Description and Phylogenetic Relationships of Anhingatrema n. gen. (Digenea: Diplostomidae) with Two New Species from New World Anhingas (Aves: Anhingidae).

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.

Molecular phylogeny supports invalidation of Didelphodiplostomum and Pharyngostomoides (Digenea: Diplostomidae) and reveals a Tylodelphys from mammals.

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.

Molecular phylogenetic analysis of Neodiplostomum and Fibricola (Digenea, Diplostomidae) does not support host-based systematics.

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.

Molecular phylogeny of Diplostomum, Tylodelphys, Austrodiplostomum and Paralaria (Digenea: Diplostomidae) necessitates systematic changes and reveals a history of evolutionary host switching events.

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.

Unravelling the diversity of the Crassiphialinae (Digenea: Diplostomidae) with molecular phylogeny and descriptions of five new species.

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 (cox1) 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.

Phylogeny and systematics of the Proterodiplostomidae Dubois, 1936 (Digenea: Diplostomoidea) reflect the complex evolutionary history of the ancient digenean group.

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.

Phylogenetic Relationships of Cardiocephaloides spp. (Digenea, Diplostomoidea) and the Genetic Characterization of Cardiocephaloides physalis from Magellanic Penguin, Spheniscus magellanicus, in Chile.

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.

Phylogenetic relationships, expanded diversity and distribution of Crassiphiala spp. (Digenea, Diplostomidae), agents of black spot disease in fish.

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.

Phylogenetic relationships and systematic position of the enigmatic Urotrema Braun, 1900 (Platyhelminthes: Digenea).

The systematic position of Urotrema Braun, 1900 and the family Urotrematidae Poche, 1926 have always been controversial. Due to its unusual morphological characteristics, lack of knowledge of the life cycle or details of its excretory system, this family was placed within different higher taxonomic groups of digeneans. Despite being one of the most enigmatic digenean families in terms of its phylogenetic affinities, DNA sequence data for Urotrematidae were lacking. Here, we evaluate the phylogenetic relationships of Urotrema using newly obtained partial sequences of the 28S rRNA gene from Urotrema specimens collected in North, Central and South America including the type species U. scabridum Braun, 1900, as well as previously published sequences of digeneans. Our study has demonstrated that Urotrema is phylogenetically closest (100% branch support) to members of Parabascus Looss, 1907 belonging to the family Pleurogenidae Looss, 1899. Thus, the family Urotrematidae becomes a junior synonym of the Pleurogenidae. Urotrema forms a 100% supported clade among the Pleurogenidae, parasitic in warm-blooded vertebrates. However, the phylogenetic relationships and exact systematic position of the remaining 3 genera currently placed in the Urotrematidae remains unclear and requires additional studies as their allocation is mostly based on the terminal posterior position of the genital pore and cirrus-sac. According to our results the genus Parabascus appears to be paraphyletic and requires further detailed phylogenetic and morphological analyses.

Molecular phylogeny of the Cyathocotylidae (Digenea, Diplostomoidea) necessitates systematic changes and reveals a history of host and environment switches.

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.

On the systematics of some marine haploporids (Trematoda) with the description of a new species of Megasolena Linton, 1910.

Megasolena mikra sp. nov. is described from the queen angelfish, Holacanthus ciliaris (Linnaeus), off Florida, USA. The new species can be differentiated from all other species of Megasolena Linton, 1910 except Megasolena littoralis Muñoz, George-Nascimento, and Bray, 2017 in possessing testes that are smaller in diameter than the ovary. The new species can be differentiated from M. littoralis in lacking tegumental spines and possessing oral sucker papillae. Molecular data are provided for two species each of Cadenatella Dollfus, 1946, Hapladena Linton, 1910, and Megasolena Linton, 1910. Bayesian inference analysis of concatenated internal transcribed spacer region-2 (ITS2) and partial 28S rDNA sequences of 50 haploporoids revealed 1) a monophyletic Atractotrematidae Yamaguti, 1939 sister to the rest of the haploporoids tested; 2) a paraphyletic Megasoleninae Manter, 1935 - if Hapladena is included; and 3) a monophyletic Cadenatellinae Gibson and Bray, 1982 sister to the 'mugilid' haploporids. The 'mugilid' haploporids formed a monophyletic clade consisting of the subfamilies Chalcinotrematinae Overstreet and Curran, 2005, Forticulcitinae Blasco-Costa, Balbuena, Kostadinova, and Olson, 2009, Haploporinae Nicoll, 1914, and Waretrematinae Srivastava, 1937. Based on our analysis we restrict the Megasoleninae to include Megasolena, Vitellibaculum Montgomery, 1957, and Metamegasolena Yamaguti, 1970, all of which have species with two testes. To accommodate the former megasolenine taxa with a single testis, we erect the Hapladeninae subf. nov. for species in Hapladena and tentatively, Myodera Montgomery, 1957. Our results further support that haploporoids had a common marine ancestor with two testes, and that members of the Haploporoidea Nicoll, 1914 underwent diversification following a shift from a primarily marine life history with eupercarian hosts to a more euryhaline one with diadromous hosts (namely mullet).

