Darwin acknowledged that there were important holes in his theory of natural selection. What is the "hole" described in the film?

The hole is lack of knowledge of the concept of natural selection, especially its physical properties and how it operates. That is what made those organisms with fitted traits to resit the selective pressures and therefore survive than others with less resistance.

Basically, he lacked the knowledge of the concept of variation as the driving mechanisms of natural selection he theorised. Darwin did not understand how variation among different species of organisms physically emerged to made these organisms resistant to the selective pressure, and therefore emerge as the survival of the fittest in the population.