Convoluted history and confusing morphology: Molecular phylogenetic analysis of dicrocoeliids reveals true systematic position of the Anenterotrematidae Yamaguti, 1958 (Platyhelminthes,Digenea)

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.     

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.     

Filling the gaps in the classification of the Digenea Carus, 1863: systematic position of the Proterodiplostomidae Dubois, 1936 within the superfamily Diplostomoidea Poirier, 1886, inferred from nuclear and mitochondrial DNA sequences

The Diplostomida Olson, Cribb, Tkach, Bray & Littlewood, 2003 is the less diverse order of the two orders within the subclass Digenea Carus, 1863 and is currently classified into three superfamilies, i.e. Brachylaimoidea Joyeux & Foley, 1930, Diplostomoidea Poirier, 1886, and Schistosomatoidea Stiles & Hassall, 1898. Although the suprageneric-level relationships have been elucidated with the use of molecular markers, the lack of representation of some groups obscure the phylogenetic relationships among families, rendering the classification unstable. Here, we tested the phylogenetic position of the family Proterodiplostomidae Dubois, 1936 based on partial 28S rDNA and complete 18S rDNA sequences for Crocodilicola pseudostoma (Willemoes-Suhm, 1870), a crocodile parasite that has been found as a progenetic metacercaria parasitising the pale catfish Rhamdia guatemalensis (Günther) in Mexico and in other siluruforms in the Neotropics. We augmented the representation of the species, genera and families within the Diplostomida, including mostly representatives of the superfamily Diplostomoidea, and assembled a dataset that contains 49 species for the 28S rRNA gene, and 45 species for the 18S rRNA gene. Additionally, we explored the phylogenetic signal of the mitochondrial gene cox1 in reconstructing the phylogenetic relationships of selected members of the superfamily. Our analyses showed that the family Proterodiplostomidae is the sister taxon to the paraphyletic Diplostomidae Poirier, 1886 and Strigeidae Railliet, 1919, with Cyathocotylidae Mühling, 1898 + Brauninidae Wolf, 1903 as their sister group. Analysis of concatenated 18S + 28S sequences revealed the Liolopidae Odhner, 1912 as the basal group of the superfamily Diplostomoidea, although analyses of independent datasets showed that the position of this family remains uncertain. Analysis based on cox1 unequivocally resolved the Proterodiplostomidae as the sister taxon to the Diplostomidae and Strigeidae, although the Cyathocotylidae was nested in a different clade, along with brachylaimoids and schistosomatoids.     

Phylogenetic relationships of Ochoterenatrema Caballero, 1943 (Digenea: Lecithodendriidae) with descriptions of two new species

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.     

Morphological and molecular differentiation of Parastrigea (Trematoda: Strigeidae) from Mexico,with the description of a new species

Parastrigea plataleae n. sp. (Digenea: Strigeidae) is described from the intestine of the roseate spoonbill Platalea ajaja (Threskiornithidae) from four localities on the Pacific coast of Mexico. The new species is mainly distinguished from the other 18 described species of Parastrigea based on the ratio of its hindbody length to forebody length. A principal component analysis (PCA) of 16 morphometric traits for 15 specimens of P. plataleae n. sp., five of Parastrigea cincta and 11 of Parastrigea diovadena previously recorded in Mexico, clearly shows three clusters, which correspond to the three species. DNA sequences of the internal transcribed spacers (ITSs) of ribosomal DNA and the mitochondrial gene cytochrome c oxidase subunit I (cox 1) were used to corroborate this morphological distinction. The genetic divergence estimated among P. plataleae n. sp., P. cincta and P. diovadena ranged from 0.5 to 1.48% for ITSs and from 9.31 to 11.47% for cox 1. Maximum parsimony (MP) and maximum likelihood (ML) analyses were performed on the combined datasets (ITSs + cox 1) and on each dataset alone. All of the phylogenetic analyses indicated that the specimens from the roseate spoonbill represent a clade with strong bootstrap support. The morphological evidence and the genetic divergence in combination with the reciprocal monophyly in all of the phylogenetic trees support the hypothesis that the digeneans found in the intestines of roseate spoonbills represent a new species.     

