Table 1.
Annotated genes in the mitochondrial genome of Dirofilaria repens.
Fig 1.
Schematic map of the 13,672 bp Dirofilaria repens mitochondrial genome.
On the outer circle, positions of restriction enzymes recognition sites (with positions in the genome) are indicated for enzymes that cut only once in the genome. The position of tRNA genes and the AT-rich control region (CR) are also shown on this circle. The next circle shows the position of the genes encoding proteins (gray) or rRNAs (blue). In the center, the positions on the map is provided in base pairs (bp). All genes, including the genes encoding rRNAs are orientated in clockwise direction. The map was drawn using the sequence with the accession no KX265047.
Fig 2.
Phylogenetic analysis of spirurid nematodes using complete mitochondrial genomes.
The mitochondrial genomes of Dirofilaria repens (from top to bottom: KX265048, KX265047, KX265049), Dirofilaria hongkongensis (KX265047), Dirofilaria immitis, Onchocerca volvulus, Onchocerca flexuosa, Wuchereria bancrofti (from top to bottom: JN367461, JF775522, JQ316200, HQ184469), Brugia malayi, Chandlerella quiscali, Acanthocheilonema vitae, Setaria digitate, Heliconema longissimum, Spirocerca lupi, Thelazia callipedia and Gongylonema pulchrum were included in the analysis. The mitochondrial genomes of Ascaris lumbricoides and Dracunculus medinensis, clade III parasitic nematode not belonging to the Spirurida were used as outgroup. Concatenated alignments were analyzed with RAxML 8.1.11 with optimized models for each gene partition using the rapid bootstrapping algorithm. The Shimodaira-Hasegawa likelihood ratio test as implemented in RAxML was used as an alternative, unrelated method to obtain support values for all individual internal notes of this tree. Support values obtained by bootstrapping and by the likelihood ratio test are provided before and after the slash. If only one number is shown, the results of the tests were identical. Small letters were used if the space was too limited to write the support values directly at the branch. At the right of the scheme, different “classical” families within the Spirurida as defined by morphological criteria [81] are indicated as bars in different gray scales. As colored bars, different “classical” subfamilies within the Onchocercidae are indicated.
Fig 3.
Analysis of sequence variability along the sequence of the mitochondrial genomes of Onchocercidae.
(A) All sequences of mitochondrial genomes from Onchocercidae were aligned with MUSCLE and nucleotide diversity was determined in DnaSP using a Jukes-Cantor corrected distance. In order to avoid that the result is skewed due to overrepresentation of a single species, only one of four Wuchereria bancrofti (JN367461) and Dirofilaria repens genomes (KX265047) were used. Nucleotide diversity was calculated for a 300 bp window and plotted against the mid-point of this window. The window was moved in 10 bp steps across the alignment. Since the software is not able to handle circular DNA molecules, results at the beginning and the end of the alignment should be considered with care. Above the plot, the position of the different protein and rRNA coding genes as well as the AT-rich control-region (CR) are indicated. The PCR fragment 4, which was selected for further analysis in a larger set of samples from different geographical origins is shown at the top of the figure. (B) The same analysis was carried out comparing the three D. repens and the C. D. hongkongensis mitochondrial genomes (KX265047- KX265050).
Fig 4.
Phylogenetic analysis of Dirofilaria repens like sequences from samples collected in Europe and Asia.
Sequences of an approximately 2.5 kb mitochondrial DNA fragment including parts of the ctc-3 and the trnF genes, the AT-rich control region, the complete protein coding genes for ndfl4 and nduo-1, the ribosomal RNA gene rrnS and the genes for the tRNAs trnA, trnLUUR, trnN, trnM and trnK. Sample origin in terms of countries are shown behind an in-house sample number/code. All sequences have been deposited in GenBank under the accession nos. KX265047—KX265093. In addition, samples of human origin are indicated, all other samples were from dogs. Numbers at the internal nodes represent results for the Shimodaira-Hasegawa and a Bayesian transformation of the likelihood ratio test before and after the slash, respectively. If only one number is shown, the results of the tests were identical. Small letters were used if the space was too limited to write the support values directly at the branch. The branches labelled with “Dirofilaria repens”, “Candidatus Dirofilaria (C. D.) hongkongensis” and Dirofilaria (D.) sp. ‘Thailand II’ contain only the respective (putative) species. The branch labeled with “Candidatus Dirofilaria (C. D.) hongkongensis like” clusters together this proposed species with sequences of C. D. sp. ‘Thailand II’ that might belong to the same species or represent an additional species. The yellow area indicates all sequences that belong to an apparently monophyletic group containing a large number of samples from Poland and Hungary. The green color labels a second apparently monophyletic group with most samples coming from South-Western Europe and Hungary.