Each one of the HGT candidate amino acid sequences was used as a query against the NCBI Protein Reference Sequence Database (Ref Seq)  with the BLASTp algorithm , using default parameters.
The top 50 blast hits were collected for further analyses. nuttallii was included in the analyses to calibrate the HGT divergence time estimates. histolytica HGT candidate was used as a query against the E.
(1999) Errors in Generating Time-Series and in Dating Events at Late Quaternary Millenial (Radiocarbon) Time-Scales: Examples from Baffin Bay, NW Labrador Sea, and East Greenland, in Mechanisms of Global Climate Change at Millennial Time Scales (eds P.
Keigwin), American Geophysical Union, Washington, D.
Moreover, it has been proposed as a candidate to revive the name E.
HGT may be inferred amiss due to substitution saturation and it must be taken into account on every phylogenetic analysis.Accession numbers of the 68 well supported candidates were obtained from the list in Clark et al., 2007, and then searched in the Amoeba DB database, , in order to get the amino acid and coding sequences.The following genes were selected to carry out the Entamoeba divergence time estimations: DNA-directed RNA polymerase I subunit RPA2 (EHI_186020), elongation factor 2 (EHI_189490), actin (EHI_131230), tubulin gamma chain (EHI_008240), and clathrin heavy chain (EHI_201510) since they are single-copy, housekeeping genes that were not obtained by HGT.The divergence time estimation for protozoan species is commonly a challenging endeavor, especially at the node calibration step.Even though some protozoan taxa might have fossil record, the most common strategy to calibrate date estimates is the indirect calibration based on animal or plant fossils with a specific underlying biological hypothesis .