2.4. Phylogenetic analyses
To determine the phylogenetic status of these two Taenia spp., the
phylogenetic trees were constructed using Bayesian methods in MrBayes v3
with the tandem DNA sequences and amino acid sequences of 12 encoding
genes in their mt genomes and other 32 taeniid mt genome sequences
downloaded from GenBank, while the sequences of Schistosoma
japonicum was used as outgroup (Supplementary Table S2). For the amino
acid data set, the mixed model was applied (prset aamodelpr = mixed);
two chains (temp = 0.2) were run for 3,000,000 generations and sampled
every 1,000 generations. For the nucleotide data set, Modeltest 3.7 maxX
(Posada and Crandall, 1998) was used to estimate a suitable model for
nucleotide substitution; this was equivalent to GTR+I+G and settings
were nst = 6, rates = invgamma, ngammacat = 4. Four chains (temp = 0.2)
were run for 1,000,000 generations and sampled every 1,000 generations.
The first 25% of trees were omitted as burn-in and the remaining trees
were used to calculate Bayesian posterior probabilities. The best
Bayesian tree was then compiled and processed by FigTree.v1.4.4.
2.5. Divergence times
analysis
The phylogenetic trees were used as a reference for species selection in
divergence times analysis. Echinococcus multilocularis andE. shiquicus were also
selected because the parasitism of their larvae is also found in Plateau
pika (O. curzoniae ) (Wang et al., 2018), besides E.
multilocularis is the sister species of E. shiquicus (Lymbery,
2017). Divergence times were calculated from the concatenated CDS
alignment of the 12 mitochondrial protein-coding genes by BEAST2
(v2.6.2). The Strict Clock model was chosen to ignore the rate
differences between the branches in the mode. The gamma category count
was set to 4, and HKY substitution model was selected with the empirical
setting from the frequencies in site model. Other settings, such as
substitution rate and shape, in the site model were evaluated in the
analysis. The calibrated Yule model was used as the tree prior. Time
calibration was calibrated with the previously estimated date betweenT. saginata and T. asiatica (~1.14 Mya)
(Michelet & Dauga, 2012; Wang et al., 2016). Samples from the posterior
were drawn every 1,000 steps over a total of 10,000,000 steps per MCMC
run. Other options were run on their default values. The convergence of
likelihood values was determined by Tracer (v1.7.1). Trees were
annotated by TreeAnnotator (v2.1.2) using maximum clade credibility tree
and median heights settings with 50% burn-in. The evolutionary
divergence time of the intermediate host, Qinghai vole (N.
fuscus ), was also calculated with the concatenated CDS alignment of 13
mt protein-coding genes of the
rodents, and the species involved were selected from our previous report
(Li et al., 2019) (Supplementary Table S2). The time calibration was
based on the divergence time of Mus and Rattus (11-13 Mya)
(Wang et al., 2020), and other parameters were the same as above.
Results