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Competing Interests: Declaration of interests The authors declare no competing interests.

Proalarioides Yamaguti, 1933 (Digenea Carus, 1863: Diplostomoidea Poirier, 1886) is a small genus of proterodiplostomids parasitic in the intestines of snakes in Asia. Only two species are considered valid: Proalarioides serpentis Yamaguti, 1933 and Proalarioides tropidonotis Vidyarthi, 1937. Unlike other proterodiplostomids, Proalarioides spp. possess pseudosuckers and lack the paraprostate, otherwise extremely characteristic of the Proterodiplostomidae Dubois, 1936. In the present study, we describe the morphology of progenetic metacercariae of a Proalarioides sp. from bicolored frog, Clinotarsus curtipes (Jerdon), collected in India and provide the first DNA sequences from any member of the genus. These specimens differ from previously described metacercariae and adults of P. serpentis and P. tropidonotis in several ways, including body and organ sizes, sucker ratios, and distribution of vitellarium. The newly generated partial large ribosomal subunit (28S) rRNA gene sequence was used to test the phylogenetic position of the genus among other major lineages of diplostomoideans. Our 28S phylogeny clearly demonstrated Proalarioides sp. to be well-separated from other members of the Proterodiplostomidae. Based on morphological and molecular evidence, we transfer Proalarioides out of the Proterodiplostomidae into the Diplostomidae Poirier, 1886., Competing Interests: Declaration of competing interest All authors disclose that there was no conflict of interest of any type from the beginning of the work to the submission of manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Cyathocotylidae Mühling, 1896 is a small but broadly distributed family of digeneans parasitic in a wide range of vertebrate definitive hosts, from fish to mammals. Despite existing taxonomic questions, only a few studies have generated DNA sequence data from cyathocotylids, and only a single publication included sequence data from a cyathocotylid parasitic in snakes. Four genera are known to infect snakes: GogateaLutz, 1935, Szidatia Dubois, 1938, MesostephanoidesDubois, 1951, and SerpentostephanusSudarikov, 1961. Members of these genera were known from only Asia and Africa. In the present study, we describe 2 new species of Gogatea from snakes in Australia and 1 from Vietnam. The new species from Vietnam described herein is the first member of the genus that lacks a ventral sucker. We used partial sequences of the nuclear large ribosomal subunit (28S) and mitochondrial cytochrome c oxidase subunit I (COI) genes to explore phylogenetic relationships among cyathocotylids and species differentiation. In addition, this is the first report of a cyathocotylid from snakes in Australia, thus posing interesting questions regarding the dispersal and historical biogeography of these parasites. Cyathocotylid genera from snakes have a long, convoluted taxonomic history. The genera Gogatea, Mesostephanoides, and Szidatia were distinguished from each other based on very small morphological differences. Therefore, the validity of Szidatia and Mesostephanoides was often questioned in the literature. Based on the detailed morphological analysis of our freshly collected high-quality specimens and comparison with published information, we synonymize Mesostephanoides and Szidatia with Gogatea., (© American Society of Parasitologists 2024.)

The detection of severe limb malformations in metamorphosing northern leopard frogs ( Rana pipiens ) from a Colorado pond in August 2022 prompted questions about the cause(s) and concern over the implications. Northern leopard frogs, which are a Tier 1 Species of Greatest Conservation Need in Colorado, have declined over much of their range in the state, particularly along the Front Range. Although malformations in amphibians have been reported in other parts of the USA, they are rare in Colorado, and the current case represents the most severe hotspot reported in the state for over 70 years. Across three survey events in late summer and early fall of 2022, approximately 68% of captured leopard frogs (late-stage larvae and metamorphic frogs) exhibited one or more malformations. Malformations exclusively affected the hind limbs and were dominated by skin webbings (51.7% of the total), bony triangles (32.2%), and extra limbs or digits (11%). Many animals had multiple malformations that limited the movement of one or both limbs (average of 2.3 malformations per malformed frog). Dissection of a subset of animals coupled with 28S rDNA genetic sequencing revealed the occurrence of the trematode Ribeiroia ondatrae at an average of 75.2 trematode cysts (metacercariae) per frog. The parasite was also detected in 2.6% of dissected snails ( Helisoma trivolvis ), which function as the trematode's first intermediate host. The relatively high loads of infection detected here - coupled with the similarity of observed malformations to those previously linked to R. ondatrae in experimental studies and from other malformation hotspots in the USA - offer compelling evidence that the current case is the result of parasite infection. Unresolved questions include why malformation prevalence was so high in 2022 and the degree to which such abnormalities will affect population persistence for local leopard frogs, particularly if malformations continue., Competing Interests: The authors declare no conflict of interest. All authors have approved the manuscript, and we hereby attest the manuscript is not under consideration for publication elsewhere., (© 2024 The Authors.)

