The effect of the amino acid substitutions was predicted based on sequence homology and the physical properties of amino acids using Sorting Intolerant From Tolerant (SIFT) program [26]. For distinguishing whether the fragments of DNA sequences were neutrally evolved or derived under selection processes, the Tajima’s D was calculated using DnaSP version 5 [25]. Tajima’s D statistic determines the difference between two nucleotide variation BV-6 order parameters, the average number of BI 10773 concentration polymorphisms between all pairs of sequences (π) and the total number of polymorphic sites of all sequences in the dataset (θ). The greater value of π implies positive selection while the

greater value of θ implies negative selection [27]. In order to test for recombination, gdh gene sequences of G. duodenalis available from GenBank on March 2010 were additionally included in the analysis. Because the region and the length of the gdh sequences deposited in GenBank varied depending on selleck inhibitor the primers used by individual research studies, the

75 sequences originated from 14 countries were selected with the minimum coverage at 75% to the fragment size used for analysis in this study (Table 1). The phylogenetic network tree was used to visualize the extent of networked evolution among the sequences which preliminarily indicate possible locations of recombination events [28]. Principally, the phylogenetic tree and phylogenetic network tree are each constructed on a different basis. The phylogenetic tree is constructed under the assumption that once two lineages are created, they will subsequently not interact with each other again, whereas

the phylogenetic network assumes the evolutionary process in a more relaxed manner and constructs the tree under the assumption that the interaction between these two lineages might have occurred again later on. To present the data according to the aims of this study, this method is more appropriate than a conventional bifurcating phylogenetic tree. The analysis was undertaken with the SplitsTree program version 4 [29], through the Neighbor-Net method [30]. This method draws networks between sequences if there are potentially multiple evolutionary Calpain pathways linking them. The analysis was performed using sequences of all isolates presented in this study together with the sequences selected from GenBank. For the isolates that carried the heterozygous polymorphic sites identified by cloning, the standard one-letter code for combining nucleotides defined by the International Union of Pure and Applied Chemistry nomenclature (IUPAC) was used. Table 1 Characteristics and sources of the isolates from GenBank No. Accession No. Isolates Assemblage Geographical origin % coverage 1 EU594667.1 Cub-G81 BIII Cuba 100 2 EU594666.1 Cub-G12 BIV Cuba 100 3 EU594665.1 Cub-G89 BIII Cuba 100 4 EU594664.1 Cub-G33 BIII Cuba 100 5 EU594663.