Classifying species can be a difficult and particularly gray area. The key to this question, however, is that mules are sterile. This is an example of post-zygotic reproductive isolation. Hybrids that are sterile are not considered unique species.

Simply identifying traits or chromosome number is not enough to determine species. The defining factor of speciation is that the organism can no longer reproduce with the parent species, but can reproduce with other individuals of the species. Note that all breeds of dog still fall in the same species, despite the variety of different traits displayed across breeds, because they can produce offspring. Chromosome number also cannot solely determine species. Some genetic disorders result in aneuploidy in humans, in which individuals have too many or too few chromosomes.

Out of the choices, only temporal isolation can explain this phenomenon. Temporal isolation describes a type of reproductive isolation that comes about because the two species mate at different times.

Habitat isolation cannot explain this occurrence because we are told that the two species live in the same area. Hybrid breakdown also cannot be correct because the researcher was able to generate a viable hybrid (so he should reasonably be able to find first generation hybrids in the wild). Gametic isolation is also not correct because a hybrid was successfully generated. 

A hybrid zone is a geographic region where allopatric populations happen to come back in contact with one another. There are three potential outcomes for the hybrid zone over time. It is possible for the two species to re-integrate due to a weakening of reproductive barriers. In this scenario, the two species become one again. It is also possible that the reproductive barriers will strengthen, resulting in an inability to produce the hybrid offspring and a retention of species identity. The final option is a stable intermediate in which the species remain separate, but are still naturally able to create hybrid offspring.

This is an example of temporal isolation. Temporal isolation means that different species are kept reproductively isolated because they mate at different times.

Reproductive barriers can be divided into two major categories: pre-zygotic or post zygotic. The former is an issue that arises before fertilization such as two animals not being compatible anatomically or a behavioral incompatibility (think of bird mating rituals, or imagine a mouse trying to reproduce with a whale). A Post-zygotic barrier is a reproductive barrier that prevents the zygote from forming, or hampers the developmental process. It does not prevent the animals from mating however. Thus, The sterile mule example falls under the post-zygotic category since the animals were not prevented from mating. 

Barriers to reproduction are behaviors and physiological processes that maintain distinct species and prevent hybridization. There are two major classifications of barriers to reproduction: pre-zygotic barriers and post-zygotic barriers. Pre-zygotic barriers prevent hybridization before fertilization and post-zygotic barriers prevent it after fertilization.

Temporal isolation is a pre-zygotic barrier to reproduction. In instances of temporal isolation, fertile individuals do not meet one another, thereby isolating them reproductively. This includes distinct habitats, physical barriers, and different timing of sexual maturity.

Behavioral isolation is a type of pre-zygotic barrier to reproduction that isolates species through different mating rituals. “Species of fish living in different areas of a pond due to differential salt concentrations” is not an example of behavioral isolation because there is not difference in mating behaviors that reproductively isolates the two species of fish. The rest of these examples demonstrate barriers to reproduction that originate with mating behavior.

Mechanical isolation is a type of pre-zygotic barrier that maintains distinct species through genital incompatibility. In this type of reproductive isolation, the genitals of a male and female of the same species allow for copulation and reproduction, while the genitals of individuals of different species mechanically will not allow for reproduction.

Gametic isolation is a type of pre-zygotic reproductive isolation in which gametes of different species are unable to hybridize due to genetic incompatibility. In coral reef ecosystems, large numbers of gametes are released by many species of marine reefs, making gametic isolation important in preventing hybridization.