Did. nunezae into Tylodelphys as T. variabilis (Chandler, 1932) comb. nov. and Tylodelphys nunezae (Dubois, 1976) comb. nov., respectively. The partial 28S and cox1 sequences of T. variabilis and Tylodelphys sp. VVT1 of Achatz et al. (In press) are identical. It is clear that the larval specimens of Tylodelphys sp. VVT1 from the mole salamander Ambystoma talpoideum Holbrook, 1838 collected in Mississippi are conspecific with T. variabilis. An amended diagnosis of Tylodelphys is provided below., Published as part of Achatz, Tyler J, Chermak, Taylor P, Martens, Jakson R, Woodyard, Ethan T, Rosser, Thomas G, Pulis, Eric E, Weinstein, Sara B, Mcallister, Chris T, Kinsella, John M & Tkach, Vasyl V, 2022, Molecular phylogeny supports invalidation of Didelphodiplostomum and Pharyngostomoides (Digenea: Diplostomidae) and reveals a Tylodelphys from mammals, pp. 124-136 in Zoological Journal of the Linnean Society 196 (1) on page 133, DOI: 10.1093/zoolinnean/zlab114, http://zenodo.org/record/7031477, {"references":["Harris A, Harkema R, Miller G. 1967. Life history and taxonomy of Diplostomum variabile (Chandler, 1932) (Trematoda: Diplostomatidae). Journal of Parasitology 53: 577 - 583.","Niewiadomska K. 2002. Family Diplostomidae Poirier, 1886. In: Gibson DI, Jones A, Bray RA, eds. Keys to the Trematoda, Vol. 1. London: CAB International and the Natural History Museum, 167 - 196.","Chandler AC. 1932. Notes on the helminth parasites of the opossum (Didelphis virginiana) in southeast Texas, with descriptions of four new species. Proceedings of the United States National Museum 81: 1 - 15.","Dubois G. 1976. Description de l'adulte presume de Bursotrema tetracotyloides Szidat, 1960, et d'une nouvelle espece du genre Didelphodiplostomum Dubois, 1944, parasites de Didelphis azarae Temminck, 1825 (Trematoda: Alariinae). Annales de Parasitologie 51: 341 - 347."]}

REMARKS ON TYLODELPHYS Based on our analysis, Tylodelphys spp. belong to at least three distinct clades (Fig. 1). Achatz et al. (In press) recently suggested that Tylodelphys americana (Dubois, 1936) (see Dubois, 1936b) and Tylodelphys sp. 4 may need to be placed in a novel genus. However, the inclusion of the DNA sequence of T. excavata in the present analysis has further complicated the situation. It is possible that Tylodelphys as currently recognized may represent a complex of genera and requires the establishment of at least two new genera. DNA sequences from adult specimens of T. clavata (von Nordmann, 1832) are necessary for a conclusive decision regarding the status of Tylodelphys. The majority of Tylodelphys spp. and members of the closely related Austrodiplostomum and Diplostomum are known to primarily parasitize piscivorous birds (Achatz et al., In press). Achatz et al. (In press) recently revealed the presence of two Diplostomum spp. parasitizing North American river otters Lontra canadensis (Schreber, 1777) in the USA. Based on the results of the present study, T. variabilis represents the first species of Tylodelphys that secondarily switched from avian to mammalian definitive hosts. Transitions between birds and mammals may happen when hosts occur in the same environments and have overlapping diets; similar to many aquatic birds, otters and raccoons feed on fishes and amphibians., Published as part of Achatz, Tyler J, Chermak, Taylor P, Martens, Jakson R, Woodyard, Ethan T, Rosser, Thomas G, Pulis, Eric E, Weinstein, Sara B, Mcallister, Chris T, Kinsella, John M & Tkach, Vasyl V, 2022, Molecular phylogeny supports invalidation of Didelphodiplostomum and Pharyngostomoides (Digenea: Diplostomidae) and reveals a Tylodelphys from mammals, pp. 124-136 in Zoological Journal of the Linnean Society 196 (1) on page 133, DOI: 10.1093/zoolinnean/zlab114, http://zenodo.org/record/7031477, {"references":["Dubois G. 1936 b. Nouveaux principes de classification des trematodes du groupe des Strigeida (notes preliminaires). Revue Suisse de Zoologie 43: 507 - 515.","von Nordmann A. 1832. Mikrographische Beitrage zur Naturgeschichte der wirbellosen Thiere. Berlin: G Reimer."]}