Integrative taxonomic study of mononchid nematodes from riparian habitats in Bulgaria. I. Genera Mononchus Bastian, 1865 and Coomansus Jairajpuri & Khan, 1977 with the description of Mononchuspseudoaquaticus sp. nov. and a key to the species of Mononchus.

The species diversity of the genera Mononchus Bastian, 1865 and Coomansus Jairajpuri & Khan, 1977 was assessed in a study of the mononchid nematodes from a wide range of riparian habitats in Bulgaria. Four species were identified based on morphological and morphometric data: Coomansusparvus (de Man, 1880), Mononchustruncatus Bastian, 1865, Mononchuspseudoaquaticus sp. nov., and Mononchus sp. The first three species were characterised both morphologically and molecularly (18S and 28S rRNA gene sequences) and the integration of these data and phylogenetic analyses provided support for their distinct species status. This paper provides detailed descriptions, morphometric data for multiple species populations, drawings and photomicrographs, and the first taxonomically verified sequences for C.parvus (n = 6), M.truncatus (sensu stricto) (n = 4) and M.pseudoaquaticus sp. nov. (n = 3). Comparative sequence and phylogenetic analyses suggested that the utility of the 18S rRNA gene for species delimitation is rather limited at least for some species complexes within the genus Mononchus. At the generic and suprageneric level, the 18S and 28S rDNA phylogenies both recovered the three genera represented by two or more species (Mononchus, Mylonchulus, and Parkellus) as monophyletic with strong support, the Mononchidae as paraphyletic, the Anatonchidae as monophyletic, and there was no support for a sister-group relationship between Mylonchulus and Mononchus. A key to the species of Mononchus is provided to facilitate the identification of the currently recognised 31 species.

The Systematics of the Trematoda.

The platyhelminth class Trematoda comprises two subclasses with largely disparate species diversity, with the small Aspidogastrea with c.80 species and the speciose Digenea with c.18,000 species, which has attracted much effort towards our understanding of evolutionary relationships among suprageneric taxa. This chapter focuses on insights into the classification of the Digenea, that have become apparent from our advanced understanding of both morphological and molecular data. The field of molecular systematics of the Digenea has experienced significant advances over the past 15 years. Phylogenetic analyses of sequence data predominantly from the 18S and 28S rRNA genes have incorporated a considerable diversity of taxa, thus increasing the accuracy of phylogenetic inferences at higher taxonomic levels. As a result, the status of long-standing supraspecific taxa has been revised, new higher-level taxa have been defined, and inferences made in association with morphological and life-cycle evidence. A substantial effort has been made towards a classification reflecting a natural system of the Digenea by considering morphological evidence in conjunction with phylogenies inferred from molecular data; this has resulted in considerable congruence. However, limited taxon sampling in the phylogeny of the Digenea still remains relevant, especially in relation to some higher-level taxa, and an outline of these omissions is presented. A framework that has led to robust estimates of phylogeny is outlined, and the application of advanced morphological and molecular approaches in digenean taxonomy and systematics is illustrated using the most comprehensively studied digenean superfamilies.

Parasite communities in a marine fish indicate ecological recovery from the impacts of the Prestige oil-spill 12-13 years after the disaster.

The Prestige oil-spill (2002) is one of the major marine accidents resulting in contamination of virtually all types of marine habitat along c.900 km of coastline in the North-East Atlantic. Environmental pollution affects parasite populations and communities, both directly and through effects on intermediate and definitive hosts. However, the effects of oil-spills on shelf benthic communities are poorly known. This study addressed the hypothesis of recovery of parasite communities in a marine sparid teleost, the bogue Boops boops (L.) (Teleostei: Sparidae), as indicators of environmental pollution and its effects on benthic/pelagic invertebrate communities in an impacted area off the Galician coast, Spain, 12-13 years after the Prestige oil-spill. Novel data for the metazoan parasite communities collected during 2014-2015 were analysed in association with two unique datasets, one comprising baseline data collected in 2001, one year before the Prestige oil-spill, and one comprising data collected 3-4 years post-spill (2005-2006). Using the taxonomically consistent data on parasites in a series of fish seasonal samples, we found significant differences between the two seasonal post-spill datasets taken at a 9-year time interval (3-4 years and 12-13 years post-spill) in most community metrics and infection parameters of the common species. This was in sharp contrast with the few differences between the long-term post-spill dataset of 2014-2015 and the pre-spill dataset of 2001. Multivariate community similarity analyses confirmed that these differences reflected in the substantial differentiation of parasite community composition and structure of the post-spill communities and the significant homogenisation of communities sampled 12-13 years post-spill and baseline communities. Overall, the novel analyses demonstrated a long-term directional trend in parasite community succession towards ecological recovery irrespective of the natural seasonal variability. These findings suggest longer-term oil-spill impacts on shelf benthic/pelagic invertebrate communities lasting over 10 years.

