24Removing the requirement for cell culture has led to a substantial increase in the number 25 of lineages of Entamoeba recognized as distinct. Surveying the range of potential host 26 species for this parasite genus has barely been started and it is clear that additional 27 sampling of the same host in different locations often identifies additional diversity. In 28 this study, using small subunit ribosomal RNA gene sequencing, we identify four new 29 lineages of Entamoeba, including the first report of Entamoeba from an elephant, and 30 extend the host range of some previously described lineages. Additionally, examination 31 of microbiome data from a number of host animals suggests that substantial Entamoeba 32 diversity remains to be uncovered. 33 34Keywords: Diversity; next generation sequencing; ribosomal RNA; phylogeny; 35 36 37Over the past 25 years, our understanding of diversity in the genus Entamoeba has 38 increased significantly as a result of complementary developments in DNA amplification, 39 purification and sequencing. Traditionally, naming of species in Entamoeba was based on 40 a mixture of host identity and parasite morphology, the latter being rather limited in these 41 amoeboid organisms and the former being of debatable value due to uncertainty over host 42 ranges of the parasites. DNA sequencing allows quantitative measurement of similarity 43 that is not dependent on such characters and although it is not without its own limitations, 44 it has fundamentally changed our approach to studying diversity in organisms such as 45Entamoeba. 46In this time period, the study of Entamoeba has gone from being dependent on 47 stable laboratory cultures of parasites, preferably in the axenic form, to DNA analysis of 48 organisms directly from stool samples in the absence of even microscopic investigation. 49The latter aspect has been problematic as it is not possible to assign new sequences to 50 previously named species where the original description is dependent on morphology. 51For this reason, many new and distinct Entamoeba sequences have been assigned to 52 'ribosomal lineages' rather than species to reflect the absence of morphological 53 information (Stensvold et al. 2011). 54The new approach is dependent on the reliability of DNA purification from stool 55 samples, which are notorious for the presence of enzyme inhibitors, and the specificity of 56 the primers used for PCR amplification. In addition, investigation of Entamoeba in such 57 samples is largely limited to the ribosomal RNA genes due to the complexity of the 58 DNAs extracted from stool, which often contains DNA from multiple other parasites and 59 may include multiple Entamoeba species. The elimination of culture dependency has led 60 to a dramatic expansion in the number of genetically distinct Entamoeba organisms being 61 recognized but also to a greater understanding of sequence variability within species due 62 to the relative ease with which multiple samples can be studied in parallel.