Molecular phylogeny supports invalidation of Didelphodiplostomum and Pharyngostomoides (Digenea: Diplostomidae) and reveals a Tylodelphys from mammals 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 Zoological Journal of the Linnean Society 2022 2022-09-01 196 1 124 136 7NNFD Schrank 1788 [507,590,1690,1714] Trematoda Diplostomidae Alaria Animalia Diplostomida 8 132 Platyhelminthes genus  The members of  Alariain the two phylogenies based on 28S had only slight differences in topology ( Figs 1, 2). At the same time, the phylogenies of 28S and cox1limited to members of  Alariashowed more pronounced differences in branch topology ( Figs 2, 3).  Alaria mustelaewas positioned as a sister taxon to the other  Alariaspp.in the second 28S analysis ( Fig. 2), while in the cox1phylogeny,  A. ovalisand  A. procyonisformed an unsupported clade that was placed as a sister group to the other members of  Alaria( Fig. 3). The positions of  A. alata+  Alariasp. 1and  A. marcianae+  Alariasp. 3varied 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  Alariaspp.in the present study, except for  A. alata, were collected from North America. The nested phylogenetic position of  A. alataclearly suggests a geographic expansion from the Nearctic into the Palaearctic ( Figs 1–3). It is difficult to address questions related to host switching of  Alariaspp., 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  Alariaspp.identifications in previous reports is questionable considering that most publications lack DNA sequence data and many  Alariaspp.are morphologically similar. Some  Alariaspp., 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  Alariaspp.( 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  Alariasp. 3from the cougar  Puma concolor(Linnaeus, 1758)is immature; hence, additional collection of well-fixed, mature specimens of  Alariasp. 3is 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 cox1sequences of our specimens are only 1.9–2.6% different from material identified as  Alaria americana Hall & Wigdor, 1918by 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. americanadescribed by Hall & Wigdor (1918). For instance,  A. americanawas originally described with vitellarium that does not extend anteriorly beyond the level of the ventral sucker. The vitellarium of  A. americanafrom 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. americanaby Locke et al.(2018) are likely misidentified specimens of  A. arisaemoides.