Novel molecular data for monogenean parasites of sparid fishes in the Mediterranean and a molecular phylogeny of the Microcotylidae Taschenberg, 1879.

During a study of the monogeneans of four sparid fishes (Diplodus vulgaris, Pagellus bogaraveo, Pagrus pagrus and Sparus aurata) from the Western Mediterranean off Algeria, a large collection of parasites was characterised molecularly (28S rRNA and cox1 genes). A total of 46 partial sequences (23 for each gene) were generated from 38 isolates of monogeneans which included four species (Atrispinum acarne, Microcotyle erythrini (sensu stricto), Sparicotyle chrysophrii and Prostatomicrocotylinae gen. sp.) of the family Microcotylidae, two putative species of the family Capsalidae (Encotyllabe spp.), and one species (Choricotyle chrysophryi) of the family Diclidophoridae. Our study provides (i) the first molecular data for the Capsalidae in the Mediterranean; (ii) the first record of a member of the Prostatomicrocotylinae in the Mediterranean and in a sparid fish (D. vulgaris); (iii) the first cox1 sequences for A. acarne; (iv) the second record of M. erythrini (s.s.) from P. pagrus; and (v) the second confirmed by molecular data record of S. chrysophrii in wild populations of S. aurata. The first phylogenetic hypotheses for the family Microcotylidae developed here, revealed the monophyly of the subfamily Prostatomicrocotylinae and the genus Microcotyle but the relationships among the subfamilies were still largely unresolved with the best represented subfamily Microcotylinae being polyphyletic. Our results highlight the importance of molecular methods in the assessment of monogenean diversity and the need for a thorough taxon-sampling approach to increase the accuracy of phylogenetic reconstruction of the relationships of the large and taxonomically complex polyopisthocotylean family Microcotylidae.

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.

Molecular and morphological characterisation of Diplostomum phoxini (Faust, 1918) with a revised classification and an updated nomenclature of the species-level lineages of Diplostomum (Digenea: Diplostomidae) sequenced worldwide.

We characterised morphologically and molecularly Diplostomum phoxini (Faust, 1918) based on cercarial isolates from the snail Ampullaceana balthica (L.) (Gastropoda: Lymnaeidae) and metacercariae from the Eurasian minnow, Phoxinus phoxinus (L.) (Cypriniformes: Leuciscidae), and provided molecular evidence for the identification of the snail intermediate host. Phylogenetic analyses based on the cytochrome c oxidase subunit 1 (cox1) gene depicted 44 molecularly characterised species and genetically distinct lineages of Diplostomum, and resulted in: (i) a re-identification/re-classification of 98 isolates plus D. baeri sampled in North America; (ii) re-definition of the composition of the D. baeri species complex which now includes nine molecularly characterised species/lineages; (iii) re-definition of the composition of the D. mergi species complex which now includes seven molecularly characterised species/lineages; and (iv) an updated nomenclature for the molecularly characterised species-level lineages of Diplostomum.

Hidden parasite diversity in a European freshwater system.

Parasites comprise a huge part of the biodiversity on earth. However, on a local scale, not much is known about their diversity and community structure. Here, we assess the diversity of larval trematode communities in an interconnected freshwater system of the River Ruhr in Germany and analyse how the parasites are spatially and temporally distributed in the ecosystem. A total of 5347 snail hosts belonging to six species revealed a highly diverse parasite fauna with 36 trematode species. More abundant snail species harboured more species-rich trematode faunas and communities, with the two dominant snail species, Radix auricularia and Gyraulus albus, accounting for almost 90% of the trematode diversity and harbouring spatially and temporally stable parasite communities. The results highlight the important role of stable keystone host populations for trematode transmission, structure and diversity. This local trematode diversity reveals information on definitive host occurrence and trophic interactions within ecosystems.

