There are many ways to play with the understood (and therefore unexamined) conventions of the comic strip genre. And it seems like Mark Tatulli is going to explore them all in his strip Lio.
In this 4/14/16 sequence, the gag depends on something the reader knows intuitively, but never thinks about:
Lio isn't a real person, and neither is his world. He's just a two-dimensional drawing. That's the punchline delivered by the final panel. We know it -- but we're seldom reminded of it.
Note what Tatulli does to get maximum effect out of his gag. In the first panel, Lio's stepping cautiously; the second, he's walking and happy; the third he's running and joyful. In emotion and motion, Lio's building momentum. So when in the fourth panel, he's not just motionless, he's been stopped cold and all that energy slams us into the panel.
And note that there is no fourth panel. Not really. Nothing exists in the infinite blankness of the page until the hand draws it. The end of the branch has been drawn, but the ground, and indeed the borders of the fourth panel haven't. So Lio's arrived in an area of the comic that's still under construction.
The gag is brilliant -- and it's the delivery that makes it so. And that's why I write these appreciations of the creator's artistry. Because like the conventions of the medium, they're often invisible.