Occurrence and Molecular Identification of Microcotyle sebastis Isolated from Fish Farms of the Korean Rockfish,Sebastes schlegelii

Microcotyle sebastis is a gill monogenean ectoparasite that causes serious problems in the mariculture of the Korean rockfish, Sebastes schlegelii. In this study, we isolated the parasite from fish farms along the coasts of Tongyeong, South Korea in 2016, and characterized its infection, morphology and molecular phylogeny. The prevalence of M. sebastis infection during the study period ranged from 46.7% to 96.7%, and the mean intensity was 2.3 to 31.4 ind./fish, indicating that the fish was constantly exposed to parasitic infections throughout the year. Morphological observations under light and scanning electron microscopes of the M. sebastis isolates in this study showed the typical characteristics of the anterior prohaptor and posterior opisthaptor of monogenean parasites. In phylogenetic trees reconstructed using the nuclear 28S ribosomal RNA gene and the mitochondrial cytochrome c oxidase I gene (cox1), they consistently clustered together with their congeneric species, and showed the closest phylogenetic relationships to M. caudata and M. kasago in the cox1 tree.     

Phylogenetic analysis of nuclear and mitochondrial DNA reveals a complex of cryptic species in Crassicutis cichlasomae (Digenea: Apocreadiidae), a parasite of Middle-American cichlids

We obtained nuclear ITS-1 and mitochondrial cox1 sequences from 225 Crassicutis cichlasomae adults collected in 12 species of cichlids from 32 localities to prospect for the presence of cryptic species. This trematode is commonly found in species of cichlids over a wide geographic range in Middle-America. Population-level phylogenetic analyses of ITS-1 and cox1, assessments of genetic and haplotype diversity, and morphological observations revealed that C. cichlasomae represents a complex of seven cryptic species for which no morphological diagnostic characters have been discovered thus far. Bayesian and Maximum Likelihood analyses of concatenated datasets (906 bp) recovered eight lineages of C. cichlasomae, all with high posterior probabilities and bootstrap branch support. Values of genetic divergence between clades ranged from 1.0% to 5.2% for ITS-1, and from 7.2% to 30.0% for cox1. Morphological study of more than 300 individuals did not reveal structural diagnostic traits for the species defined using molecular evidence. These observations indicate that some traditional morphological characters (e.g., testes position) have substantial intra-specific variation, and should be used with caution when classifying C. cichlasomae and their sister taxa. Additionally, phylogenetic analyses did not reveal a strict correlation between these cryptic species and their host species or geographic distribution, however it appears that genetic distinctiveness of these cryptic species was influenced by the diversification and biogeographical history of Middle-American cichlids.     

Phylogenetic Positioning of a Strongyloides stercoralis Isolate Recovered from a Korean Patient and Comparison with Other Asian Isolates

Strongyloidiasis is caused by Strongyloides stercoralis and is one of the most neglected tropical diseases in tropical and subtropical regions. Although several strongyloidiasis cases have been reported in Korea, genetic analysis of Korean isolates is still incomplete. In this study, a parasite was isolated from a 61-year-old man diagnosed with strongyloidiasis during the treatment of lymphoma on his retroperitoneal lymph node. Diffuse symmetric wall thickening from the ascending to descending colon and a nematode-infected intestine was observed following microscopic examination. Genomic DNA was isolated from a patient tissue block, and S. stercoralis was identified by PCR and sequencing (18S rDNA). In order to determine phylogenetic location of a Korean isolate (named KS1), we analyzed cox1 gene (500-bp) and compared it with that from 47 previous S. stercoralis isolates (28 human isolates and 19 canid isolates) from Asian countries. Our results showed that phylogenetic tree could clearly be divided into 5 different groups according to hosts and regions. KS1 was most closely related with the Chinese isolates in terms of genetic distance.     

Morphological differences and molecular phylogenetic relationship of two tapeworm species,Moniezia expansa and Moniezia benedeni,collected from domestic ruminants in northern Vietnam