Ochoterenatrema Caballero, 1943 is a genus of lecithodendriid digeneans that prior to this study included 8 species parasitic in bats in the Western Hemisphere. Species of Ochoterenatrema possess a unique morphological feature in form of the pseudogonotyl on the sinistral side of the ventral sucker. In this study, we describe 2 new species of Ochoterenatrema from bats in Ecuador. The new species are readily differentiated from their congeners by a combination of morphological characters, including the distribution of vitelline follicles, length of oesophagus, sucker ratio and the body shape, among other features. We have generated partial nuclear 28S rDNA and mitochondrial cox1 gene DNA sequences from both new species. The newly obtained sequences were used to differentiate among species and study the phylogenetic interrelationships among Ochoterenatrema spp. The internal topology of the clade was weakly supported, although the cox1 tree was much better resolved than the 28S tree. Comparison of sequences revealed 0-1.2% interspecific divergence in 28S and 3.3-20.5% interspecific divergence in cox1 among Ochoterenatrema spp. The new findings demonstrate that bats in South America likely harbor multiple additional undescribed species of Ochoterenatrema. More extensive sampling from broader geographic and host ranges, especially in North America, should allow for a better understanding of the evolution of host associations and morphological traits of this lineage of lecithodendriid digeneans., (© 2024. The Author(s).)

Digenean trematodes in the strigeid genus Nematostrigea are parasites of various birds, most often ospreys (Pandion haliaetus). Nematostrigea currently contains 2 species, Nematostrigea serpens and Nematostigea hepatica. Nematostrigea serpens is divided into 2 subspecies: N. serpens serpens from the Palearctic and Indomalayan realms and N. serpens annulata from the Nearctic realm. In the present work, we studied the type material of N. serpens annulata and collected new specimens from an osprey in Wisconsin close to the type locality. The original description and illustration of N. serpens annulata were incomplete. Herein we provide a detailed redescription and new illustrations of N. serpens annulata on the basis of the type material as well as newly collected and sequenced specimens. Nematostrigea serpens annulata has a distinct tegumental thickening near the mid-length of the genital cone, which is absent in its congeners. Partial sequences of the large ribosomal subunit (28S) DNA and cytochrome c oxidase subunit 1 (COI) mitochondrial DNA gene sequences were generated from our specimens of N. serpens annulata. Comparisons with previously published sequences of N. serpens serpens from the Palearctic demonstrated 2.2% difference in 28S and 3.7% in COI. On the basis of morphological and molecular comparisons, we elevate N. serpens annulata to species status and recognize it as Nematostrigea annulata Dubois and Rausch, 1948., (© American Society of Parasitologists 2023.)

Purpose: The Diplostomidae is a globally distributed family of digeneans that parasitize a wide variety of tetrapod definitive hosts. Recent molecular phylogenetic studies have revealed unknown diplostomid diversity in avian hosts throughout the New World. Herein, we provide descriptions of a novel genus of diplostomids with two new species., Methods: Two species of diplostomids belonging to the new genus were collected from anhinga birds in Mississippi (USA) and Brazil. Partial nuclear 28S ribosomal and mitochondrial cox1 genes were sequenced. Ribosomal data were used for phylogenetic inference., Results: Both species of Anhingatrema n. gen. were positioned in a 100% supported, monophyletic clade in the phylogenetic tree. The molecular phylogenetic position and a combination of morphological features (e.g., presence of pseudosuckers, testes shape and orientation) supported erection of the new genus. Anhingatrema overstreeti n. sp. and Anhingatrema cararai n. sp. are morphologically similar, but differ in size of and ratios associated with pseudosuckers. The two species differ by 2% of 28S sequences and 13.8% of cox1 sequences. Comparison of DNA sequences revealed that Diplostomidae gen. sp. in GenBank (MZ314151) is conspecific with An. overstreeti n. sp., Conclusion: Anhingatrema n. gen. is the sixth genus of diplostomids known from anhingas worldwide. Anhingatrema cararai n. sp. is the first diplostomid to be reported from anhingas in South America. Combined with previous studies, the molecular phylogenies revealed at least two host switches to anhingas from other birds during the evolutionary history of the Diplostomidae., (© 2022. The Author(s) under exclusive licence to Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

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 ( cox 1) 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.

The Diplostomidae Poirier, 1886 is a large family of digeneans within the superfamily Diplostomoidea Poirier, 1886. Members of the family are distributed worldwide and parasitize a diversity of tetrapod definitive hosts. Notably, only 2 mature diplostomids are known from crocodilians and both are suggested to be accidental infections. In this study, we use morphological and molecular data to describe Neofibricola n. gen. from a Nile crocodile Crocodylus niloticus collected in South Africa. We provide a description of adults and metacercariae of the type species, Neofibricola smiti n. sp., and metacercariae of a likely congeneric species. We generated partial 28S and internal transcribed spacer region ribosomal deoxyribonucleic acid (DNA) and cytochrome c oxidase 1 subunit mitochondrial DNA for both species and utilized the newly generated 28S sequences to examine phylogenetic affinities of these new taxa. In addition, we provide a new key to diplostomid genera, considering the substantial systematic changes and newly erected genera since the previously published key to diplostomid genera., (© American Society of Parasitologists 2022.)

