Mitochondrial Genome Sequences and Structures Aid in the Resolution of Piroplasmida phylogeny

Abstract

The taxonomy of the order Piroplasmida, which includes a number of clinically and economically relevant organisms, is a hotly debated topic amongst parasitologists. Three genera (Babesia, Theileria, and Cytauxzoon) are recognized based on parasite life cycle characteristics, but molecular phylogenetic analyses of 18S sequences have suggested the presence of five or more distinct Piroplasmida lineages. Despite these important advancements, a few studies have been unable to define the taxonomic relationships of some organisms (e.g. C. felis and T. equi) with respect to other Piroplasmida. Additional evidence from mitochondrial genome sequences and synteny should aid in the inference of Piroplasmida phylogeny and resolution of taxonomic uncertainties. In this study, we have amplified, sequenced, and annotated seven previously uncharacterized mitochondrial genomes (Babesia canis, Babesia vogeli, Babesia rossi, Babesia sp. Coco, Babesia conradae, Babesia microti-like sp., and Cytauxzoon felis) and identified additional ribosomal fragments in ten previously characterized mitochondrial genomes. Phylogenetic analysis of concatenated mitochondrial and 18S sequences as well as cox1 amino acid sequence identified five distinct Piroplasmida groups, each of which possesses a unique mitochondrial genome structure. Specifically, our results confirm the existence of four previously identified clades (B. microti group, Babesia sensu stricto, Theileria equi, and a Babesia sensu latu group that includes B. conradae) while supporting the integration of Theileria and Cytauxzoon species into a single fifth taxon. Although known biological characteristics of Piroplasmida corroborate the proposed phylogeny, more investigation into parasite life cycles is warranted to further understand the evolution of the Piroplasmida. Our results provide an evolutionary framework for comparative biology of these important animal and human pathogens and help focus renewed efforts toward understanding the phylogenetic relationships within the group.

Fig 1. 18S sequence alone is unable to completely resolve phylogeny of the Piroplasmida.

Summary topologies of Piroplasmida phylogenetic trees based on 18S rRNA sequence analyses in four previously reported studies [, –11]. The nomenclature of sub-groupings assigned in each study is maintained in each individual tree. Stars denote nodes that had less than 70% bootstrap support (A-C) or less than 95% Bayesian posterior probability (C-D); cut-off values for bootstrap values and posterior probabilities reflect those utilized by Lack et al. Individual species included in each respective study whose mitochondrial genomes were utilized in this study are noted within each clade. Mitochondrial genomes first characterized in this study are underlined. +Low support for species included within clade. *Although Lack et al. found strong support for a node uniting Clade I-III, positioning of Clade II with respect to Clades I and III within this node was unresolved. **White clades in 1C indicate those for which no representative species were characterized in this study; Clade IV includes Babesia benneti, while Clade VII include Babesia poelea.

Fig 2. Mitochondrial genome structures of Piroplasmida species characterized in this study.

Genes shown above the central line are coded on the sense strand, while those below are on the antisense strand. Protein-coding genes (cox1, cox3, and cytb) are indicated in white. Large subunit rRNA fragments are in light gray, small subunit rRNA fragments are in dark gray, and miscellaneous conserved RNA fragments are in black. A) Mitochondrial genome sequences of C. felis, B. rossi, B. vogeli, B. canis, and Babesia sp. Coco maintained the mitochondrial genome structure that is characteristic of traditional Babesia sensu stricto and Theileria species, while B) the inferred Babesia microti-like sp. mitochondrial genome structure appears to be similar to that of B. microti and B. rodhaini, suggesting it has a “flip-flop” mitochondrial genome structure. Assumed inverted repeats A and B (indicated as IR-A and IR-B) were not confirmed due to lack of relevant sequence for phylogenetic analysis. C) Babesia conradae had a unique mitochondrial genome, which lacked cox3 and had a duplicated inversion that included the 3’ end of cox1 and RNA17 and RNA18. Additionally, a collection of rRNA fragments (RNA6, RNA7, RNA15, LSUC, and SSUF) found in Babesia sensu stricto, C. felis, and Theileria mitochondrial genomes was conserved but inverted as a unit adjacent to the duplicated inversion.

Fig 3. Phylogenetic analysis of concatenated mitochondrial genome and 18S nucleotide sequence identifies five distinct lineages within Piroplasmida.