Moniezia expansa and M. benedeni are two common tapeworm species of domestic ruminants over the world. However, their morphological and molecular data are available for limited specimens from a few countries. In the present study, we compared morphological characteristics of these two species collected from goats and cattle in northern Vietnam and analyzed their phylogenetic relationship based on the 5.8S and second internal transcribed spacer (ITS2) of nuclear ribosomal DNA and the mitochondrial cytochrome c oxidase subunit I gene (cox1). The two species are clearly distinguishable from one another not only in the morphological appearance of the interproglottidal glands but also in the gross appearance of mature and gravid proglottids. Molecular analyses revealed that the 5.8S-ITS2 sequences of Vietnamese M. expansa were highly similar (99.7%) to the sequences from Japan and India, and made a common clade, which was clearly distinct from M. benedeni of Vietnam. For cox1 sequences, Vietnamese M. expansa showed a high similarity to and were grouped with the sequences from Ethiopia and some sequences from Senegal and China to make a common clade, which was separated from the remaining clades of Senegal and China. The cox1 sequences of M. benedeni from China, Vietnam, and Senegal were far distant (10.0–15.9%) from each other. The results of this study suggest that more sequence data of Moniezia species with details of morphological features from various geographical locations should be obtained to clarify the taxonomic status of Moniezia species.     

Multigene phylogenetic analysis reveals non-monophyly of Anisakis s.l. and Pseudoterranova (Nematoda: Anisakidae)

The nematode genera Anisakis s.l. and Pseudoterranova (Anisakidae) include causative agents of anisakiasis and pseudoterranovosis, parasitic diseases resulting from eating undercooked or raw fish or squid. Species in both genera have thus attracted considerable attention especially in public health and taxonomic studies. The phylogenetic relationships of these genera within the subfamily Anisakinae, however, remain to be investigated with dense taxonomic sampling. In this study, we collected an anisakid third-stage larva, and identified it morphologically and molecularly as Pseudoterranova ceticola. Phylogeny of 15 anisakine species, including the newly collected specimen of Ps. ceticola, was reconstructed based on sequences of three mitochondrial (cox1, cox2, and 12S rRNA) and two nuclear (ITS and 28S rRNA) regions. The obtained tree suggested the non-monophyly of Anisakis s.l. and Pseudoterranova. Anisakis s.l. was divided into two groups, which are distinguished from each other by the shape of the ventriculus. Based on phylogenetic relationships and morphology, three species with a shorter ventriculus (“A.” brevispiculata, “A.” paggiae, and “A.” physeteris) were assigned to the genus Skrjabinisakis, as recently proposed. Pseudoterranova ceticola was distantly related to the monophyletic Ps. decipiens species complex. Although the phylogenetic position of the type species Ps. kogiae has not been investigated due to a lack of sequence data, this species may morphologically and ecologically resemble Ps. ceticola, inferring a close kinship between the two species.     

Morphology and molecules reveal the alien <Emphasis Type="Italic">Posthodiplostomum centrarchi</Emphasis> Hoffman, 1958 as the third species of <Emphasis Type="Italic">Posthodiplostomum</Emphasis> 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.     

Morphological and genetic characterization of Porrocaecum angusticolle (Molin, 1860) (Nematoda: Ascaridomorpha) from the common buzzard Buteo buteo (Linnaeus) (Accipitriformes: Accipitridae) in Czech Republic

Porrocaecum angusticolle is a nematode species mainly parasitic in the birds of Accipitriformes and Strigiformes. However, some aspects of the morphology of P. angusticolle remain insufficiently known. In the present study, the detailed morphology of P. angusticolle was studied using light and, for the first time, scanning electron microscopy, based on newly collected specimens from the common buzzard Buteo buteo (Linnaeus) (Accipitriformes: Accipitridae) in Czech Republic. Some previously unreported morphological features of taxonomic significance were observed. The nuclear and mitochondrial DNA markers, including partial large ribosomal DNA (28S), complete internal transcribed spacer (ITS-1 + 5.8S + ITS-2), cytochrome c oxidase subunit 1 (cox1) and subunit 2 (cox2) of P. angusticolle were sequenced for molecular identification of this species. There was no intraspecific genetic variation detected in the 28S and ITS regions among different individuals of P. angusticolle, but low level of intraspecific nucleotide divergence was found in the cox1 (0.26–0.78%) and cox2 regions (1.0%). The 28S and cox2 of P. angusticolle were sequenced for the first time. Our molecular evidence supported the validity of both P. angusticolle and P. depressum. The newly obtained genetic data are helpful for further studies of DNA-based taxonomy, population genetics and phylogeny of the genus of Porrocaecum.     

