There are spoilers in this question:

I am playing the game for a second time and noticed that in Black Velvetopia, there's an Ammo upgrade that wasn't where it should have been...and then later on it appeared. So is its appearance triggered by something? Or was this just a glitch in the game and it should have been there all along?

I got three of the Queen cards and entered the Matador's apartment (next to the last card) and entered the secret area through the fireplace. I exited out the window at the end of the secret fireplace area and was walking on the wood railing towards the end of the alley (that has the fruit stand in it), I noticed that the Ammo Upgrade that should have been on a nearby balcony wasn't there like it was the first time I played through the game.
But after I got the last card and re-tried the wood railing, the Ammo Upgrade (the glowing Exclamation Point) was there on the balcony. Of course, this was also after getting the Confusion Badge from the Matador.
So what triggered the Ammo Upgrade to appear? The 4th card? Or was it the Confusion Merit Badge?
I'm just curious so if I play it for a third time, I'll know how to make sure the Ammo Upgrade is there.

I'm pretty sure that if you get a new power that involves ammo, then any ammo upgrades for that power will appear. The exact same thing happened to me -- I got Confusion, and then after going back to Black Velvetopia, found that ammo upgrade. It also appeared in other places where I haven't seen it before.