Sarcocystis spp

Adri, Hern, an, andez-Ortiz, Emilie, Bouchard, Snyman, Louwrens P., Al-Adhami, Batol H., Gouin, eraldine-G., Neelin, Mikhaela & Jenkins, Emily J., 2023, Toxoplasma gondii and related Sarcocystidae parasites in harvested caribou from Nunavik, Canada, International Journal for Parasitology: Parasites and Wildlife 21, pp. 246-254 : 249-251

3.4. Sequencing results and phylogeny for Sarcocystis spp

In total, 32 sequences were generated as a subsample of the 71 positive heart samples. On average, the sequences shared a 99.4 (±1.24) percentage of identity (%ID) with S. grueneri using BLAST. Sixteen sequences were successfully generated from 38 positive skeletal muscle samples. Thirteen of these sequences represent 5 different Sarcocystis spp . as follows: five sequences had an average of 99 %ID (±1.11) with S. tarandi , three had 99.6 %ID (±0.44) with S. tarandivulpes , two had 99.8 %ID with S. rangi , two had 100 %ID to S. rangiferi , and one had 99.8 %ID with S. scandinavica . Additionally, 3 samples from the 18S rRNA PCR generated sequences identical to Besnoitia spp ; these samples were tested with ITS-1 nested PCR and the sequences had 100%ID with Besnoitia spp .

The aligned matrix consisted of 74 ingroup and one outgroup sequence with a final length of 658 characters. The assignment of 8

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groups was intended for discussion puposes rather than for taxonomic designation. The subfamily Toxoplasmatinae (Group VIII) was supported as a monophyletic clade across both analyses (DDN bs: 100, ML bs: 95) including Isospora belli in the grouping ( Figs. 4 and 5 ). Sequences of Besnoitia spp . obtained from GenBank, as well as those generated in this study were included within the Group VIII. It was not possible to differentiate among B. besnoiti , B. tarandi and B. jellisoni using 18S sequences or ITS-1 sequences. The subfamily Sarcocystinae was recovered as monophyletic in the ML analysis, although it lacked support with the inclusion of S. neurona (ML bs: 67) ( Fig. 4 ). However, when S. neurona is excluded, the clade enjoys bootstrap support of 100. All 32 sequences recovered from heart samples grouped with a S. grueneri reference sequence in a well supported monophyly (DDN bs: 100, ML bs: 96) (Group I). Sequences generated from muscle tissue were more diverse and represented members of groups II through VII (red dots in Figs. 4 and 5 ). Group II comprised a well-supported S. tarandivulpes grouping inclusive of sequences generated here (DDN bs: 95, ML bs: 97), sister to a S. alces and S. capracanis clade. Two more samples grouped alongside S. rangi forming a well-supported monophyly in Group III sister to S. alcestrans, completing Group III. Despite forming a monophyletic clade, Group IV lacked support in the ML analysis and was rendered paraphyletic by Group V in the network analysis. The sister grouping of Group V to Group IV was also not supported in the ML analysis. Group IV consisted of a polyphyletic grouping of reference

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sequences designated as S. tarandi , S. cf. tarandi and S. elongata as well as sample sequences generated here. Inferring identity of these samples beyond members of Group IV is thus not possible. Group V, consisting of S. silva sequences, was confidently supported as a monophyletic group in both methods (DDN bs: 90, ML bs: 89). Group VI comprised two sample sequences from this study alongside S. rangiferi reference sequences in a monophyletic group in both the network (bs 94) and ML phylogeny (bs 99). A single sample sequence grouped alongside two reference sequences of S. scandinavica forming Group VII. We could therefore confidently infer species level identification through phylogenetic and network reconstruction for most sample sequences and group level identification to the remaining sequences (see supplementary excel file).

Figures

Fig. 4. Maximum likelihood topology for tissue dwelling coccidians (mostly Sarcocystis spp.) generated from 18S rDNA sequence data analyzed in RAxML 8 under the GTRCAT approximation. Group names bear no taxonomic designation but merely assigned for discussion purposes. Bootstrap values> 60 are displayed above branches as branch/node support. Red dots indicate sequences generated in this study. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. A data-display network constructed from uncorrected 18S rDNA p-distances, using all characters, for tissue dwelling coccidians (mostly Sarcocystis spp.). Group names bear no taxonomic designation but merely assigned for discussion purposes. Bootstrap supports are displayed by the gray curves and associated values imposed on the network. Red dots indicate sequences generated in this study. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)