Ochoterenatrema is a small genus of lecithodendriid digeneans that includes six species parasitic in Neotropical bats in North and South America. Members of this genus are characterized by the presence of a pseudogonotyl formed by thickened tegument on the left side of the ventral sucker. We examined morphology of specimens belonging to five species of Ochoterenatrema from Brazil, Ecuador and the USA and describe two new Ochoterenatrema spp. (Ochoterenatrema sphaerula n. sp. and Ochoterenatrema gracilis n. sp.). These new species are readily differentiated from their congeners by a combination of morphological characters, including the distribution of vitelline follicles and the body shape. We have provided the first DNA sequences for Ochoterenatrema. Newly generated partial nuclear 28S rDNA and mitochondrial cox1 gene sequences were used to differentiate among species and study the phylogenetic affinities of Ochoterenatrema spp., including one of the new species, O. gracilis n. sp., as well as Ochoterenatrema diminutum, Ochoterenatrema fraternum and Ochoterenatrema cf. labda. The phylogeny of the Microphalloidea based on 28S had well-supported topology, particularly at the family level. The Ochoterenatrema clade was strongly supported; however, the internal topology of the clade was weakly supported. Comparison of sequences revealed 0.4-1.3% interspecific divergence in 28S and 9.1-19.7% interspecific divergence in cox1 among Ochoterenatrema spp. We hypothesize that extremely diverse fauna of bats in South and Central America harbors multiple undescribed species of Ochoterenatrema. Several lecithodendriid genera from bats and other hosts are yet to be included in future molecular phylogenetic analyses to test the monophyly of this extremely diverse digenean family and analyze evolutionary affinities of its constituent taxa., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Fibricola and Neodiplostomum are diplostomid genera with very similar morphology that are currently separated based on their definitive hosts. Fibricola spp. are normally found in mammals, while Neodiplostomum spp. typically parasitize birds. Previously, no DNA sequence data was available for any member of Fibricola. We generated nuclear ribosomal and mtDNA sequences of Fibricola cratera (type-species), Fibricola lucidum and 6 species of Neodiplostomum. DNA sequences were used to examine phylogenetic interrelationships among Fibricola and Neodiplostomum and re-evaluate their systematics. Molecular phylogenies and morphological study suggest that Fibricola should be considered a junior synonym of Neodiplostomum. Therefore, we synonymize the two genera and transfer all members of Fibricola into Neodiplostomum. Specimens morphologically identified as Neodiplostomum cratera belonged to 3 distinct phylogenetic clades based on mitochondrial data. One of those clades also included sequences of specimens identified morphologically as Neodiplostomum lucidum. Further study is necessary to resolve the situation regarding the morphology of N. cratera. Our results demonstrated that some DNA sequences of N. americanum available in GenBank originate from misidentified Neodiplostomum banghami. Molecular phylogentic data revealed at least 2 independent host-switching events between avian and mammalian hosts in the evolutionary history of Neodiplostomum; however, the directionality of these host-switching events remains unclear.

