Table 1.
Details of PCR primers used in this study.
| Organism | Gene | Primers | Amplification conditions | Amplicon length (bp) | References |
|---|---|---|---|---|---|
| B. studeri | 18SrRNA |
| denaturation at 94ºC for 3 min; 30 cycles of 94ºC for 30 s; 55ºC for 1min; 72ºC for 1 min; final extension at 72ºC for 10 min | 412 | Medlin et al., 1988 |
| B. studeri | COX1 |
| denaturation at 94ºC for 1 min 30 s; 30 cycles of 94ºC for 50 s; 45ºC for 1 min 30 s; 72ºC of 1 min 30 s; final extension at 72ºC for 7 min | 448 | Okamoto et al., 1997 |
| B. studeri | ITS1-5.8S |
| denaturation at 94ºC for 2 min; followed by first cycle; 94°C for 2 min; 63°C for 2 min; 72°C for 1 min; 34 cycles of 94°C for 20 s; 63°C 20 s; 72°C 45 s; final extension at 72ºC for 7 min | 806 | MacNish et al., 2002 |
Fig. 1.
Proglottids of B. studeri.
A – mature segment of B. studeri, B – gravid segment of B. studeri
Fig. 2.
Phylogenetic tree constructed using 18SrRNA gene sequence of B. studeri.
The evolutionary history was inferred by using the Maximum Likelihood method and Hasegawa-Kishino-Yano model. The tree with the highest log likelihood (−1247.04) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. This analysis involved 16 nucleotide sequences. There was a total of 315 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.
Fig. 3.
Phylogenetic tree constructed using COX1 gene sequence of B. studeri.
The evolutionary history of B. studeri was inferred by using the Maximum Likelihood method and Hasegawa-Kishino-Yano model. The tree with the highest log likelihood (−1017.95) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. This analysis involved 13 nucleotide sequences.
Fig.4.
Phylogenetic tree constructed using ITS1-5.8S gene sequence of B. studeri.
The evolutionary history was inferred by using the Maximum Likelihood method and Hasegawa-Kishino-Yano model. The tree with the highest log likelihood (−1819.29) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. This analysis involved 9 nucleotide sequences. There was a total of 604 positions in the final dataset.
Fig.5.
Minimum spanning network of B. studeri species complex determined by A) 18SrRNA, B) COX1, C) ITS1-5.8S genes using POPART program. The size of a circle indicates the relative frequency of sample such as B. studeri and Bertiella spp.
Hatch marks (numbers) along the branches indicate the numbers of mutations. Each colour indicates a different geographic area.
Table 2.
Frequency of haplotypes of Bertiella spp. in different countries.
| 18SrRNA | COX1 | ITS1-5.8 S | ||||||
|---|---|---|---|---|---|---|---|---|
| Haplotype | Frequency | Country | Haplotype | Frequency | Country | Haplotype | Frequency | Country |
| Hap_1 | 2 | India, Mauritius, | Hap_1 | 1 | India | Hap_1 | 1 | India |
| Hap_2 | 2 | Argentina | Hap_2 | 5 | Sri Lanka | Hap_2 | 2 | Japan |
| Hap_3 | 4 | Sri Lanka | Hap_3 | 2 | Sri Lanka | Hap_3 | 2 | Argentina |
| Hap_4 | 1 | Spain | Hap_4 | 1 | Argentina | Hap_4 | 3 | Uganda |
| Hap_5 | 5 | Guinea-Bissau, Uganda, Rwanda, Brazil, Peru | Hap_5 | 1 | Argentina | |||
| Hap_6 | 1 | Central African Republic | Hap_6 | 1 | Kenya | |||
| Hap_7 | 1 | Uganda | ||||||
| Hap_8 | 1 | Peru | ||||||
| Hap_9 | 1 | Indonesia | ||||||