The Systematics of the Trematoda.

The platyhelminth class Trematoda comprises two subclasses with largely disparate species diversity, the small Aspidogastrea with c.80 species and the speciose Digenea with c.18,000 species, which has attracted much effort towards our understanding of evolutionary relationships among suprageneric taxa. This chapter focuses on insights into the classification of the Digenea that have become apparent from our advanced understanding of both morphological and molecular data. The field of molecular systematics of the Digenea has experienced significant advances over the past 15 years. Phylogenetic analyses of sequence data predominantly from the 18S and 28S rRNA genes have incorporated a considerable diversity of taxa thus increasing the accuracy of phylogenetic inferences at higher taxonomic levels. As a result, the status of long-standing supraspecific taxa has been revised, new higher-level taxa have been defined, and inferences made in association with morphological and life-cycle evidence. A substantial effort has been made towards a classification reflecting the natural system of the Digenea by considering morphological evidence in conjunction with phylogenies inferred from molecular data; this has resulted in considerable congruence. However, limited taxon sampling in the phylogeny of the Digenea still remains relevant especially in relation to some higher-level taxa, and an outline of these omissions is presented. A framework that has led to robust estimates of phylogeny is outlined, and the application of advanced morphological and molecular approaches in digenean taxonomy and systematics is illustrated using the most comprehensively studied digenean superfamilies.

Drivers of parasite community structure in fishes of the continental shelf of the Western Mediterranean: the importance of host phylogeny and autecological traits.

We explored the relationships between features of host species and their environment, and the diversity, composition and structure of parasite faunas and communities using a large taxonomically consistent dataset of host-parasite associations and host-prey associations, and original environmental and host trait data (diet, trophic level, population density and habitat depth vagility) for the most abundant demersal fish species off the Catalonian coast of the Western Mediterranean. Altogether 98 species/taxa belonging to seven major parasite groups were recovered in 683 fish belonging to 10 species from seven families and four orders. Our analyses revealed that (i) the parasite fauna of the region is rich and dominated by digeneans; (ii) the host parasite faunas and communities exhibited wide variations in richness, abundance and similarity due to a strong phylogenetic component; (iii) the levels of host sharing were low and involved host generalists and larval parasites; (iv) the multivariate similarity pattern of prey samples showed significant associations with hosts and host trophic guilds; (v) prey compositional similarity was not associated with the similarity of trophically transmitted parasite assemblages; and (vi) phylogeny and fish autecological traits were the best predictors of parasite community metrics in the host-parasite system studied.

Species of Lepidapedon Stafford, 1904 (Digenea: Lepidapedidae) from deep-sea fishes in the Western Mediterranean: molecular and morphological evidence.

The species diversity of Lepidapedon Stafford, 1904 (Lepidapediae) in the Western Mediterranean was assessed based on samples from five deep-sea gadiform fishes collected between the Balearic Islands and the Catalonian coast of Spain: Lepidion lepidion (Risso) and Mora moro (Risso) (both Moridae); Coelorinchus mediterraneus Iwamoto & Ungaro and Trachyrincus scabrus (Rafinesque) (both Macrouridae); and Phycis blennoides (Brünnich) (Phycidae). Integration of morphological and molecular evidence (28S rRNA gene and the mitochondrial nad1 gene) revealed that the deep-sea fishes in the Western Mediterranean share two species of Lepidapedon. Lepidapedon desclersae Bray & Gibson, 1995 was recovered in all five fish species of three families [Moridae (L. lepidion and M. moro); Macrouridae (C. mediterraneus and T. scabrus); and Phycidae (P. blennoides)] and L. guevarai Lopez-Roman & Maillard, 1973 was recovered in three fish species of three families [Phycidae (P. blennoides); Macrouridae (T. scabrus); and Moridae (L. lepidion)]. Therefore, both species are considered to be euryxenic but restricted to gadiforms. The voucher material for the two species from different fish hosts is described in detail and the host-related variability based on the morphometric data is assessed.