Gauging the influence of increased search effort on reporting rates of bottlenose dolphin (Tursiops truncatus) strandings following the deepwater horizon oil spill.

The co-occurrence of the Deepwater Horizon oil spill and the northern Gulf of Mexico cetacean Unusual Mortality Event have raised questions about the stability of inshore bottlenose dolphin (Tursiops truncatus) populations throughout the region. Several factors could have contributed to the ongoing event, but little attention has been paid to the potential effects of increased search effort and reporting of strandings associated with oil spill response activities, which were widespread for an extended period. This study quantified the influence of increased search effort by estimating the number of bottlenose dolphin strandings reported by oil spill responders and comparing monthly stranding rates with and without response-related records. Results showed that response teams reported an estimated 58% of strandings during the Active Response period within the study area. Comparison of Poisson rates tests showed that when responder-influenced stranding records were removed, the monthly stranding rates from the Active Response period (May 2010 -April 2014) were similar to the Post-Removal Actions Deemed Complete period (May 2013 -March 2015) (e.g., p = 0.83 for remote areas in Louisiana). Further, analyses using the Getis-Ord Gi* spatial statistic showed that when response-related stranding reports were removed from the Active Response period, significant spatial clustering of strandings (p < 0.05) was reduced by 48% in coastal Louisiana. Collectively, these results suggest that increased search effort resulting from the Deepwater Horizon oil spill response throughout remote portions of the Unusual Mortality Event geographic region had the capacity to increase reporting and recovery of marine mammal strandings to unusually high levels. To better understand how stranding data relates to actual mortality, more work is needed to quantify dolphin population size, population trends, and carcass detection rates including the role of search effort. This is vital for understanding the status of a protected species within the northern Gulf of Mexico.

Two New Species of Saccocoelioides (Digenea: Haploporidae) with Phylogenetic Analysis of the Family, Including Species of Saccocoelioides from North, Middle, and South America.

Two cryptic species of haploporid digeneans belonging in Saccocoelioides are described from Costa Rica: one from a poeciliid fish, Poecilia gillii, and the other from a characid fish, Astyanax aeneus. Traditional morphological features are examined and found to be largely inefficient for differentiating among the new species and 20 New World congeners. Comparison of ribosomal DNA sequences among the 2 new species, 2 North American, 4 Middle American, and 3 South American species of Saccocoelioides, including the type-species Saccocoelioides nanii from Argentina and Saccocoelioides sogandaresi from Texas, is effective in differentiating among these species. A Bayesian inference analysis is conducted using a concatenated alignment of the same 2 ribosomal gene regions from 35 species belonging in the Haploporoidea and rooted by the atractotrematid Isorchis anomalus. The analysis provides stronger support for a close relationship between species in Saccocoelioides and Intromugil; thus, Intromugil is transferred from the Waretrematinae to the Chalcinotrematinae. Additionally, interrelationships among 11 species of Saccocoelioides are resolved and reveal a distinct genetic rift suggesting the presence of 2 distinct lineages within the genus, 1 containing the type-species and the other containing a group of species that more closely resemble other genera in the subfamily. Taxonomy of Saccocoelioides is discussed, and observations made during the present study justify taking several taxonomic actions: new combinations are proposed for Saccocoelioides tilapiae n. comb., formerly in the now-defunct Culuwiya, and Saccocoelioides ruedasueltensis n. comb., formerly in Chalcinotrema; Saccocoelioides guaporense nomen novem is proposed for Lecithobotrioides elongatus; Saccocoelioides papernai is considered a junior subjective synonym of Saccocoelioides overstreeti; Saccocoelioides godoyi is considered a junior subjective synonym of Saccocoelioides szidati; Saccocoelioides magnorchis and Saccocoelioides saccodontis are considered species inquirendae; and Saccocoelioides adelae is considered a nomen nudum. Four new hosts are reported for Saccocoelioides cichlidorum: Amphilophus lyonsi, Amatitlania nigrofasciatus, Amatitlania septemfasciatus, and Hypsophrys nicaraguensis. Molecular data call into question some existing species identifications in Saccocoelioides and reveal that molecular tools combined with traditional taxonomy are required for accurately identifying species in the genus. Twenty-two species of Saccocoelioides are formally accepted, but it is noted that as new molecular data become available, some of these species may be transferred to other genera in the subfamily.

