We argue that the mode of reproduction of microorganisms in nature can only be decided by population genetic information. The evidence available indicates that many parasitic protozoa and unicellular fungi have clonal rather than sexual population structures, which has major consequences for medical research and practice. Plasmodium falciparum, the agent of malaria, is a special case: the scarce evidence available is contradictory, some suggesting that uniparental lineages may exist in nature. This is puzzling (because P. falciparum is known to have a sexual stage) and poses a challenge that can be readily settled by ascertaining the frequency distribution of genotypes in natural populations.Sexual reproduction is generally assumed to be a common mode of reproduction of eukaryotes. In the case of parasitic protozoa, the assumption of sexual reproduction relies largely on the presumption that these organisms are diploid, as well as on the occurrence of sexual recombination in the laboratory under appropriate circumstances (review in ref. 1), rather than on relevant evidence obtained from nature. Yet, whether or not sexual reproduction prevails in these organisms is of considerable medical and agronomic consequence as well as of scientific interest. These eukaryotic microorganisms include the agents of malaria, sleeping sickness, Chagas disease, and other parasitic diseases that affect more than 10% of the world population. The strategies for developing vaccines or curative drugs as well as for diagnosis and treatment are different for clonal and for sexual organisms.That sexual reproduction may occur in laboratory cultures or even occasionally in nature does not by itself settle the issue, since that simply manifests that the potentiality for sexual reproduction has not been lost. What remains to be determined is the prevailing mode of reproduction of these organisms in natural circumstances. The evidence to settle the matter exists for some of these organisms and could be obtained for others without massive investment of resources or new scientific or medical advances. We herein advance a sustained argument to show that population genetic evidence and population genetic theory is all that is needed to ascertain the extent to which, if at all, these (or any other) organisms reproduce sexually in nature. We have already reviewed the evidence for Trypanosoma cruzi, the agent of Chagas disease (2), and some other protozoa (3). Here we develop further the argument and present the results of a survey of the available evidence for parasitic protozoa and unicellular fungi. CLONALITY IN MICROBIAL EUKARIOTESThe two genetic consequences of sexual reproduction are segregation and recombination. Population genetic methods make it possible to ascertain whether or not the distribution of genotypes in natural populations is consistent with the occurrence of segregation and recombination. The kind of evidence that is needed is the frequency distribution ofgenotypes rather than the direct observation of sexual or clonal r...

Trypanosoma cruzi, the causative agent of Chagas disease, presents wide genetic diversity. Currently, six discrete typing units (DTUs), named TcI to TcVI, and a seventh one called TcBat are used for strain typing. Beyond the debate concerning this classification, this systematic review has attempted to provide an inventory by compiling the results of 137 articles that have used it. A total of 6,343 DTU identifications were analyzed according to the geographical and host origins. Ninety-one percent of the data available is linked to South America. This sample, although not free of potential bias, nevertheless provides today’s picture of T. cruzi genetic diversity that is closest to reality. DTUs were genotyped from 158 species, including 42 vector species. Remarkably, TcI predominated in the overall sample (around 60%), in both sylvatic and domestic cycles. This DTU known to present a high genetic diversity, is very widely distributed geographically, compatible with a long-term evolution. The marsupial is thought to be its most ancestral host and the Gran Chaco region the place of its putative origin. TcII was rarely sampled (9.6%), absent, or extremely rare in North and Central America, and more frequently identified in domestic cycles than in sylvatic cycles. It has a low genetic diversity and has probably found refuge in some mammal species. It is thought to originate in the south-Amazon area. TcIII and TcIV were also rarely sampled. They showed substantial genetic diversity and are thought to be composed of possible polyphyletic subgroups. Even if they are mostly associated with sylvatic transmission cycles, a total of 150 human infections with these DTUs have been reported. TcV and TcVI are clearly associated with domestic transmission cycles. Less than 10% of these DTUs were identified together in sylvatic hosts. They are thought to originate in the Gran Chaco region, where they are predominant and where putative parents exist (TcII and TcIII). Trends in host-DTU specificities exist, but generally it seems that the complexity of the cycles and the participation of numerous vectors and mammal hosts in a shared area, maintains DTU diversity.