Similarly, our data reflects the genetic variability of a set of isolates grown as promastigotes in axenic culture in vitro, a very different environment, and different life stage of the parasite to that present in patients. This means that we may miss variation present within host parasite populations that are lost during parasite isolation or subsequent growth, and that our results may be affected by selection to in vitro environments: In particular aneuploidy patterns in vectors and mammalian hosts were shown to differ from that in culture (Domagalska et al., 2019; Dumetz et al., 2017), and have other variants in particular during long term in vitro adaptation (e.g. Sinha et al., 2018; Bussotti et al., 2018). Given the breadth of global isolate collection used in our study it was not possible for us to ensure that common culture conditions were used for all the isolates. A recent approach to directly sequence Leishmania genomes in clinical samples has given some first insights into the effects of parasite culture in vitro and will allow future studies of Leishmania genome variation to avoid this potential bias (Domagalska et al., 2019).