A new species of leech of the genus Placobdella (Hirudinida, Glossiphoniidae) from the American alligator (Alligator mississippiensis) in Mississippi, USA.

To date, the only species of leech reported from the American Alligator, Alligator mississippiensis is Placobdella multilineata. Seven specimens of a previously undescribed species of Placobdella were collected from the feet and lower jaw of a single female alligator from the Pascagoula River Wildlife Management Area, George County, Mississippi. The new species was named Placobdella siddalli Richardson & Moser, sp. n., in honor of the contributions of Dr. Mark Siddall to our understanding of the biology of leeches. Placobdella siddalli Richardson & Moser is similar to other papillated members of the genus Placobdella, but differs from Placobdella ali Hughes & Siddall, 2007, Placobdella rugosa (Verrill, 1874), Placobdella multilineata Moore, 1953, and Placobdella papillifera (Verrill, 1872) in coloration, papillation, ventral striping, and in the possession of a relatively large caudal sucker. In addition, molecular comparison of 626 nucleotides of CO-I between the new species and other papillated leeches (P. ali, P. multilineata, Placobdella ornata, P. papillifera, P. rugosa) revealed interspecific differences of 14.0-18.0% (88-113 nucleotides).

The Psilostomidae Looss, 1900 (sensu stricto) (Digenea: Echinostomatoidea): description of three new genera and a key to the genera of the family.

Three new psilostomid genera, Byrdtrema n. g., Longisaccus n. g. and Macracetabulum n. g., each with a single species, are described from ducks, Aix sponsa (L.) and Bucephala albeola (L.) in North America. Byrdtrema n. g. and Macracetabulum n. g. possess a bipartite seminal vesicle and share this character with four psilostomid genera, Grysoma Byrd, Bogitsh & Maples, 1961, Neopsilotrema Kudlai, Pulis, Kostadinova & Tkach, 2016, Psilostomum Looss, 1899 and Psilotornus Byrd & Prestwood, 1969. Byrdtrema n. g. differs from Macracetabulum n. g. in the shape of the body (elongate vs elongate-oval); the position of the ventral sucker (in first third of body vs just pre-equatorial); the shorter forebody; as well as in the smaller size of the eggs in relation to body length. Both new genera differ from (i) Grysoma by the nature of the vitellarium (large, compact follicles with small vitelline cells vs weakly defined follicles with large vitelline cells, respectively) and the smaller size of the eggs in relation to body length; (ii) Psilostomum in the posterior extend of the cirrus-sac in relation to ventral sucker (slightly posterior vs more posterior), the location of the genital pore (at the level of oesophagus vs just postbifurcal), the shorter length of uterine and longer post-testicular fields in relation to body length, and the anterior limits of vitellarium (at the level of ventral sucker vs posterior to ventral sucker); (iii) Psilotornus by the presence of a muscular pharynx (vs absent or rudimentary) and the location of the cirrus-sac (antero-dorsal to ventral sucker or more posterior vs entirely anterior to ventral sucker) and ovary (in hindbody vs in forebody). Byrdtrema n. g. differs from Neopsilotrema in the shape of the body (elongate vs subspherical to elongate-oval) and ventral sucker (elongate-oval vs subspherical to transversely oval), the shorter forebody and smaller eggs in relation to body length. Macracetabulum n. g. differs from Neopsilotrema by the shape of the ventral sucker (elongate-oval vs subspherical to transversely oval), the anterior limits of vitellarium (level of middle of ventral sucker vs level of intestinal bifurcation or anterior testis); and the slightly smaller size of eggs in relation to body length. Among the psilostomid genera, Longisaccus n. g. shows close affinities to Psilochasmus Lühe, 1909 in the presence of the long cirrus-sac and tubular internal seminal vesicle but can be clearly distinguished from the latter by the absence of the retractile tail-like process. In combination with molecular data, the above differences justify the recognition of three new genera. A key to the genera of the Psilostomidae is provided.

