Sequencing and phylogenetic analysis

The obtained 350-bp ITS1-PCR products were purified using exonuclease I/shrimp alkaline phosphatase (GE Healthcare, US). The sequencing reaction was performed in both directions using BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA, USA). The resulting forward and reverse sequences were aligned. A total of nine partial ITS1 DNA sequences, in addition to the studied sequences, were selected from GenBank, including five sequences from L. infantum ({"type":"entrez-nucleotide","attrs":{"text":"AJ634361","term_id":"79677070","term_text":"AJ634361"}}AJ634361, {"type":"entrez-nucleotide","attrs":{"text":"GQ367486","term_id":"255762237","term_text":"GQ367486"}}GQ367486, {"type":"entrez-nucleotide","attrs":{"text":"KX664452","term_id":"1135520279","term_text":"KX664452"}}KX664452, {"type":"entrez-nucleotide","attrs":{"text":"AJ634339","term_id":"79677009","term_text":"AJ634339"}}AJ634339 and {"type":"entrez-nucleotide","attrs":{"text":"AJ634355","term_id":"79677053","term_text":"AJ634355"}}AJ634355), four sequences from L. tropica ({"type":"entrez-nucleotide","attrs":{"text":"KP202104","term_id":"785936579","term_text":"KP202104"}}KP202104, {"type":"entrez-nucleotide","attrs":{"text":"KU194923","term_id":"1101267304","term_text":"KU194923"}}KU194923, {"type":"entrez-nucleotide","attrs":{"text":"KX599337","term_id":"1064859054","term_text":"KX599337"}}KX599337 and {"type":"entrez-nucleotide","attrs":{"text":"KP202102","term_id":"785936577","term_text":"KP202102"}}KP202102) and one sequence from L. major ({"type":"entrez-nucleotide","attrs":{"text":"AJ000310","term_id":"2764493","term_text":"AJ000310"}}AJ000310). Phylogenetic analysis was performed with MEGA version 7 software using the neighbour-joining and Kimura 2-parameter models. The tree topology was supported by 1000 bootstrap replicates.