Ultrastructure and molecular phylogeny of two heterolobosean amoebae, Euplaesiobystra hypersalinica gen. et sp. nov. and Tulamoeba peronaphora gen. et sp. nov., isolated from an extremely hypersaline habitat.

We isolated two amoebae, Tulamoeba peronaphora gen. et sp. nov. and Euplaesiobystra hypersalinica gen. et sp. nov. from the high salinity waters (293-300 per thousand salinity) of a Korean solar saltern. These new species show features typical of Heterolobosea - a limax form with eruptive pseudopodial formation, flattened/discoidal mitochondrial cristae, cysts with plugged pores, and no discrete, stacked dictyosomes. 18S rRNA gene phylogenies place both species within the Heterolobosea. Tulamoeba peronaphora appears to lack a flagellate phase, and has one cyst pore that penetrates the cyst wall. In 18S rRNA gene trees, Tulamoeba peronaphora is specifically related to Pleurostomum flabellatum, an extreme halophile that is observed only as a flagellate. Its next closest relatives are Naegleria and Willaertia. Euplaesiobystra hypersalinica has 2-4 cyst pores in the ectocyst wall (only), and has a bi-flagellated flagellate phase with no obvious cytostome. Its closest described relative is Heteramoeba clara, which is marine, has a cytostome, lacks cyst pores, and has a different nucleolus organization. The Euplaesiobystra hypersalinica 18S rRNA gene is 99.5% identical to a sequence accessed under the nomen nudum 'Plaesiobystra hypersalinica' - we consider them the same species. Tulamoeba peronaphora grows at 75-250 per thousand salinity, while E. hypersalinica grows at 100-300 per thousand (at least) salinity. Both amoebae seem to be 'extreme halophiles', and their ancestors invaded high salinity environments independently of each other. These results provide more evidence that there is a substantial ecological and phylogenetic diversity of heterotrophic eukaryotes capable of growing in very high salinity environments, and these ecosystems may be more complex than usually assumed.