Exploring the diversity of Diplostomum (Digenea: Diplostomidae) in fishes from the River Danube using mitochondrial DNA barcodes.

BACKGROUND: Metacercariae of Diplostomum are important fish pathogens, but reliable data on their diversity in natural fish populations are virtually lacking. This study was conducted to explore the species diversity and host-parasite association patterns of Diplostomum spp. in a large riverine system in Europe, using molecular and morphological data. METHODS: Twenty-eight species of fish of nine families were sampled in the River Danube at Nyergesújfalu in Hungary in 2012 and Stúrovo in Slovakia in 2015. Isolates of Diplostomum spp. were characterised morphologically and molecularly. Partial sequences of the 'barcode' region of the cytochrome c oxidase subunit 1 (cox1) and complete sequences of the nicotinamide adenine dinucleotide dehydrogenase subunit 3 (nad3) mitochondrial genes were amplified for 76 and 30 isolates, respectively. The partial cox1 sequences were used for molecular identification of the isolates and an assessment of haplotype diversity and possible host-associated structuring of the most prevalent parasite species. New primers were designed for amplification of the mitochondrial nad3 gene. RESULTS: Only lens-infecting Diplostomum spp. were recovered in 16 fish species of five families. Barcoding of representative isolates provided molecular identification for three species/species-level genetic lineages, D. spathaceum, D. pseudospathaceum and 'D. mergi Lineage 2', and three single isolates potentially representing distinct species. Molecular data helped to elucidate partially the life-cycle of 'D. mergi Lineage 2'. Many of the haplotypes of D. spathaceum (16 in total), D. pseudospathaceum (15 in total) and 'D. mergi Lineage 2' (7 in total) were shared by a number of fish hosts and there was no indication of genetic structuring associated with the second intermediate host. The most frequent Diplostomum spp. exhibited a low host-specificity, predominantly infecting a wide range of cyprinid fishes, but also species of distant fish families such as the Acipenseridae, Lotidae, Percidae and Siluridae. The nad3 gene exhibited distinctly higher levels of interspecific divergence in comparison with the cox1 gene. CONCLUSIONS: This first exploration of the species diversity and host ranges of Diplostomum spp., in natural fish populations in the River Danube, provided novel molecular, morphological and host-use data which will advance further ecological studies on the distribution and host ranges of these important fish parasites in Europe. Our results also indicate that the nad3 gene is a good candidate marker for multi-gene approaches to systematic estimates within the genus.

New molecular and morphological data for opecoelid digeneans in two Mediterranean sparid fishes with descriptions of Macvicaria gibsoni n. sp. and M. crassigula (Linton, 1910) (sensu stricto).

Molecular and morphological data were gathered for specimens of species of Macvicaria Gibson & Bray, 1982 and Pseudopycnadena Saad-Fares & Maillard, 1986 (Digenea: Opecoelidae) collected from two sparid fishes, Diplodus vulgaris (Geoffroy Saint-Hilaire) and Sparus aurata L., off the Algerian coast of the Western Mediterranean. Phylogenetic analyses based on ITS1-5.8S-ITS2 and partial 28S rDNA sequences provided evidence for the distinct species status of eight Mediterranean species of Macvicaria. Novel molecular data are provided for four species, M. gibsoni n. sp. and M. crassigula (Linton, 1910) (sensu stricto) based on specimens from D. vulgaris, and M. mormyri (Stossich, 1885) and M. maamouriae Antar, Georgieva, Gargouri & Kostadinova, 2015 based on specimens from S. aurata; descriptions of the molecular voucher material of the former three species are provided. Two species were delineated within the "crassigula" species complex of Macvicaria, M. gibsoni n. sp. and M. crassigula (s. str.), the distinctive differentiating features being the distribution of the dorsal vitelline fields in the forebody, confluent in M. gibsoni n. sp. and non-confluent in M. crassigula (s. str.), and the molecular data for both markers. Sequences associated with morphological description are also provided for the type-species of Pseudopycnadena, P. fischthali Saad-Fares & Maillard, 1986, based on material from D. vulgaris.

