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

Making the most of mitochondrial genomes - Markers for phylogeny, molecular ecology and barcodes in Schistosoma (Platyhelminthes : Digenea)

An increasing number of complete sequences of mitochondrial(mt) genomes provides the opportunity to optimise the choice of molecular markers for phylogenetic and ecological studies. This is particularly the case where mt genomes from closely related taxa have been sequenced; e.g., within Schistosoma. These blood flukes include species that are the causative agents of schistosomiasis, where there has been a need to optimise markers for species and strain recognition. For many phylogenetic and population genetic studies, the choice of nucleotide sequences depends primarily on suitable PCR primers. Complete mt genomes allow individual gene or other mt markers to be assessed relative to one another for potential information content, prior to broad-scale sampling. We assess the phylogenetic utility of individual genes and identify regions that contain the greatest interspecific variation for molecular ecological and diagnostic markers. We show that variable characters are not randomly distributed along the genome and there is a positive correlation between polymorphism and divergence. The mt genomes of African and Asian schistosomes were compared with the available intraspecific dataset of Sehistosoma mansoni through sliding window analyses, in order to assess whether the observed polymorphism was at a level predicted from interspecific comparisons. We found a positive correlation except for the two genes (cox1 and nad1) adjoining the putative control region in S. mansoni. The genes nad1, nad4, nad5, cox1 and cox3 resolved phylogenies that were consistent with a benchmark phylogeny and in general, longer genes performed better in phylogenetic reconstruction. Considering the information content of entire mt genome sequences, partial cox1 would not be the ideal marker for either species identification (barcoding) or population studies with Schistosoma species. Instead, we suggest the use of cox3 and nad5 for both phylogenetic and population studies. Five primer pairs designed against Schistosoma mekongi and Schistosoma malayensis were tested successfully against Schistosoma japonicum. In combination, these fragments encompass 20-27% of the variation amongst the genomes (average total length similar to 14,000 bp), thus providing an efficient means of encapsulating the greatest amount of variation within the shortest sequence. Comparative mitogenomics provides the basis of a rational approach to molecular marker selection and optimisation. (c) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.status: publishe