First, if you don't know, the .iso is basically all the files for the game. If you take them, compile them, and slap it on a disk, you get the entire game. But here is something interesting people on Smashboards found out.
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..ahem. The evidence that we will find more on the 2nd layer is the following:
-People that burned the .iso to play the game on modded Wiis could not access 1P Modes or Videos
-There are gaps in the musical tracks we all downloaded the other night and demo players confirmed, in videos and otherwise, music that did NOT make the "final cut"
-A few playable characters are missing trophy data in the .iso that was floating around, for example Yoshi's 2nd Smash trophy
There is also talk that a lot of ATs have yet to be seen in gameplay videos for whatever reason. Ridley is listed in the original .iso but not found anywhere; Andross and Rosalina have not been shown in any form; and characters that were confirmed at Whobby, E For All, and Jumpfesta such as Sothe and Onix have yet to be seen. It's silly to think they cut that much content from the game just before release, though still possible.
And as far as the character .pac files go, they are the same file size as Sheik's data and actually require their textures, motions, sounds, and other files to be full-fledged fighters. However, the size of such data is miniscule enough to be a "VC Comparable" download. In other words, the "missing files" can be received through a download. To add fuel to the fire, one of the missing tracks folks discovered but were unable to rip was titled "Purchase" and accompanied by "New Feature!" fanfares that were unheard of. Is this ghost data, or is there something Nintendo is hiding?
The argument against this is, of course, that it very well could be ghost data and that although it is 6 more fighters it doesn't fit the roster selection screen all too well.
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Here
Also, those characters are Dr. Mario, Roy, Mewtwo, Dixie Kong, Toon Zelda/Sheik.