Alaria , Didelphodiplostomum and Pharyngostomoides are among genera of diplostomid digeneans known to parasitize mammalian definitive hosts. Despite numerous recent molecular phylogenetic studies of diplostomids, limited DNA sequence data is available from diplostomids parasitic in mammals. Herein, we provide the first 28S rDNA and cox1 mtDNA sequences from morphologically identified, adult specimens of Didelphodiplostomum and Pharyngostomoides . Newly generated 28S sequences were used to infer the phylogenetic interrelationships of these two genera among other major lineages of diplostomoideans. The phylogeny based on 28S and a review of morphology clearly suggests that Pharyngostomoides should be considered a junior synonym of Alaria , while Didelphodiplostomum should be considered a junior synonym of Tylodelphys . Pharyngostomoides procyonis (type species), Pharyngostomoides adenocephala and Pharyngostomoides dasyuri were transferred into Alaria as Alaria procyonis comb. nov. , Alaria adenocephala comb. nov. and Alaria dasyuri comb. nov. ; Didelphodiplostomum variabile (type species) and Didelphodiplostomum nunezae were transferred into Tylodelphys as Tylodelphys variabilis comb. nov. and Tylodelphys nunezae comb. nov. In addition, Alaria ovalis comb. nov. (formerly included in Pharyngostomoides ) was restored and transferred into Alaria based on a morphological study of well-fixed, adult specimens and the comparison of cox1 DNA sequences among Alaria spp. The diplostomid genus Parallelorchis was restored based on review of morphology., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Linnean Society of London. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Diplostomidae Poirier, 1886 is a large, globally distributed family of digeneans parasitic in intestines of their definitive hosts. Diplostomum and Tylodelphys spp. are broadly distributed, commonly reported, and the most often sequenced diplostomid genera. The majority of published DNA sequences from these genera originated from larval stages only, which typically cannot be identified to the species level based on morphology alone. We generated partial large ribosomal subunit (28S) rRNA and cytochrome c oxidase subunit 1 (cox1) mtDNA gene sequences from 14 species/species-level lineages of Diplostomum, six species/species-level lineages of Tylodelphys, two species/species-level lineages of Austrodiplostomum, one species previously assigned to Paralaria, two species/species-level lineages of Dolichorchis and one unknown diplostomid. Our DNA sequences of 11 species/species-level lineages of Diplostomum (all identified to species), four species/species-level lineages of Tylodelphys (all identified to species), Austrodiplostomum compactum, Paralaria alarioides and Dolichorchis lacombeensis originated from adult specimens. 28S sequences were used for phylogenetic inference to demonstrate the position of Paralaria alarioides and Dolichorchis spp. within the Diplostomoidea and study the interrelationships of Diplostomum, Tylodelphys and Austrodiplostomum. Our results demonstrate that two diplostomids from the North American river otter (P. alarioides and a likely undescribed taxon) belong within Diplostomum. Further, our results demonstrate the non-monophyly of Tylodelphys due to the position of Austrodiplostomum spp., based on our phylogenetic analyses and morphology. Furthermore, the results of phylogenetic analysis of 28S confirmed the status of Dolichorchis as a separate genus. The phylogenies suggest multiple definitive host-switching events (birds to otters and among major avian groups) and a New World origin of Diplostomum and Tylodelphys spp. Our DNA sequences from adult digeneans revealed identities of 10 previously published lineages of Diplostomum and Tylodelphys, which were previously identified to genus only. The novel DNA data from this work provide opportunities for future comparisons of larval diplostomines collected in ecological studies., (Copyright © 2021 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The Proterodiplostomidae Dubois, 1936 is a family of digeneans within the superfamily Diplostomoidea Poirier, 1886. Members of the family are distributed mostly in the tropics and subtropics, primarily in crocodilians, although some species are known from other reptiles. Despite their broad geographical distribution, the knowledge of proterodiplostomid diversity remains limited, mostly because a number of potential host species and regions of the world have not been sufficiently explored for these parasites. In this study, we use morphological and molecular data to describe four new genera ( Afroproterodiplostomum gen. nov., Dungalabatrema gen. nov., Australiadiplostomum gen. nov. and Nattererodiplostomum gen. nov.) and five new species ( Afroproterodiplostomum ingwenyae sp. nov., Australiadiplostomum blairi sp. nov., Dungalabatrema kostadinovae sp. nov., Dungalabatrema snyderi sp. nov. and Pseudoneodiplostomum angustus sp. nov.) of proterodiplostomids from crocodilians in Australia, South Africa and South America. Nattererodiplostomum gen. nov. has been established upon re-evaluation of the status of Proterodiplostomum medusae (Dubois, 1936) from caimans in Brazil using combined morphological and molecular evidence. Only a few previous studies provided DNA sequence data of proterodiplostomids. We generated partial 28S rDNA and cytochrome c subunit ( cox 1) mtDNA for three previously undescribed proterodiplostomids collected from Crocodylus spp. in Australia and South Africa. The newly generated 28S sequences were used to examine phylogenetic affinities of these taxa. All three newly sequenced proterodiplostomid species appeared in the phylogenetic tree in a strongly supported monophyletic clade comprising exclusively parasites of Crocodylus ., Competing Interests: Disclosure statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Crassiphialinae Sudarikov, 1960 is a large subfamily of the Diplostomidae Poirier, 1886 with a complex taxonomic history. It includes a diversity of species parasitic in the intestines of avian and mammalian definitive hosts worldwide. Posthodiplostomum Dubois, 1936 is a large and broadly distributed crassiphialine genus notorious for its association with diseases in their fish second intermediate hosts. In this study, we generated partial 28S rDNA and cytochrome c oxidase subunit 1 ( cox 1) mtDNA gene sequences of digeneans belonging to seven crassiphialine genera. The 28S sequences were used to study the interrelationships among crassiphialines and their placement among other major diplostomoidean lineages. Our molecular phylogenetic analysis and review of morphology does not support subfamilies currently recognized in the Diplostomidae; therefore, we abandon the current subfamily system of the Diplostomidae. Molecular phylogenetic analyses suggest the synonymy of Posthodiplostomum , Ornithodiplostomum Dubois, 1936 and Mesoophorodiplostomum Dubois, 1936; morphological study of our well-fixed adult specimens and review of literature revealed lack of consistent differences among the three genera. Thus, we synonymize Ornithodiplostomum and Mesoophorodiplostomum with Posthodiplostomum . Our phylogenetic analyses suggest an Old World origin of Posthodiplostomum followed by multiple dispersal events among biogeographic realms. Furthermore, our analyses indicate that the ancestors of these digeneans likely parasitized ardeid definitive hosts. Four new species of Posthodiplostomum collected from birds in the New World as well as one new species of Posthodiplostomoides Williams, 1969 from Uganda are described., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Neopsilotrema is a small genus of psilostomid digeneans parasitic in the intestine of birds in the Palearctic and Nearctic. At present, the genus includes 4 species: Neopsilotrema lisitsynae from the Palearctic and Neopsilotrema affine, Neopsilotrema lakotae, and Neopsilotrema marilae from the Nearctic. Herein, we describe a new species, Neopsilotrema itascae n. sp., from lesser scaup Aythya affinis collected in Minnesota. The species can be distinguished from congeners on the basis of the ventral sucker:oral sucker width ratio, body width:length ratio, and cirrus sac size, along with other characters. We generated new 28S ribosomal deoxyribonucleic acid (DNA) and NADH dehydrogenase (ND1) mitochondrial DNA sequence data of a variety of psilostomids from the Palearctic and Nearctic along with sequences of the ribosomal internal transcribed spacer (ITS) region (ITS1 + 5.8S + ITS2) from 3 Neopsilotrema species. The molecular phylogenetic affinities of a variety of psilostomid taxa were studied using 28S sequence data. The 28S sequences of psilostomids demonstrated 1-7.9% intergeneric divergence, whereas the sequences of ND1 had 17.7-34.1% intergeneric divergence. The interspecific divergence among members of Neopsilotrema was somewhat lower (0.2-0.5% in 28S; 0.3-0.4% in ITS; 12-15.7% in ND1). Our comparison of DNA sequences along with morphologic study suggests Holarctic distribution of N. lisitsynae., (© American Society of Parasitologists 2021.)

