Molecular approaches to trematode systematics: 'best practice' and implications for future study

To date, morphological analysis has been the cornerstone to trematode systematics. However, since the late-1980s we have seen an increased integration of genetic data to overcome problems encountered when morphological data are considered in isolation. Here, we provide advice regarding the ‘best molecular practice’ for trematode taxonomy and systematic studies, in an attempt to help unify the field and provide a solid foundation to underpin future work. Emphasis is placed on defining the study goals and recommendations are made regarding sample preservation, extraction methods, and the submission of molecular vouchers. We advocate generating sequence data from all parasite species/host species/geographic location combinations and stress the importance of selecting two independently evolving loci (one ribosomal and one mitochondrial marker). We recommend that loci should be chosen to provide genetic variation suitable to address the question at hand and for which sufficient ‘useful’ comparative sequence data already exist. Quality control of the molecular data via using proof-reading Taq polymerase, sequencing PCR amplicons using both forward and reverse primers, ensuring that a minimum of 85% overlap exists when constructing consensus sequences, and checking electropherograms by eye is stressed. We advise that all genetic results are best interpreted using a holistic biological approach, which considers morphology, host identity, collection locality, and ecology. Finally, we consider what advances next-generation sequencing holds for trematode taxonomy and systematics

Description of a new species of Crassicutis Manter, 1936, parasite of Cichlasoma beani Jordan (Osteichthyes: Cichlidae) in Mexico, based on morphology and sequences of the ITS1 and 28S ribosomal RNA genes.

A new species of Crassicutis Manter, 1936 is described from the Sinaloan cichlid Cichlasoma beani (Jordan) (Osteichthyes: Cichlidae) in the upper Rıo Santiago basin. Crassicutis choudhuryi n. sp. differs from most of the other nominal species by having testes located in a symmetrical position. The only other species of the genus that includes some specimens exhibiting this trait is Crassicutis intermedius (Szidat 1954), a species found in 5 species of siluriforms and 1 species of characiform in South America. However, this species differs from Cr. Choudhuryi n. sp. by having testes almost half of the size, and vitelline follicles extending anteriorly to the region between the acetabulum and the intestinal bifurcation. The new species is morphologically very similar to Crassicutis cichlasomae Manter, 1936, but clearly differs from this species because of the constantly symmetrical position of the testes. Additionally, Cr. Choudhuryi n. sp. is found in the Santiago River basin on the Pacific slope of Mexico, parasitizing specifically the endemic Ci. Beani that does not co-occur with any other cichlid. Cr. Cichlasomae exhibits more hosts (about 25 species of cichlids only in Mexico) and a wider distribution range that extends from northeastern Mexico southward to Central America, Cuba, and Brazil. To corroborate that our specimens were not conspecific with Cr. Cichlasomae, sequences of the internal transcribed spacer (ITS1) and the 28S ribosomal RNA genes of individuals from several populations (recently collected in southeastern Mexico) were obtained and compared to the species described herein. Sequence divergence (1.3% for the 28S and 4.0% for the ITS1) gives further support to the erection of a new species

Gynichthys diakidnus n.g., n.sp. (Digenea: Cryptogonimidae) from the grunt Plectorhinchus gibbosus (Lacepede, 1802) (Perciformes: Haemulidae) off the Great Barrier Reef

Gynichthys diakidnus n. g., n. sp. (Digenea: Cryptogonimidae) is described from the fish Plectorhinchus gibbosus (Lac,pSde) (Perciformes: Haemulidae) off Heron and Lizard Islands on the Great Barrier Reef, Australia. The monotypic Gynichthys n. g. is distinguished from all other cryptogonimid genera by the combination of a fusiform body, the lack of oral spines, a forebody that occupies approximately half or more of the body length, a deeply lobed ovary, opposite to slightly oblique testes, a seminal vesicle that is confined mainly in the forebody and the presence of multiple gonotyls in the form of two small slightly muscular pores or pseudosucker-like structures in the mid-line well anterior to the ventral sucker. Bayesian inference analysis of LSU rDNA data revealed that G. diakidnus n. sp. grouped relatively distant to species of the cryptogonimid genus Oligogonotylus Watson, 1976, which also have multiple gonotyls, suggesting that the presence of multiple gonotyls is homoplasious and has thus at least evolved twice in the family. The secondary structure of the internal transcribed spacer 2 (ITS2) rDNA region was inferred for G. diakidnus using minimum free energy and homology modelling algorithms. A four helix model was inferred with helices I and IV being relatively short (< 30 nucleotides) and helix three being the longest; this structure is homologous with that observed for other digeneans and eukaryotes in general

Host specificity, molecular phylogeny and morphological differences of Phyllodistomum pseudofolium Nybelin, 1926 and Phyllodistomum angulatum Linstow, 1907 (Trematoda: Gorgoderidae) with notes on Eurasian ruffe as final host for Phyllodistomum spp.

Abstract Background Host-specificity patterns are not well-defined for trematodes of the genus Phyllodistomum Braun, 1899. The Eurasian ruffe, Gymnocephalus cernuus L., has been recorded as a definitive host for Phyllodistomum folium (Olfers, 1816), P. angulatum Linstow, 1907 and P. megalorchis Nybelin, 1926 and as the type-host for P. pseudofolium Nybelin (1926). A wide range of other host fishes have been recorded for these species as well. All present host records have been based on light microscopy and the life-cycles of P. pseudofolium, P. angulatum and P. megalorchis are unknown. The validity of P. pseudofolium and P. megalorchis require verification. In this study, rDNA sequences generated from adult Phyllodistomum spp., as well as from larval stages developing in Pisidium amnicum Müller, were analysed to establish the real number of Phyllodistomum species utilizing G. cernuus, and to associate larvae with the corresponding adult forms. Results Phylogenetic analyses of adult and larval stages of Phyllodistomum spp. based on ITS2 and partial 28S rDNA data allowed the confirmation of the validity of P. pseudofolium. A macrocercous cercaria, known as Phyllodistomum sp. from P. amnicum is genetically identical to adult P. pseudofolium. Phyllodistomum megalorchis obtained from its type-host, Lota lota L., showed no genetic differences from P. angulatum parasitizing Sander lucioperca L. In our analysis, P. pseudofolium, P. angulatum and P. macrocotyle formed a highly supported clade despite the fact that these species appear to be associated with distinct patterns of first intermediate host identity and cercarial morphology. Some morphological differences between gravid specimens of P. pseudofolium and P. angulatum were observed and their SEM tegumental surface topography is described. Conclusions The results lead us to the perception that macroevolutionary host switching in the genus Phyllodistomum is independent of host phylogeny. This study suggests strict host-specificity (oioxeny) for P. pseudofolium using one first intermediate host species (P. amnicum) and one definitive host species (G. cernuus). Phyllodistomum megalorchis is to be regarded as a synonym of P. angulatum. The close phylogenetic relatives, P. pseudofolium and P. angulatum, can be differentiated by morphological traits, the micromorphology and tegumental surface topography of these two species is intended to provide useful data for their identification and support the use of such features as a valuable taxonomic criterion. Molecular data showed that G. cernuus is a definitive host for two species: the oioxenous P. pseudofolium and the euryxenous P. folium