How does Scapelli's crew constantly manage to beat the Mario brothers to various plumbing jobs after the Marios are hired? Scapelli is very likely tapping their phone lines.
When Koopa is walking, you might notice that he keeps his hands at chest level, curling them. This is likely a minor evolutionary holdover as Koopa descended from Tyrannosaurus rex who were known to have relatively small arms kept close to their bodies. He is momentarily de-evolved into a Tyrannosaurus in the climax.
Also, check out the Tyrannosaurus's coloring, just before it turns into slime. Yellow muzzle and belly, green elsewhere... it's Bowser's color scheme!
Scapelli is Mario's biggest enemy before he meets Koopa. Scapelli is turned into an ape. Scapelli is Donkey Kong. Another thing Scapelli and Donkey Kong have in common? They both sabotaged a construction site in progress...
When Mario and Luigi are escaping via police car, they accidentally ramp on top of another car. Meaning their escape is aided by doubling in height.
That Mario and Luigi's last name is just "Mario" seems lazy, but then you remember that you call them the Mario Brothers, meaning the last name comes from the whole series' name.
As mentioned on the film page in Evolutionary Levels, there are separate settings on the de-evolution ray for physical and intellectual transformation. This means Koopa intentionally let the King retain his intelligence as a way of tormenting him. This comes back to bite him when the King intentionally chokes the city and helps the Mario Bros.
Related to the above, the scene at the Boom-Boom Bar when Lena is downing a drink with a small talking creature who begs for his life. At first, it seems like a minor Black Comedy gag, but then you remember the de-evolution chamber.
Why is Dinohatten such a desolate, dystopian place in comparison to the Mushroom Kingdom, Sarasaland, or even Dinosaur Land from the games? It's because this is a world where Bowser won and Mario and Luigi weren't there to save it so Bowser was allowed to turn the whole Kingdom into the Dark Lands.