Anenterotrema is a small genus of dicrocoeliids (Digenea: Dicrocoeliidae) containing 6 species found in Neotropical bats. Members of this genus are characterized by the lack of digestive system organs and, unlike the majority of dicrocoeliids, parasitize the intestine of their definitive hosts. In this study, we have morphologically examined newly collected specimens belonging to four species of Anenterotrema from Brazil, Ecuador, and Panama. Based on the data in original descriptions and our analysis of quality new specimens, we resurrected Anenterotrema freitasi, previously synonymized with A. eduardocaballeroi. We also described a new species of Anenterotrema from Molossus molossus in the Brazilian Amazon. The new species differs from congeners in several morphological features, most prominently in the size and structure of its highly muscular, protuberant ventral sucker. It is also characterized by the lack of the semi-circular thickening of the tegument around the oral sucker typical for some members of the genus. We used partial DNA sequences of the large ribosomal subunit gene (28S) and mitochondrial cytochrome c oxidase subunit 1 gene (cox1) to test the monophyly of Anenterotrema, and study the interrelationships and determine the inter- and intraspecific variation of three Anenterotrema spp. collected from different bat species in Brazil, Ecuador and Panama.

As presently recognized, Herpetodiplostomum is a small genus of proterodiplostomid digeneans parasitic in crocodilians. Most members of Cheloniodiplostomum, a genus of proterodiplostomids that parasitize chelonians, were originally placed within Herpetodiplostomum. The 2 genera were distinguished based on the presence/absence of papillae on the holdfast organ and anterior extent of vitellarium. Our study of Herpetodiplostomum and Cheloniodiplostomum revealed a lack of consistent morphological differences between the genera. Therefore, we consider Cheloniodiplostomum to be a junior synonym of Herpetodiplostomum. Recent molecular phylogenetic studies included a number of proterodiplostomid taxa; however, DNA sequence data are not available for any Herpetodiplostomum or Cheloniodiplostomum species. Herein, we describe a new Herpetodiplostomum species from Geoffroy's side-necked turtle Phrynops geoffroanus from Mato Grosso State, Brazil. The new species can be distinguished from other Herpetodiplostomum species based on the distribution of vitellarium, level of development of holdfast papillae, and ratio of prosoma:opisthosoma width, among other characters. We provide the first molecular phylogeny of the Proterodiplostomidae that includes a Herpetodiplostomum species. The limited geographic distribution of Herpetodiplostomum spp. within the geographic ranges of caimans suggests a secondary host switching event from crocodilians to chelonian definitive hosts in the evolution of Herpetodiplostomum., (© American Society of Parasitologists 2021.)

