Characterization of the complete mitochondrial genomes of Coronocyclus labiatus and Cylicodontophorus bicoronatus: Comparison with Strongylidae species and phylogenetic implication.

Coronocyclus labiatus and Cylicodontophorus bicoronatus are two significant horse parasitic nematodes which are classified into subfamily Cyathostominae, family Strongylidae, however, the classification of these nematodes has been controversial for more than a century. Mitochondrial (mt) genomes are considered valuable sources for parasite taxonomy, population genetics, and systematics studies. In the present study, the mt genomes of Co. labiatus and Cd. bicoronatus (type species) were determined and subsequently compared with those from closely related species by phylogenetic analysis based on concatenated datasets of amino acid sequences predicted from mt protein-coding genes. The complete mt genomes of Co. labiatus and Cd. bicoronatus were circular with 13,827 bp and 13,753 bp in size, respectively. Both mt genomes consisted of a total of 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and two non-coding regions. All protein coding genes were transcribed in the same direction, and the gene order in both mt genomes belonged to the gene arrangement type 3 (GA3). There were 19 intergenic spacers with 1 bp to 35 bp and one overlap with 4 bp in mt genome of Co. labiatus, and 22 intergenic spacers with 1-29 bp in size but no overlap in the mt genome of Cd. bicoronatus. The A + T content of Co. labiatus and Cd. bicoronatus mt genomes were 75.87 % and 75.16 %, respectively. Similar to mt genones of other Strongylidae species published in GenBank, they also exhibited a strong A + T bias not only in the nucleotide composition but also in codon usage. Comparative analyses of mt genomes nucleotide sequence showed that mt genomes of Co. labiatus and Cd. bicoronatus had higher identities to that of Cylicostephanus goldi (90.3 % and 86.9 %, respectively), followed by those of two Cyathostomum species (89.9∼90.0 %; 86.4 %), respectively. Phylogenetic analyses using mt genomes of 26 Strongyloidea nematodes revealed that Co. labiatus was closely related to Cyathostomum species, and Cd. bicoronatus formed a distinct branch with Cyathostominae species, which was closer to Triodontophorus than Poteriostomum imparidentatum. We concluded Coronocyclus might be closely related with Cyathostomum but represent a distinct genus based on comparative mt genome sequences and phylogenetic analyses. The availability of complete mt genome sequences of Co. labiatus and Cd. bicoronatus provides new and useful genetic markers for further studies on Strongylidae nematodes.

Comparative analysis of mitochondrial DNA datasets indicates that Cylicostephanus minutus represents a species complex.

Cyathostomins are one kind of the most important parasites in equids. Cylicostephanus minutus is a member of the subfamily Cyathostominae. In the present study, we determined the complete mitochondrial (mt) genomes from four Cs. minutus isolates and reconstructed the phylogenetic relationship of Strongylidae to test the hypothesis that Cs. minutus represents a species complex. The complete mt genome sequences of Cs. minutus were 13,772-13,822 bp in length, and contained 36 genes (12 protein coding genes, 22 tRNA genes, two rRNA genes), and two non-coding regions (NCRs). The intraspecific identity of nucleotide sequences and amino acid sequences in Cs. minutus (1-4) were 89.3-97.9% and 97.0-98.8%, respectively. Two operational taxonomic units (OTUs) were determined based on the mt genome sequences, OTU 2 (Csm 1 and Csm 2) and OTU 3 (Csm 3 and Csm 4). Sequence analysis showed the divergence between OTU 2 and OTU 3 was 8.9-10.7%. Pairwise comparisons of 12 protein coding genes between OTU 2 and OTU 3 showed a difference of 3.0-13.3% at the nucleotide level and 0-6.7% at the amino acid level. Phylogenetic analysis showed the separation of Cs. minutus isolates from the same host into different distinct clades based on mt genomes. Comparisons of partial mt cox1, nad5, and cytb and ITS2 sequences from 20 Cs. minutus isolates from the same host and the same geographical location with other Cs. minutus sequences available in GenBank revealed significant nucleotide differences. Phylogenetic analysis showed a separation of Cs. minutus into three distinct clades. Thus, the comparative and phylogenetic analyses of mtDNA datasets indicated that Cs. minutus represents a complex of at least three species. Our results have further confirmed the existence of a cryptic Cs. minutus species, and provides a reference for the taxonomical, population genetics, and systematics studies of other cyathostomin species.