During the process of allopatric speciation, after geographic separation has occurred, what process would be a necessary last step before reproductive isolation is established?

Occurrence and accumulation in time of new mutations in each of the separated populations. This leads to a gradual differentiation.

Explanation:

Allopatric speciation consists of the geographic separation of an original population so that it can give place to two or more new geographically isolated populations. These separations might be due to migration, extinction of geographically intermediate populations, or geological events. In this speciation, some barriers impede genetic interchange, or genetic flux, as the two new populations that are separated can not get together and mate anymore. These barriers might be geographical or ecological.

The process of allopatric speciation involves different steps:

The emergence of the barrier, Interruption in the genetic interchange, The occurrence of new mutations and their accumulation in time in each population. Slow and gradual differentiation, Genetic divergence by natural selection and reproductive isolation, which makes it impossible for the two groups to cross even if the barrier disappears, Prezigotic isolation mechanisms favored by selection once it occurs a secondary contact between the new species in formation.