Classical theory suggests that parasites will exhibit higher fitness in sympatric relative to allopatric host populations (local adaptation). However, evidence for local adaptation in natural host-parasite systems is often equivocal, emphasizing the need for infection experiments conducted over realistic geographic scales and comparisons among species with varied life history traits. Here, we used infection experiments to test how two trematode (flatworm) species (Paralechriorchis syntomentera and Ribeiroia ondatrae) with differing dispersal abilities varied in the strength of local adaptation to their amphibian hosts. Both parasites have complex life cycles involving sequential transmission among aquatic snails, larval amphibians and vertebrate definitive hosts that control dispersal across the landscape. By experimentally pairing 26 host-by-parasite population infection combinations from across the western USA with analyses of host and parasite spatial genetic structure, we found that increasing geographic distance-and corresponding increases in host population genetic distance-reduced infection success for P. syntomentera, which is dispersed by snake definitive hosts. For the avian-dispersed R. ondatrae, in contrast, the geographic distance between the parasite and host populations had no influence on infection success. Differences in local adaptation corresponded to parasite genetic structure; although populations of P. syntomentera exhibited ~10% mtDNA sequence divergence, those of R. ondatrae were nearly identical (<0.5%), even across a 900 km range. Taken together, these results offer empirical evidence that high levels of dispersal can limit opportunities for parasites to adapt to local host populations., (© 2020 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2020 European Society For Evolutionary Biology.)

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.

The Dicrocoeliidae is a highly diverse and broadly distributed family of digeneans typically parasitic in the gall bladder and liver of their tetrapod hosts. So far, no study has reported dicrocoeliids, or any digeneans, from the ancient marsupial family Caenolestidae. Herein, we describe a new genus of dicrocoeliids (Otongatrema n. gen.) from Tate's shrew opossum Caenolestes fuliginosus and a new species of Metadelphis (Metadelphis cesartapiai n. sp.) from a phyllostomid bat Anoura peruana collected in Ecuador. Otongatrema can be readily distinguished from the morphologically closest dicrocoeliid genera Concinnum, Conspicuum and Canaania based on the position of the genital pore, distribution/position of the uterus and extent of vitellarium. Metadelphis cesartapiai can be easily differentiated from other Metadelphis spp. based on a combination of morphological characters including body shape and size, distribution of vitellarium, shape of the gonads as well as size of suckers and cirrus sac. In addition, we used newly generated partial sequences of the nuclear 28S rRNA gene and mitochondrial cox1 genes to examine phylogenetic affinities of the new taxa within the Dicrocoeliidae. Both the 28S and cox1 phylogenies confidently positioned Otongatrema as a sister/basal group to all other dicrocoeliids sequenced so far. The phylogenetic position of Otongatrema may be explained by a close co-evolutionary relationship with Caenolestidae, one of the most basal and most ancient groups of marsupials. In addition, our 28S phylogeny provides evidence that the complete or partial loss of intestinal structures has likely occurred independently at least 3 times in the evolutionary history of the Dicrocoeliidae., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Proterodiplostomidae Dubois, 1936 is a relatively small family of diplostomoidean digeneans parasitising the intestines of reptilian hosts associated with freshwater environments in tropical and subtropical regions. The greatest diversity of proterodiplostomids is found in crocodilians, although some parasitise snakes and turtles. According to the most recent revision, the Proterodiplostomidae included 17 genera within 5 subfamilies. Despite the complex taxonomic structure of the family, availability of testable morphology-based phylogenetic hypotheses and ancient hosts, molecular phylogenetic analyses of the group were practically lacking. Herein, we use novel DNA sequence data of the nuclear lsrRNA gene and mitochondrial cox1 gene from a broad range of proterodiplostomid taxa obtained from crocodilian, fish, and snake hosts on four continents to test the monophyly of the family and evaluate the present morphology-based classification system of the Proterodiplostomidae in comparison with the molecular phylogeny. This first detailed phylogeny for the Proterodiplostomidae challenges the current systematic framework. Combination of molecular phylogenetic data with examination of freshly collected quality specimens and re-evaluation of morphological criteria resulted in a number of systematic and nomenclatural changes along with a new phylogeny-based classification of the Proterodiplostomidae. As the result of our molecular and morphological analyses: (i) the current subfamily structure of the Proterodiplostomidae is abolished; (ii) three new genera, Paraproterodiplostomum n. g., Neocrocodilicola n. g. and Proteroduboisia n. g., are described and Pseudoneodiplostomoides Yamaguti, 1954 is restored and elevated from subgenus to genus level; (iii) two new species, Paraproterodiplostomum currani n. g., n. sp. and Archaeodiplostomum overstreeti n. sp., are described from the American alligator in Mississippi, USA. Comparison of the structure of terminal ducts of the reproductive system in all proterodiplostomid genera did not support the use of these structures for differentiation among subfamilies (or major clades) within the family, although they proved to be useful for distinguishing among genera and species. Our study includes the first report of proterodiplostomids from Australia and the first evidence of a snake acting as a paratenic host for a proterodiplostomid. A key to proterodiplostomid genera is provided. Questions of proterodiplostomid-host associations parasitic in crocodilians are discussed in connection with their historical biogeography. Our molecular phylogeny of the Proterodiplostomidae closely matches the current molecular phylogeny of crocodilians. Directions for future studies of the Proterodiplostomidae are outlined.

