Phenotypic tests
The reproductive modes of NZ Daphnia clones were determined using a sexuality test. Because sexual Daphnia need sperm fertilization to produce diapausing embryos, the sexuality test is to check whether, in the absence of males, diapausing embryos are deposited into ephippia by females. The consistent presence of embryos in ephippia in the absence of males implies an obligately asexual clone, whereas consistent results of no ephippial embryos from at least three consecutive rounds of tests was used to infer a male-requiring cyclical parthenogen. We decapsulated a total of 32 ephippial embryos produced by clones from Lake Alexandrina in the absence of males. All 30 contained embryos and some of these hatched, consistent with the clones from this lake being obligate asexuals.
Male production was induced by adding the hormone methyl farnesoate (MF) to the medium. Adult females for each clone were isolated and placed into 50-mL tubes containing methyl farnesoate at a concentration of 400 nM (changed daily). We examined the sex of offspring using a dissection microscope. Males can be visually distinguished from females based on the enlarged antennules and flattened ventral carapace margin. Successful production of males by ≥5 individuals was taken to be evidence for a male-producing clone.