Molecular Identification And Mitochondrial Genomes Of Six Trematodes From Birds In Pakistan

Taxonomy and systematics are essential tools for the fundamental understanding of biodiversity.Molecular data in conjunction with morphological approach for the taxonomy/systematics of trematodes should be able to maximize phylogenetic information.The use of nuclear ribosomal DNA(rDNA)region and mitochondrial(mt)genomes offer rich sources of genetic markers.The partial or entire rDNA sequences have been extensively used for trematode identification and phylogenetic studies.However,the mt genome data presently available are only from limited number of families of trematodes.Therefor,the mt genome sequences are needed from those trematode parasites for which they are still missing or their taxonomic positions are controversial.Despite the fact that birds are the most common definitive hosts of trematodes,no attempt has been made to elucidate the phylogenetic relationship of trematodes inhabiting birds based on mt genome sequences.In order to fill this gap of knowledge,this study aimed to determine the rDNA(region/gene(s))and mt genomes of several trematodes parasitizing birds of different feeding habits in Pakistan,and to assess their phylogenetic relationships with other trematodes.The internal transcribed spacer(ITS)and/or larger subunit rDNA(LSU/28S),and complete or nearly complete mt genomes were amplified by polymerase chain reaction(PCR)using several universal and self-designed primers,then sequenced and determined from a total of six species of trematodes parasitizing different avian hosts.The rDNA data sets were used for specific identification,discrimination,genetic divergence,and when needed used for the phylogenetic analyses between species within a family.The mt genomes were annotated,their gene arrangements,nucleotide composition and skewness,codon usage and relative synonymous codon usage(RSCU)for 12 protein-coding genes(PCGs)were described and used for comparative analyses.The newly sequenced mt genomes of the studied digenetic trematodes contain 36 genes(12 PCGs,22 transfer RNA(tRNA)and 2 ribosomal RNA(rRNA)genes)plus a 1-2 non-coding region(s).Similar to other flatworm mt genomes,the atp8 gene is missing.The concatenated amino acid and/or nucleotide sequences of 12 PCGs,with the combination of nucleotide sequences of mitochondrial rRNAs and tRNAs were used for the higher level systematics of each species(family)within selected digenetic trematodes.The phylogenetic analyses based on mt genomes assigned the examined trematodes to three suborders and four families of the order Plagiorchiida.The phylogenetic analyses demonstrated that Paragonomidae is genetically closer to the Plagiorchiidae and Brachycladiidae than to the Dicrocoeliidae,which is a disagreement to the previously published data based on partial 28 S rDNA.The systematic position of the Cyclocoelidae within Echinostomata is consistent with recent molecular phylogenetic studies based on partial 28 S rDNA gene.The higher sequence similarity across the ITS rDNA and mtDNA between Hepatiarius sudarikovi and Opisthorchis felineus(Opisthorchiidae)suggested that H.sudarikovi to be a member of the genus Opisthorchis.The mt genomes of the studied trematodes were not available to date.The Plagiorchis maculosus(Plagiorchiidae)and Uvitellina sp.(Cyclocoelidae)are first representatives of each family,and Brachylecithum sp.,Brachydistomum sp.,and Lyperosomum longicauda(Dicrocoeliidae)are first representatives of each genus whose mt genomes were sequenced and annotated.Thus,the present study provided novel and useful source of molecular markers from the nuclear and mt genomes of trematodes,which provided foundation for further investigation of the genetic relationship of trematodes within the Digenea.