Molecular data reveal a new species of Seuratascaris Sprent, 1985 (Nematoda: Ascaridoidea) from Quasipaa exilispinosa (Liu & Hu) (Amphibia: Anura)

The genus Seuratascaris Sprent, 1985 is a group of obligate nematode parasites of amphibians. In the present study, a new species of Seuratascaris, S. physalis sp. n. was described using light and scanning electron microscopy based on specimens collected from Quasipaa exilispinosa (Liu & Hu) (Amphibia: Anura) in China. The new species differs from S. numidica (Seurat, 1917) by the cuticle of the cervical region distinctly inflated to form a cephalic vesicle-like structure and the absence of single medio-ventral precloacal papilla. The molecular characterization of the nuclear large ribosomal DNA (28S) and internal transcribed spacer (ITS) and the mitochondrial cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2) and 12S small subunit ribosomal RNA gene of S. physalis sp. n., together with the 28S, cox2 and 12S of S. numidica are provided for the first time. Molecular analysis revealed the presence of high level of interspecific genetic variation between the two species in the ITS (5.50%), cox1 (13.3%), cox2 (10.6%) and 12S regions (10.5%), which strongly supported that S. physalis sp. n. represented a different species from S. numidica. Angusticaecum ranae Wang, Zhao & Chen, 1978 reported from the frog Quasipaa spinosa (David) (Anura: Dicroglossidae) in China was transferred into the genus Seuratascaris as S. ranae (Wang, Zhao & Chen, 1978) comb. n. based on the morphology of lips and the presence of very short and robust spicules without alae and small numbers of precloacal papillae. The present study provided useful genetic data for molecular identification of species of Seuratascaris and provides the foundation for being able to determine if S. numidica represents a species complex of some sibling species or a single species.     

MOLECULAR PHYLOGENY OF GRACILARIA SPECIES INFERRED FROM MOLECULAR MARKERS BELONGING TO THREE DIFFERENT GENOMES1

The nucleotide sequence data of molecular markers 18S rRNA, RUBISCO spacer, and cox2‐3 intergenic spacer were integrated to infer the phylogeny of Gracilaria species, collected from the western coast of India, reducing the possibility of misidentification and providing greater phylogenetic resolution. A phylogenetic tree was constructed using cox2‐3 and RUBISCO spacer sequences, exhibiting the same clustering but differing slightly from that of the rRNA‐based phylogenetic tree. The phylogeny inferred from the combined data set confers an analogous pattern of clustering, compared with those of trees constructed from individual data sets. The combined data set resulted in a phylogeny with better resolution, which supported the clade with higher consistency index, retention index, and bootstrap values. It was observed that Gracilaria foliifera (Forssk.) Børgesen is closer to G. corticata (J. Agardh) J. Agardh varieties, while G. salicornia (C. Agardh) E. Y. Dawson and G. fergusonii J. Agardh both originated from the same clade. The position of G. textorii (Suringar) De Toni faltered and toppled between G. salicornia and G. dura (C. Agardh) J. Agardh; however, G. gracilis (Stackh.) M. Steentoft, L. M. Irvine et W. F. Farnham was evidently distant from the rest of the species.     

Characterization of the Cystoid Nematode Meloidoderita kirjanovae (Nemata: Sphaeronematidae) from Southern Italy

A population of the cystoid nematode Meloidoderita kirjanovae was detected parasitizing water mint (Mentha aquatica) in southern Italy. The morphological identification of this species was confirmed by molecular analysis using the internal transcribed spacer 1 (ITS1) and 5.8S gene sequences of nuclear ribosomal DNA (rDNA), which clearly separated it from the closely related species Meloidoderita polygoni. A phylogenetic analysis of M. kirjanovae with species of related genera was conducted using sequences of the D2-D3 expansion segments of the 28S nuclear ribosomal RNA gene. The resulting phylogenetic tree was congruent with trees from an extended dataset for Criconematina and Tylenchida. The basal position of the genus Meloidoderita together with Sphaeronema within the Criconematina clade in this tree may indicate their close relationships. The anatomical changes induced by M. kirjanovae population from Italy in water mint were similar to those reported for a nematode population infecting roots of M. longifolia in Israel. Nematode feeding caused the formation of a stellar syncytium that disorganized the pericycle and vascular root tissues.     

