Background: The Dicrocoeliidae are digenetic trematodes mostly parasitic in the bile ducts and gall bladder of various avian and mammalian hosts. Until recently their systematics was based on morphological data only. Due to the high morphological uniformity across multiple dicrocoeliid taxa and insufficient knowledge of relative systematic value of traditionally used morphological characters, their taxonomy has always been unstable. Therefore, DNA sequence data provide a critical independent source of characters for phylogenetic inference and improvement of the system. Methods: We examined the phylogenetic affinities of three avian dicrocoeliids representing the genera Brachylecithum, Brachydistomum and Lyperosomum, using partial sequences of the nuclear large ribosomal subunit (28S) RNA gene. We also sequenced the complete or nearly complete mitogenomes of these three isolates and conducted a comparative mitogenomic analysis with the previously available mitogenomes from three mammalian dicrocoeliids (from 2 different genera) and examined the phylogenetic position of the family Dicrocoeliidae within the order Plagiorchiida based on concatenated nucleotide sequences of all mitochondrial genes (except trnG and trnE). Results: Combined nucleotide diversity, Kimura-2-parameter distance, non-synonymous/synonymous substitutions ratio and average sequence identity analyses consistently demonstrated that cox1, cytb, nad1 and two rRNAs were the most conserved and atp6, nad5, nad3 and nad2 were the most variable genes across dicrocoeliid mitogenomes. Phylogenetic analyses based on mtDNA sequences did not support the close relatedness of the Paragonimidae and Dicrocoeliidae and suggested non-monophyly of the Gorgoderoidea as currently recognized. Conclusions: Our results show that fast-evolving mitochondrial genes atp6, nad5 and nad3 would be better markers than slow-evolving genes cox1 and nad1 for species discrimination and population level studies in the Dicrocoeliidae. Furthermore, the Dicrocoeliidae being outside of the clade containing other xiphidiatan trematodes suggests a need for the re-evaluation of the taxonomic content of the Xiphidiata.

Background: The Eucotylidae Cohn, 1904 (Superfamily: Microphalloidea), is a family of digeneans parasitic in kidneys of birds as adults. The group is characterized by the high level of morphological similarities among genera and unclear systematic value of morphological characters traditionally used for their differentiation. In the present study, we sequenced the complete or nearly complete mitogenomes (mt genome) of two eucotylids representing the genera Tamerlania (T. zarudnyi) and Tanaisia (Tanaisia sp.). They represent the first sequenced mt genomes of any member of the superfamily Microphalloidea. Methods: A comparative mitogenomic analysis of the two newly sequenced eucotylids was conducted for the investigation of mitochondrial gene arrangement, contents and genetic distance. Phylogenetic position of the family Eucotylidae within the order Plagiorchiida was examined using nucleotide sequences of mitochondrial protein-coding genes (PCGs) plus RNAs using maximum likelihood (ML) and Bayesian inference (BI) methods. BI phylogeny based on concatenated amino acids sequences of PCGs was also conducted to determine possible effects of silent mutations. Results: The complete mt genome of T. zarudnyi was 16,188 bp and the nearly complete mt genome of Tanaisia sp. was 13,953 bp in length. A long string of additional amino acids (about 123 aa) at the 5′ end of the cox1 gene in both studied eucotylid mt genomes has resulted in the cox1 gene of eucotylids being longer than in all previously sequenced digeneans. The rrnL gene was also longer than previously reported in any digenean mitogenome sequenced so far. The TΨC and DHU loops of the tRNAs varied greatly between the two eucotylids while the anticodon loop was highly conserved. Phylogenetic analyses based on mtDNA nucleotide and amino acids sequences (as a separate set) positioned eucotylids as a sister group to all remaining members of the order Plagiorchiida. Both ML and BI phylogenies revealed the paraphyletic nature of the superfamily Gorgoderoidea and the suborder Xiphidiata. Conclusions: The average sequence identity, combined nucleotide diversity and Kimura-2 parameter distances between the two eucotylid mitogenomes demonstrated that atp6, nad5, nad4L and nad6 genes are better markers than the traditionally used cox1 or nad1 for the species differentiation and population-level studies of eucotylids because of their higher variability. The position of the Dicrocoeliidae and Eucotylidae outside the clade uniting other xiphidiatan trematodes strengthened the argument for the need for re-evaluation of the taxonomic content of the Xiphidiata. Graphical

Our present genetic data of Acanthocephala, especially the mitochondrial (mt) genomes, remains very limited. In the present study, the nearly complete mt genome sequences of Sphaerirostris lanceoides (Petrochenko, 1949) was sequenced and determined for the first time based on specimens collected from the Indian pond heron Ardeola grayii (Sykes) (Ciconiiformes: Ardeidae) in Pakistan. The mt genome of S. lanceoides is 13 478 bp in size and contains 36 genes, including 12 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs) and two ribosomal RNA genes (rRNAs). Moreover, in order to clarify the phylogenetic relationship of the genera Centrorhynchus and Sphaerirostris, and to test the systematic position of S. lanceoides in the Centrorhynchidae, the phylogenetic analyses were performed using Bayesian inference and maximum likelihood methods, based on concatenated nucleotide sequences of 12 PCGs, rRNAs and tRNAs. The phylogenetic results further confirmed the monophyly of the order Polymorphida and the paraphyly of the order Echinorhynchida in the class Palaeacanthocephala. Our results also challenged the validity of the genus Sphaerirostris (Polymorphida: Centrorhynchidae) and showed a sister relationship between S. lanceoides and S. picae (Rudolphi, 1819).