Statistical support for clades are indicated at each node: posterior probabilities from Bayesian analysis (10 million generations of Markov chain Monte Carlo) are listed above nodes, while bootstrap values from Maximum Likelihood analysis (200 bootstrap replicates) are listed below nodes. Trees are drawn to scale, with branch lengths measured in the number of substitutions per site. A) Analysis of concatenated mitochondrial and 18S nucleotide sequences (6006 total characters); see S8 Table for specific sequences included in analysis. The five lineages identified by analysis of concatenated mitochondrial and 18S nucleotide sequences are depicted in B (branch lengths not to scale).

Fig 4. Removal of cox3 and 18S sequence for phylogenetic analysis identifies same five distinct lineages within Piroplasmida but with less statistical support.

Statistical support for clades are indicated at each node: posterior probabilities from Bayesian analysis (10 million generations of Markov chain Monte Carlo) are listed above nodes, while bootstrap values from Maximum Likelihood analysis (100 bootstrap replicates) are listed below nodes. Trees are drawn to scale, with branch lengths measured in the number of substitutions per site. A) Analysis of concatenated mitochondrial and 18S nucleotide sequences with cox3 sequences excluded (5292 total characters) and B) mitochondrial nucleotide sequence alone (4395 total characters). Maximum Likelihood analysis of mitochondrial sequences alone did not recover Group 3 (T. equi) as a distinct clade, which is denoted with an asterisk (*).

Fig 5. Mitochondrial genome structures further support recognition of the five groups identified by phylogenetic analysis of concatenated mitochondrial and 18S sequences.

Groups are indicated by yellow circles to the left of respective clades. Genes are indicated with names (cox1, cox3, cytb) and ribosomal sequences are indicated in gray with “L” for large subunit and “S” for small subunit. Genes placed above the black central line on the diagram are coded on the sense strand of DNA, while those below the line are coded on the anti-sense strand. The presence or absence of TIRs in B. conradae’s mitochondrial genome has not been confirmed. Branches not drawn to scale.

Fig 6. Biology of Piroplasmida organisms is consistent with phylogeny inferred from analysis of concatenated mitochondrial and 18S sequences.

Groups are indicated by yellow circles to the left of respective clades. A) Organisms in Group 1 (Babesia sensu stricto; red) do not infect leukocytes and can be transmitted transovarially in the tick host, two traits unique to the group. Organisms in Groups 2, 3, and 5 (gray) are thought to be limited to transstadial transmission in the tick host and infect nucleated cells prior to erythrocytes. Notably, details regarding tick hosts, transmission in the tick, and infection of nucleated cells for Group 4 (blue) remains unknown, and infection of nucleated host cells has only been demonstrated for a single species in Group 5 (24). Characteristics of species in Groups 2 and 3 (outlined with dashed red line) are further summarized in B. B) While many organisms in Group 2 and 3 have been demonstrated to infect leukocytes, the specific leukocyte infected isn’t clade-specific and hasn’t even been confirmed for some species (e.g., Cytauxzoon felis). Additionally, the shared biological features of organisms in Group 2 support their distinction from the organism in Group 3, T. equi. T. equi exclusively infects equine hosts, and disease is caused by parasite infection of erythrocytes rather than the brief schizogonous phase in leukocytes. However, there is evidence indicating that organisms in Group 2 have evolved more complex methods of host leukocyte manipulation. Species within Group 2 that diverged earliest (Cytauxzoon felis) exclusively infect carnivores and have grossly enlarged schizont-infected cells, which suggests a blocking of host cell apoptosis. The remaining Theileria species in Group 2 exclusively infect ruminants. Organisms in the next clade to diverge in Group 2, including Theileria orientalis, also have grossly enlarged schizont-infected cells. This group is commonly known as the “non-transforming” Theileria species. This is in contrast to the “transforming” Theileria species (T. annulata and parva), which reversibly transform infected host leukocytes into a proliferative neoplastic state to support the replicating parasite. Branches not drawn to scale.

Fig 7. Phylogenetic analysis of cox1 putative amino acid sequence recovers the same five Piroplasmida groups as concatenated mitochondrial and 18S nucleotide sequences.

Statistical support for clades are indicated at each node: posterior probabilities from Bayesian analysis (10 million generations of Markov chain Monte Carlo) are listed above nodes, while bootstrap values from Maximum Likelihood analysis (100 bootstrap replicates) are listed below nodes. Tree is drawn to scale, with branch lengths measured in the number of substitutions per site; 429 characters were analyzed.