Purpose: Cardiocephaloides is a small genus of strigeid digeneans with an essentially cosmopolitan distribution. Most members of Cardiocephaloides are found in larid birds, however, Cardiocephaloides physalis is an exception and parasitizes penguins in some coastal regions of South America and South Africa. No prior molecular phylogenetic studies have included DNA sequence data of C. physalis. Herein, we provide molecular phylogenetic analyses of Cardiocephaloides using DNA sequences from five species of these strigeids., Methods: Adult Cardiocephaloides spp. were obtained from larid birds and penguins collected from 3 biogeographical realms (Palearctic, Nearctic and Neotropics). We have generated sequences of the complete ITS region and partial 28S gene of the nuclear ribosomal DNA, along with partial sequences of the mitochondrial CO1 gene for C. physalis, C. medioconiger and the type species of the genus, C. longicollis and used them for phylogenetic inference., Results: Cardiocephaloides spp. appeared as a 100% supported clade in the phylogenetic tree based on 28S sequences. The position of C. physalis varied between the phylogenetic trees based on the relatively conservative 28S gene on one hand, and variable ITS1 and COI sequences on the other. Cardiocephaloides physalis was nested within the clade of Cardiocephaloides spp. in the 28S tree and appeared as the sister group to the remaining members of the genus in the ITS1 region and COI trees. We detected 0.4-1.6% interspecific divergence in 28S, 1.9-6.9% in the ITS region and 8.7-11.8% in CO1 sequences of Cardiocephaloides spp. Our 28S sequence of C. physalis from South America and a shorter sequence from Africa available in the GenBank were identical., Conclusion: Cardiocephaloides as represented in the currently available dataset is monophyletic with C. physalis parasitism in penguins likely resulting from a secondary host-switching event. Identical 28S sequences of C. physalis from South America and Africa cautiously confirm the broad distribution of this species, although comparison of faster mutating genes (e. g., CO1) is recommended for a better substantiated conclusion.

Codonocephalus is a monotypic genus of diplostomid digeneans and is the only genus in the sub-family Codonocephalinae. The type-species Codonocephalus urniger has an unusual progenetic metacercaria that uses frogs as intermediate hosts and can use snakes as paratenic hosts. Adult C. urniger parasitize ardeid wading birds in the Palearctic. Despite the broad distribution of Codonocephalus , no DNA sequence data are currently available for the genus. In this study, we generated sequence data for nuclear ribosomal and mitochondrial DNA from progenetic metacercaria of the type-species C. urniger from marsh frog, Pelophylax ridibundus , collected in Ukraine. We used partial sequences of the nuclear ribosomal 28S gene to examine for the first time the phylogenetic position of Codonocephalus among the Diplostomoidea.

Uvulifer Yamaguti, 1934, is a genus of diplostomoidean digeneans that parasitizes kingfishers worldwide. Species have a Neascus -type metacercaria that encysts in or on fish intermediate hosts, often causing black spot disease. Only 3 prior studies published DNA sequence data for Uvulifer species with only 1 including a single named species ( Uvulifer spinatus López-Jiménez, Pérez-Ponce de León, & García-Varela, 2018). Herein we describe 2 new species of Uvulifer from the green-and-rufous kingfisher, Chloroceryle inda (Linnaeus), collected in Peru ( Uvulifer batesi n. sp. and Uvulifer pequenae n. sp.). Both new species are readily differentiated from their New World congeners by a combination of morphological characters including distribution of vitelline follicles and prosoma:opisthosoma length ratios. In addition, we used newly generated nuclear 28S rRNA and mitochondrial COI gene sequence data to differentiate among species and examine phylogenetic affinities of Uvulifer . This includes the 2 new species and Uvulifer ambloplitis (Hughes, 1927), as well as Uvulifer elongatus Dubois, 1988 , Uvulifer prosocotyle (Lutz, 1928), and Uvulifer weberi Dubois, 1985 , none of which have been part of prior molecular phylogenetic studies. Our data on Uvulifer revealed 0.1-2.2% interspecific divergence in 28S sequences and 9.3-15.3% in COI sequences. Our 28S phylogeny revealed at least 6 well-supported clades within the genus. In contrast, the branch topology in the COI phylogenetic tree was overall less supported, indicating that although COI sequences are a great tool for species differentiation, they should be used with caution for phylogenetic inference at higher taxonomic levels. Our 28S phylogeny did not reveal any clear patterns of host association between Uvulifer and particular species of kingfishers; however, it identified 2 well-supported clades uniting Uvulifer species from distant geographical locations and more than 1 biogeographic realm, indicating at least 2 independent dispersal events in the evolutionary history of the New World Uvulifer . Our results clearly demonstrate that the diversity of Uvulifer in the New World has been underestimated.