Two avian schistosome cercariae from Nepal,including a Macrobilharzia-like species from Indoplanorbis exustus

As part of a global survey of schistosomes, a total of 16,109 freshwater snails representing 14 species were collected from lakes, ponds, rivers, rice fields and swamps mostly in the Terai region of southern Nepal. Only two snails were found to harbor avian schistosome cercariae even though Nepal is well known for its rich avian diversity. One schistosome infection was from an individual of Radix luteola and on the basis of phylogenetic analyses using 28S rDNA and cox1 sequences, grouped as a distinctive and previously unknown lineage within Trichobilharzia. This genus is the most speciose within the family Schistosomatidae. It includes 40 described species worldwide, and its members mostly infect anseriform birds (ducks) and two families of freshwater snails (Lymnaeidae and Physidae). The second schistosome cercaria was recovered from an individual of Indoplanorbis exustus that was also actively emerging a Petasiger-like echinostome cercaria. Although I. exustus is commonly infected with mammalian schistosomes of the Schistosoma indicum species group on the Indian subcontinent, this is the first specifically documented avian schistosome reported in this snail. Both cercariae reported here are among the largest of all schistosome cercariae recovered to date. The I. exustus-derived schistosome clustered most closely with Macrobilharzia macrobilharzia, although it seems to represent a distinct lineage. Specimens of Macrobilharzia have thus far not been recovered from snails, being known only as adult worms from anhingas and cormorants. This study is the first to characterize by sequence data avian schistosomes recovered from Asian freshwater habitats. This approach can help unravel the complex of cryptic species causing cercarial dermatitis here and elsewhere in the world.     

Synergistic effects of combining morphological and molecular data in resolving the phylogenetic position of Semispathidium (Ciliophora,Haptoria) with description of Semispathidium breviarmatum sp. n. from tropical Africa

We describe a new species, Semispathidium breviarmatum sp. n., from tropical Africa and analyse its phylogenetic position within the subclass Haptoria, using live observation, various silver impregnation methods, SEM and the 18S rRNA gene. Semispathidium breviarmatum differs from its congeners by the much higher number of ciliary rows and by the shape and size of the extrusomes, that is, extrusive organelles that kill the prey. The phylogenetic position of Semispathidium is controversial due to its ‘hybrid’ morphology. Specifically, the cylindroidal body has a more or less discoidal oral bulge indicating an enchelyodonid origin, while the anteriorly curved somatic kineties suggest a spathidiid ancestor. In order to reconstruct the evolutionary history of Semispathidium and to unravel its affinity to other haptorians, we used synergistic effects of combining morphological and molecular data coming from 34 haptorian taxa. These analyses show that Semispathidium belongs to the order Spathidiida representing a basal lineage that is far from ordinary Spathidium species, but very likely related to Protospathidium and Enchelys. Any closer phylogenetic relationship between Semispathidium and Enchelyodon spp. is not recognized in morphological and molecular phylogenies and is consistently excluded by statistical tree topology tests.     

Phylogenetic relationships among Neoechinorhynchus species (Acanthocephala: Neoechinorhynchidae) from North-East Asia based on molecular data

Phylogenetic and statistical analyses of DNA sequences of two genes, cytochrome oxidase subunit 1 (cox 1) of the mitochondrial DNA and 18S subunit of the nuclear ribosomal RNA (18S rRNA), was used to characterize Neoechinorhynchus species from fishes collected in different localities of North-East Asia. It has been found that four species can be clearly recognized using molecular markers—Neoechinorhynchus tumidus, Neoechinorhynchus beringianus, Neoechinorhynchus simansularis and Neoechinorhynchus salmonis. 18S sequences ascribed to Neoechinorhynchus crassus specimens from North-East Asia were identical to those of N. tumidus, but differed substantially from North American N. crassus. We renamed North-East Asian N. crassus specimens to N. sp., although the possibility that they represent a subspecies of N. tumidus cannot be excluded, taking into account a relatively small distance between cox 1 sequences of North-East Asian specimens of N. crassus and N. tumidus. Maximum likelihood, maximum parsimony and Bayesian inference analyses were performed for phylogeny reconstruction. All the phylogenetic trees showed that North-East Asian species of Neoechinorhynchus analyzed in this study represent independent clades, with the only exception of N. tumidus and N. sp. for 18S data. Phylogenetic analysis has shown that the majority of species sampled (N. tumidus + N. sp., N. simansularis and N. beringianus) are probably very closely related, while N. salmonis occupies separate position in the trees, possibly indicating a North American origin of this species.