Description of three species of Isorchis (Digenea: Atractotrematidae) from Australia.

Three species of Isorchis Durio and Manter, 1969 are described from Australian waters. Isorchis megas sp. nov. is described from the spotbanded scat, Selenotoca multifasciata (Richardson), off Western Australia (WA) and Northern Territory (NT); Isorchis currani sp. nov. is described from S. multifasciata off NT; and Isorchis anomalus sp. nov. is described from the milkfish, Chanos chanos Forsskål, off WA. Isorchis megas sp. nov. can be differentiated from the other species of Isorchis by possessing a single, large egg that is greater than 20% of the body length; having a shorter body (the largest specimen is less than 500 µm); and utilizing a scatophagid rather than a chanid host. Isorchis currani sp. nov. can be differentiated from species of Isorchis other than I. megas sp. nov. by utilizing a scatophagid rather than a chanid host; it is differentiated from I. megas sp. nov. in having eggs that are 11-15% of the body length. Isorchis anomalus sp. nov. can be differentiated from all other species of Isorchis in possessing an irregular shaped genital pore rather than one that is circular to oblong. A Bayesian inference analysis of partial 28S rDNA sequences of the three new species of Isorchis and 30 other haploporoids revealed 1) the monophyly of the Atractotrematidae Yamaguti, 1939, 2) the two species of Isorchis infecting S. multifasciata were each other's closest relative, and 3) that Isorchis was most closely related to Pseudomegasolena Machida and Komiya, 1976 rather than Atractotrema Goto and Ozaki, 1929 although sequence data are not yet available for a member of Pseudisorchis Ahmad, 1985.

Neopsilotrema n. g. (Digenea: Psilostomidae) and three new species from ducks (Anseriformes: Anatidae) in North America and Europe.

Neopsilotrema n. g. (Digenea: Psilostomidae) and three new species of psilostomid digeneans are described from birds in North America and Europe: Neopsilotrema lakotae n. sp. from Aythya americana (Eyton) in North Dakota, USA, Neopsilotrema affine n. sp. from Aythya affinis (Eyton) in Minnesota, USA and Neopsilotrema lisitsynae n. sp. from Anas crecca L. in Kherson Region, Ukraine. Neopsilotrema n. g. shares a bipartite seminal vesicle with only three genera within the Psilostomidae, Psilotornus Byrd & Prestwood, 1969, Psilostomum Looss, 1899 and Grysoma Byrd, Bogitsh & Maples, 1961. The new genus differs from Psilotornus in the presence of a muscular pharynx and a massive ventral sucker; the location of the cirrus-sac in relation to the ventral sucker and more posterior location of ovary; the nature of the vitellarium (i.e. comprising large, compact follicles with small vitelline cells vs weakly defined follicles with large vitelline cells); a proportionately shorter forebody; and in parasitisation in anseriform (vs passeriform) birds. Differences between the new genus and Psilostomum comprise the shape of the body, the relative size of the suckers, somewhat longer forebody and a more anterior location of the testes. Neopsilotrema n. g. differs from Grysoma in the relative size of the suckers, the degree of development of prostatic cells, the nature of the vitellarium and the size of the eggs in relation to body length. The European species Neopsilotrema lisitsynae n. sp. is distinguished from its congeners in having a longer, narrower and distinctly more elongate body with a longer post-testicular region and anterior limits of the vitelline fields posterior to ventral sucker. The two North American forms, Neopsilotrema lakotae n. sp. and Neopsilotrema affine n. sp., are cryptic species with largely overlapping metrical data; these are distinguished by comparing genetic data. The phylogenetic hypotheses for the Psilostomidae developed from sequence data analyses based on partial 28S rDNA support the erection of the new genus and the distinction of the three new species. Grysoma marilae (Price, 1942) agrees more closely with the generic diagnosis of Neopsilotrema, especially in relation to the size and shape of the body, the relative length of the forebody and post-testicular field, the structure of the vitellarium, the location of the reproductive organs and the sucker ratio. Consequently, it is here transferred to the new genus as Neopsilotrema marilae (Price, 1942) n. comb.