Skoulekia erythrini n. sp. (Digenea: Aporocotylidae): a parasite of Pagellus erythrinus (L.) (Perciformes: Sparidae) from the western Mediterranean with an amendment of the generic diagnosis.

A new aporocotylid, Skoulekia erythrini n. sp., is described from the heart, cephalic kidney and gill blood vessels of the common pandora Pagellus erythrinus (L.) collected from several localities of the western Mediterranean off Spain. The new species differs from the type- and only species of the genus, S. meningialis Alama-Bermejo, Montero, Raga & Holzer, 2011 in possessing a symmetrical body (vs laterally curved), short tegumental spines (3-4 vs 7-10 µm) without hooked ends, diffuse (vs conspicuous) oesophageal gland-cells, a relatively longer oesophagus, a testis that is shorter in relation to body length and a much smaller seminal vesicle (17-34 × 10-26 vs 33-101 × 27-97 µm). Elongated ellipsoidal eggs of S. erythrini n. sp. were found trapped in gill vessels in histological sections. Phylogenetic analyses based on partial 28S rDNA and ITS2 sequences supported the placement of the new species within Skoulekia and the close relationships of this genus with Psettarium Goto & Ozaki, 1929 and Pearsonellum Overstreet & Køie, 1986. Skoulekia meningialis is described from a new sparid host, Diplodus puntazzo (Walbaum), collected off Santa Pola, Spain. The new morphological data for the two Skoulekia spp. and a re-examination of three paratypes of S. meningialis pinpointed features amending both the description of S. meningialis and the generic diagnosis of Skoulekia.

Molecular analyses reveal high species diversity of trematodes in a sub-Arctic lake.

To identify trematode diversity and life-cycles in the sub-Arctic Lake Takvatn, Norway, we characterised 120 trematode isolates from mollusc first intermediate hosts, metacercariae from second intermediate host fishes and invertebrates, and adults from fish and invertebrate definitive hosts, using molecular techniques. Phylogenies based on nuclear and/or mtDNA revealed high species richness (24 species or species-level genetic lineages) and uncovered trematode diversity (16 putative new species) from five families typical in lake ecosystems (Allocreadiidae, Diplostomidae, Plagiorchiidae, Schistosomatidae and Strigeidae). Sampling potential invertebrate hosts allowed matching of sequence data for different stages, thus achieving molecular elucidation of trematode life-cycles and exploration of host-parasite interactions. Phylogenetic analyses also helped identify three major mollusc intermediate hosts (Radix balthica, Pisidium casertanum and Sphaerium sp.) in the lake. Our findings increase the known trematode diversity at the sub-Arctic Lake Takvatn, showing that digenean diversity is high in this otherwise depauperate sub-Arctic freshwater ecosystem and indicating that sub-Arctic and Arctic ecosystems may be characterised by unique trematode assemblages.

Molecular characterisation of four echinostomes (Digenea: Echinostomatidae) from birds in New Zealand, with descriptions of Echinostoma novaezealandense n. sp. and Echinoparyphium poulini n. sp.

