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Isoetes dubsii sp. nov. and I. santacruzensis sp. nov., two new species from lowland areas in South America, are described, illustrated and compared to similar species. Isoetes dubsii can be distinguished from other species of the Brazilian Pantanal wetlands by a set of characters including leaves that are long, flexuous and trigonal in transverse section, tri-lobate stems, rudimentary velum, pustulate megaspores of 310-390 µm diameter and laesurae of the megaspore at least four times wider than high. Isoetes santacruzensis has flexuous, filiform leaves, 0.4-0.8 mm wide at mid length and reaching up to 15 cm long, black or reddish-black sporangia, sclerified phyllopodia and sparsely verrucate megaspores of 320-390 µm in diameter. We also include a key for species from the Brazilian Pantanal wetlands and Bolivia and spore images for all species that are discussed. Isoetes dubsii and I. santacruzensis are only known from their type localities and they may deserve special attention concerning their conservation status. However, based on our current knowledge on these species and according to IUCN Red List criteria, they are assessed here as data deficient (DD). [ABSTRACT FROM AUTHOR]