Redescription and phylogenetic relationships of the rare Lyperosomum sarothrurae Baer, 1959 (Digenea: Dicrocoeliidae).

Lyperosomum sarothrurae Baer, 1959 is a rare dicrocoeliid described based on a single specimen from Belgian Congo, now Democratic Republic of the Congo, and never reported again. The original description lacked several important details of the species morphology. This work provides morphological redescription of Lyp. sarothrurae based on specimens from Kibale National Park, Uganda which is about 200 kilometres from the type locality. Results of re-examination of the holotype are also provided. Morphology of Lyp. sarothrurae shows some characteristics intermediate between those of the species rich genus Lyperosomum Looss, 1899 and Megacetabulum Oshmarin, 1964 that includes only 3 species. Due to the lack of clear morphological differences between the two genera, Megacetabulum is considered here as a junior synonym of Lyperosomum. Molecular phylogenetic analysis including Lyp. sarothrurae and all currently available partial sequences of the nuclear ribosomal 28S RNA gene of dicrocoeliid species is also included and its results are discussed.

An additional genus and two additional species of Forticulcitinae (Digenea: Haploporidae).

Forticulcita platana sp. n. and Forticulcita apiensis sp. n. are described from Mugil liza Valenciennes in Argentina, and from Mugil cephalus Linnaeus in Salt Springs, Florida, USA, respectively. Supplemental material relating to the hermaphroditic sac of Forticulcita gibsoni Blasco-Costa, Montero, Balbuena, Raga et Kostadinova, 2009 is provided from a specimen isolated from M. cephalus off Crete, Greece. Forticulcita platana can be distinguished from all species of Forticulcita Overstreet, 1982 except F. gibsoni, based on possessing small pads or gland cells along the hermaphroditic duct. It can be differentiated from that species in possessing a hermaphroditic sac that is one and a half to two times longer than wide rather than one that is approximately three times longer than wide, longer eggs (44-52 µm rather than 34-44 µm long) and a shorter post-testicular space (< 45% of the body length). Forticulcita apiensis can be differentiated from the other species of Forticulcita in possessing a testis that is shorter than or equal to the pharynx rather than one that is longer than the pharynx. Xiha gen. n. is erected for Dicrogaster fastigatus Thatcher et Sparks, 1958 as Xiha fastigata (Thatcher et Sparks, 1958) comb. n., and we tentatively consider Dicrogaster fragilis Fernández Bargiela, 1987 to be Xiha fragilis (Fernández Bargiela, 1987) comb. n. The new genus fits within the concept of Forticulcitinae Blasco-Costa, Balbuena, Kostadinova et Olson, 2009 in having a vitellarium comprised of a single elongate to subspherical mass. Xiha can be differentiated from Forticulcita in having spines lining the hermaphroditic duct, or intromittent organ. A Bayesian inference analysis of partial 28S rDNA sequences of the two New World species of Forticulcita, Xiha fastigata and previously published haploporids places Xiha fastigata within the Forticulcitinae and sister to Forticulcita. Amended diagnoses for the subfamily and for Dicrogaster Looss, 1902 are provided.

A new species of Drepanocephalus Dietz, 1909 (Digenea: Echinostomatidae) from the double-crested cormorant Phalacrocorax auritus (Lesson) (Aves: Phalacrocoracidae) in North America.

Drepanocephalus auritus n. sp. is described based on specimens from the double-crested cormorant Phalacrocorax auritus (Lesson) in North America. The new species differs from its congeners in its very narrow, elongate body, long uterine field and widely separated testes. Sequences of the nuclear rRNA gene cluster, spanning the 3' end of the nuclear ribosomal 18S rRNA gene, internal transcribed spacer region (ITS1+5.8S gene+ITS2) and partial 28S gene (2,345 bp), were identical in specimens collected from North Dakota, Minnesota and Mississippi, USA. Sequences of the 651 bp long fragment of the mitochondrial cox1 gene exhibited very low intraspecific variability (< 1%). Comparisons of the newly-generated sequences with those available in the GenBank indicate that the sequences from North America published under the name D. spathans Dietz, 1909 in fact represent D. auritus n. sp.