Morphological and molecular characterisation of echinostome specimens (Digenea: Echinostomatidae) recovered in one Anas platyrhynchos L. and one Cygnus atratus (Latham) (Anseriformes: Anatidae) from New Zealand revealed the presence of two known species, Echinostoma miyagawai Ishii, 1932 and Echinoparyphium ellisi (Johnston & Simpson, 1944) and two species new to science. Comparative morphological and phylogenetic analyses supported the distinct species status of Echinostoma novaezealandense n. sp. ex Branta canadensis (L.), A. platyrhynchos and C. atratus, and Echinoparyphium poulini n. sp. ex C. atratus. Echinostoma novaezealandense n. sp., a species of the "revolutum" species complex characterised by the possession of a head collar armed with 37 spines, keyed down to E. revolutum but was distinguished from the latter in having a much narrower body with almost parallel margins, longer oesophagus, wider cirrus-sac, larger seminal vesicle, much smaller ventral sucker, ovary, Mehlis' gland and testes, more anteriorly located ovary and testes, and distinctly smaller eggs (81-87 × 42-53 vs 106-136 × 55-70 µm). This new species appears similar to Echinostoma acuticauda Nicoll, 1914 described in Australia but differs in having a longer forebody, more posteriorly located ovary and testes, and much smaller eggs (81-87 × 42-53 vs 112-126 × 63-75 µm). Echinoparyphium poulini n. sp. is differentiated from the four species of Echinoparyphium possessing 37 collar spines considered valid as follows: from E. chinensis Ku, Li & Chu, 1964 in having a much smaller body, four (vs five) angle spines and simple seminal vesicle (vs bipartite); from E. schulzi Matevosyan, 1951 in having a less robust body at a comparable body length, much smaller ventral sucker, ovary and testes, and longer but narrower eggs (87-109 × 50-59 vs 70-85 × 60-84 µm); and from the two smaller forms, E. serratum Howell, 1968 and E. aconiatum Dietz, 1909, in a number of additional metrical features correlated with body size and especially in the possession of much larger collar spines. Partial fragments of the mitochondrial nad1 and 28S rRNA genes were amplified for representative isolates of the four species and analysed together with sequences for Echinostoma spp. and Echinoparyphium spp. available on GenBank. Phylogenetic analyses based on the mitochondrial nad1 gene revealed congruence between the molecular data and species identification/delineation based on morphology; this was corroborated by the 28S rDNA sequence data.

Morphology and molecules reveal the alien Posthodiplostomum centrarchi Hoffman, 1958 as the third species of Posthodiplostomum Dubois, 1936 (Digenea: Diplostomidae) in Europe.

Metacercariae of two species of Posthodiplostomum Dubois, 1936 (Digenea: Diplostomidae) were subjected to morphological and molecular studies: P. brevicaudatum (von Nordmann, 1832) from Gasterosteus aculeatus (L.) (Gasterosteiformes: Gasterosteidae), Bulgaria (morphology, cox1 and ITS1-5.8S-ITS2) and Perca fluviatilis L. (Perciformes: Percidae), Czech Republic (morphology, cox1, ITS1-5.8S-ITS2 and 28S); and P. centrarchi Hoffman, 1958 from Lepomis gibbosus (L.) (Perciformes: Centrarchidae), Bulgaria (morphology, cox1 and ITS1-5.8S-ITS2) and Slovakia (cox1 and ITS1-5.8S-ITS2). In addition, cercariae of P. cuticola (von Nordmann, 1832) from Planorbis planorbis (L.) (Mollusca: Planorbidae), Lithuania (morphology and cox1) and metacercariae of Ornithodiplostomum scardinii (Schulman in Dubinin, 1952) from Scardinius erythrophthalmus (L.) (Cypriniformes: Cyprinidae), Czech Republic, were examined (morphology, cox1, ITS1-5.8S-ITS2 and 28S). These represent the first molecular data for species of Posthodiplostomum and Ornithodiplostomum Dubois, 1936 from the Palaearctic. Phylogenetic analyses based on cox1 and ITS1-5.8S-ITS2, using O. scardinii as the outgroup and including the three newly-sequenced Posthodiplostomum spp. from Europe and eight published unidentified (presumably species-level) lineages of Posthodiplostomum from Canada confirmed the distinct status of the three European species (contrary to the generally accepted opinion that only P. brevicaudatum and P. cuticola occur in the Palaearctic). The subspecies Posthodiplostomum minimum centrarchi Hoffmann, 1958, originally described from North America, is elevated to the species level as Posthodiplostomum centrarchi Hoffman, 1958. The undescribed "Posthodiplostomum sp. 3" of Locke et al. (2010) from centrarchid fishes in Canada has identical sequences with the European isolates of P. centrarchi and is recognised as belonging to the same species. The latter parasite, occurring in the alien pumpkinseed sunfish Lepomis gibbosus in Europe, is also supposed to be alien for this continent. It is speculated that it colonised Europe long ago and is currently widespread (recorded in Bulgaria, Slovakia and Spain); based on the cox1 sequence of an adult digenean isolate from the Ebro Delta, Spain, only the grey heron (Ardea cinerea L.) (Ciconiiformes: Ardeidae) is known to be its definitive host in Europe.

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.

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.