You are comparing "running the logic of the function in-line" vs. "calling a function that does not execute the logic". That's not the comparison that matters. Comment out the "return" at the top of the function in the latter version, and the timing shows the function call to be more expensive than the inline code.

Another minor detail: in your inline version, you do two hash lookups for the value of  $zone_o->{_chromosome_length} whereas the function call version does only one lookup. If you change the inline version to assign that value to a "my" variable, and use that variable twice (just like it was used twice in the function call), you'll see a reduction of a few sec -- i.e. the improvement over the function call will be more evident.

Now, get back to the part that really matters.