Two enrichment libraries were prepared for each of six Z. carpinifolia specimens (population ZE053_IR, Appendix 1).
Polymorphism assessment used genomic DNA extracted from 25 Z. carpinifolia specimens (five specimens per population; Appendix 1) and fluorescently labeled forward or reverse primers (Applied Biosystems, Warrington, United Kingdom), and the same reaction conditions as in the previous step.
Genomic DNA of each individual was extracted from silica gel dried leaves using the modified CTAB method (Doyle and Doyle, 1987), and one voucher specimen per population was preserved in the IBSC herbarium (Huiyu-DA1, Huiyu-MH1, Huiyu-WN1, and Huiyu-YC1).
We also pre-specified in the protocol investigations of heterogeneity in relation to HIV status, condition of the specimens (fresh or frozen, volume of specimen), patient population (patients suspected of having MDR-TB or XDR-TB) and whether WHO-recommended critical drug concentrations were used for culture-based reference testing.
For assessing polymorphism of the microsatellite markers, genomic DNA templates from 25 A. montana specimens of five distinct populations were used (five specimens per population; Appendix 1).
Because the ITS2 region occurs in multiple copies in the same organism, it was necessary to compare its variability within single tick specimens, within populations, and between populations to ensure that, in A. cajennense, its evolution reflected concerted evolution.
To identify possible contamination between specimens the population sequences were used to reconstruct neighbor-joining phylogenetic trees with the MEGA 4.0 software using the maximum composite likelihood model with gamma distributed rates across sites (α = 0.5) [23].
Leaves from two specimens per population were dried using silica gel to preserve DNA before extraction.
After capture (> 30 specimens per population) the voles were transported to the laboratory where they were housed in individual cages with wood shavings and hay as bedding, in a 16L 8D photoperiod and 20 ± 2°C with standard food (Labfor 36, Lactamin AB, Stockholm, Sweden) and water available ad libitum.
Both analyses provide a low dimensional representation of shape space among specimens and populations.