Crassiphiala is a monotypic genus of diplostomid digeneans and is the type genus of the subfamily Crassiphialinae. The type species Crassiphiala bulboglossa parasitizes kingfishers in the Nearctic and has a Neascus-type metacercaria that encysts on fish intermediate hosts, often causing black spot disease. While recent molecular phylogenetic studies included some members of the Crassiphialinae, no DNA sequence data of Crassiphiala is currently available. Our molecular and morphological study of adult and larval crassiphialines from the Americas revealed the presence of at least three lineages of Crassiphiala from the Nearctic and two lineages from the Neotropics. This is the first record of Crassiphiala from the Neotropics. Herein, we provide the first molecular phylogeny of the Diplostomoidea that includes Crassiphiala. Our data revealed 0.2-2.4% divergence among 28S sequences and 11-19.8% among CO1 sequences of lineages of Crassiphiala. The results of our analyses did not support the monophyly of Crassiphialinae. Our results clearly demonstrated that the diversity of Crassiphiala has been underestimated.

The Cyathocotylidae is a globally distributed family of digeneans parasitic as adults in fish, reptiles, birds, and mammals in both freshwater and marine environments. Molecular phylogenetic analysis of interrelationships among cyathocotylids is lacking with only a few species included in previous studies. We used sequences of the nuclear 28S rRNA gene to examine phylogenetic affinities of 11 newly sequenced taxa of cyathocotylids and the closely related family Brauninidae collected from fish, reptiles, birds, and dolphins from Australia, Southeast Asia, Europe, North America and South America. This is the first study to provide sequence data from adult cyathocotylids parasitic in fish and reptiles. Our analyses demonstrated that the members of the genus Braunina (family Brauninidae) belong to the Cyathocotylidae, placing the Brauninidae into synonymy with the Cyathocotylidae. In addition, our DNA sequences supported the presence of a second species in the currently monotypic Braunina . Our phylogeny revealed that Cyathocotyle spp. from crocodilians belong to a separate genus ( Suchocyathocotyle , previously proposed as a subgenus) and subfamily (Suchocyathocotylinae subfam. n.). Morphological study of Gogatea serpentum indicum supported its elevation to species as Gogatea mehri . The phylogeny did not support Holostephanoides within the subfamily Cyathocotylinae; instead, Holostephanoides formed a strongly supported clade with members of the subfamily Szidatiinae ( Gogatea and Neogogatea ). Therefore, we transfer Holostephanoides into the Szidatiinae. DNA sequence data revealed the potential presence of cryptic species reported under the name Mesostephanus microbursa . Our phylogeny indicated at least two major host switching events in the evolutionary history of the subfamily Szidatiinae which likely resulted in the transition of these parasites from birds to fish and snakes. Likewise, the transition to dolphins by Braunina represents another major host switching event among the Cyathocotylidae. In addition, our phylogeny revealed more than a single transition between freshwater and marine environments demonstrated in our dataset by Braunina and some Mesostephanus .

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.

The Dicrocoeliidae is a highly diverse family of digeneans parasitic in amniotic tetrapods. Detailed molecular phylogenetic analysis of dicrocoeliids is lacking and only a few dicrocoeliids from mammals have been included in previous studies. Sequence data were previously absent for the Anenterotrematidae that shares several morphological characteristics with dicrocoeliids. We examined phylogenetic affinities of several newly sequenced (nuclear 28S rDNA) taxa of dicrocoeliids and anenterotrematids collected from small mammals in Ecuador, Panama, Peru, USA and Vietnam. Our analyses demonstrated that the two anenterotrematid genera (Anenterotrema, Apharyngotrema) belong to the Dicrocoeliidae, placing the Anenterotrematidae into synonymy with the Dicrocoeliidae. Molecular data combined with morphological examination of type and new specimens provided evidence that Parametadelphis and Apharyngotrema are junior synonyms of Metadelphis, with all Metadelphis species lacking a digestive system. Phylogenetic analysis demonstrates that reduction of the alimentary tract in Lutztrema and its loss in Anenterotrema and Metadelphis represent at least two independent evolutionary events. Genera Brachylecithum, Brachydistomum, and Lyperosomum proved to be non-monophyletic, each likely representing more than a single genus. Furthermore, phylogenetic analysis did not support monophyly of the two largest subfamilies of the Dicrocoeliidae (Dicrocoeliinae and Leipertrematinae) with the other two subfamilies not included in this study. Therefore, we propose to abandon the current subfamily division of the Dicrocoeliidae. Analysis of host associations indicates multiple host-switching events throughout evolution of dicrocoeliids. Lastly, analysis of dicrocoeliid geographic distribution revealed that nearly all major clades included taxa from more than a single zoogeographic realm with the exception of the clade Anenterotrema + Metadelphis, found only in the Neotropics., (Copyright © 2018 Elsevier B.V. All rights reserved.)