The superfamily Diplostomoidea Poirier, 1886 is a large, globally distributed group of digeneans characterized by the presence of a unique holdfast organ and parasitic in most major groups of vertebrates (birds, mammals, reptiles, fishes) as definitive hosts. A number of diplostomoideans are associated with diseases in their intermediate and, more rarely, definitive hosts. Prior to this work and upon the recent synonymization of the Brauninidae Wolf, 1903, the Diplostomoidea included 5 families: Bolbocephalodidae Strand, 1935; Cyathocotylidae Mühling, 1896; Diplostomidae Poirier, 1886; Proterodiplostomidae Dubois, 1936; and Strigeidae Railliet, 1919. The separation of these families was based primarily on the structure and shape of prosoma and holdfast organ as well as the presence/absence of cirrus sac and paraprostate. More rarely, distinguishing among families was based on life cycles and types of larval stages, excretory system or even host specificity. However, due to the inconsistent nature of most of morphological and biological characters across the Diplostomoidea and nearly universal lack of agreement on their relative value, the systematic history of the group has been extremely tumultuous, and none of many classification systems proposed over the last 140 years has become broadly accepted or supported by phylogenetic analyses. Extensive molecular phylogenetic studies of the Diplostomoidea in the last 15 years helped to partly improve the classification system and resolve multiple taxonomic questions. Notably, practically all molecular phylogenies have clearly demonstrated non-monophyly of the two largest families, the Diplostomidae and the Strigeidae and indicated it as systematic problem. We provide a brief overview of the history and current state of knowledge of diplostomoidean systematics and re-evaluate the classification system of the Diplostomoidea based on morphological and molecular evidence. We propose changes in the classification system that reconciles the traditional morphological and life cycle data with molecular phylogenies. The major element of the proposed classification system is the synonymization of the families Proterodiplostomidae and Strigeidae with the Diplostomidae as the only feasible way to resolve the problem of consistent non-monophyly of the latter two families and provide stability to the classification system.

The description and delineation of trematode species is a major ongoing task. Across the field there has been, and currently still is, great variation in the standard of this work and in the sophistication of the proposal of taxonomic hypotheses. Although most species are relatively unambiguously distinct from their congeners, many are either morphologically very similar, including the major and rapidly growing component of cryptic species, or are highly variable morphologically despite little to no molecular variation for standard DNA markers. Here we review challenges in species delineation in the context provided to us by the historical literature, and the use of morphological, geographical, host, and molecular data. We observe that there are potential challenges associated with all these information sources. As a result, we encourage careful proposal of taxonomic hypotheses with consideration for underlying species concepts and frank acknowledgement of weaknesses or conflict in the data. It seems clear that there is no single source of data that provides a wholly reliable answer to our taxonomic challenges but that nuanced consideration of information from multiple sources (the ‘integrated approach’) provides the best possibility of developing hypotheses that will stand the test of time.

Kingfishers (Alcedinidae Rafinesque) are common inhabitants of wetlands and are known to be definitive hosts to a wide range of digeneans that parasitize fish as second intermediate hosts. Among these digeneans, members of the Diplostomidae Poirier, 1886 (diplostomids) are particularly common. Recent studies of diplostomids collected from kingfishers have revealed that they are probably more diverse than currently known. This particularly concerns the genera Crassiphiala Van Haitsma, 1925 and Uvulifer Yamaguti, 1934. In the present work, we studied seven diplostomid taxa from kingfishers in Brazil, the USA and the Philippines. Partial DNA sequences of the nuclear large ribosomal subunit (28S) and mitochondrial cytochrome c oxidase I (cox1) genes were obtained, and 28S sequences were used to study the phylogenetic interrelationships of these diplostomids. We provide the first DNA sequences from Uvulifer semicircumcisus Dubois et Rausch, 1950 and a member of Subuvulifer Dubois, 1952. Pseudocrassiphiala n. gen. is erected for a previously recognized species-level lineage of Crassiphiala and a new generic diagnosis of Crassiphiala is provided. Crassiphiala jeffreybelli n. sp., Crassiphiala wecksteini n. sp. and Pseudocrassiphiala tulipifera n. sp. are described, and a description of newly collected, high-quality specimens of Crassiphiala bulboglossa Van Haitsma, 1925 (the type-species of the genus) is provided.

Sphincterodiplostomum is a monotypic genus of diplostomid digeneans that parasitize fish-eating birds in the neotropics. The type species Sphincterodiplostomum musculosum has a unique, dorsal, tubular invagination in the opisthosoma with a muscular sphincter. Whereas larvae of S. musculosum are relatively commonly reported in Neotropical fish helminth surveys, adult specimens from birds are rarely collected. Prior to our study, no DNA sequence data for S. musculosum were available. Our molecular and morphological study of mature and immature adult Sphincterodiplostomum specimens from three species of birds and one species of crocodilian revealed the presence of at least two species of Sphincterodiplostomum in the neotropics. We provide the first molecular phylogeny of the Diplostomoidea that includes Sphincterodiplostomum. In addition, this is the first record of S. musculosum from caimans, along with the first record of fully mature adult S. musculosum from green kingfisher Chloroceryle americana. The new species of Sphincterodiplostomum (Sphincterodiplostomum joaopinhoi n. sp.) can be morphologically distinguished from S. musculosum based on the anterior extent of vitelline follicles, narrower prosoma, substantially smaller holdfast organ and structure of tegumental spines. Our data revealed 0.7% interspecific divergence in 28S and 10.6–11.7% divergence in cox1 sequences between the two Sphincterodiplostomum species.

Digenetic trematodes of the genus Clinostomum are cosmopolitan parasites infecting fishes, amphibians, reptiles, and snails as intermediate hosts. Despite the broad geographical distribution of this genus, debate about the number of species and how they vary in host use has persisted. To better understand patterns of infection among host species and across life stages, we used large-scale field surveys and molecular tools to examine five species of amphibians and seven species of fishes from 125 California ponds. Among the 12,360 examined hosts, infection was rare, with an overall prevalence of 1.7% in amphibians and 9.2% in fishes. Molecular evidence indicated that both groups were infected with Clinostomum marginatum. Using generalized linear mixed effects models, host species identity and host life stage had a strong influence on infection status, such that Lepomis cyanellus (green sunfish) (49.3%) and Taricha granulosa (rough skinned newt) (9.2%) supported the highest overall prevalence values, whereas adult amphibians tended to have a higher prevalence of infection relative to juveniles (13.3% and 2.5%, respectively). Experimentally, we tested the susceptibility of two amphibian hosts (Pseudacris regilla [Pacific chorus frog] and Anaxyrus boreas [western toad]) to varying levels of cercariae exposure and measured metacercariae growth over time. Pseudacris regilla was 1.3× more susceptible to infection, while infection success increased with cercariae exposure dose for both species. On average, metacarcariae size increased by 650% over 20 days. Our study highlights the importance of integrating field surveys, genetic tools, and experimental approaches to better understand the ecology of host–parasite interactions.