Analyses of mitochondrial amino acid sequence datasets support the proposal that specimens of Hypodontus macropi from three species of macropodid hosts represent distinct species

Abstract

Background: Hypodontus macropi is a common intestinal nematode of a range of kangaroos and wallabies (macropodid marsupials). Based on previous multilocus enzyme electrophoresis (MEE) and nuclear ribosomal DNA sequence data sets, H. macropi has been proposed to be complex of species. To test this proposal using independent molecular data, we sequenced the whole mitochondrial (mt) genomes of individuals of H. macropi from three different species of hosts (Macropus robustus robustus, Thylogale billardierii and Macropus [Wallabia] bicolor) as well as that of Macropicola ocydromi (a related nematode), and undertook a comparative analysis of the amino acid sequence datasets derived from these genomes.

Results: The mt genomes sequenced by next-generation (454) technology from H. macropi from the three host species varied from 13,634 bp to 13,699 bp in size. Pairwise comparisons of the amino acid sequences predicted from these three mt genomes revealed differences of 5.8% to 18%. Phylogenetic analysis of the amino acid sequence data sets using Bayesian Inference (BI) showed that H. macropi from the three different host species formed distinct, well-supported clades. In addition, sliding window analysis of the mt genomes defined variable regions for future population genetic studies of H. macropi in different macropodid hosts and geographical regions around Australia.

Conclusions: The present analyses of inferred mt protein sequence datasets clearly supported the hypothesis that H. macropi from M. robustus robustus, M. bicolor and T. billardierii represent distinct species.

Figure 1

Schematic representation of the circular mitochondrial genome of Hypodontus macropi and Macropicola ocydromi. Three operational taxonomic units (OTUs) of H. macropi were from three different hosts (i.e., OTU-C from Macropus robustus robustus; OTU-G from Macropus bicolor; OTU-J from Thylogale billardierii) and M. ocydromi was from Macropus fuliginosus. Transfer RNA genes are designated using one-letter amino acid codes. Ribosomal genes are shaded.

Figure 2

Phylogenetic analysis of the concatenated amino acid sequences for protein coding genes ofHypodontus macropi. Concatenated amino acid sequence data for all protein coding mitochondrial genes for three operational taxonomic units of H. macropi, from Macropus robustus robustus, Macropus bicolor, Thylogale billardierii, and from Macropicola ocydromi sequenced here as well as other concatenated sequence data representing complete mitochondrial genomes of Chabertia ovina, Oesophagostomum dentatum and Strongylus vulgaris (Strongyloidea) were analyzed using Bayesian Inference. The numbers above each tree branch represent the statistical support for each node (based on posterior probability score).

Figure 3

Sliding window analysis of complete mt genome sequences ofHypodontus macropifrom different hosts. The three operational taxonomic units (OTUs) of H. macropi were from Macropus robustus robustus (OTU-C), Macropus bicolor (OTU-G) and Thylogale billardierii (OTU-J). The black line indicates nucleotide diversity comparing all the three OTUs; whereas three other symbols (a square, a circle and a plus sign) indicate pairwise comparisons across the mt genomes of OTU-C, OTU-G and OTU-J. Nucleotide diversity, measured iteratively every 10 bp over 300 bp windows of aligned mtDNA sequence data indicate peaks and troughs of sequence variability. Linearized maps of mtDNA are provided for each aligned data set, with the highest peaks of variability falling within the non-coding AT-rich regions.