Your board supports these configurations according to the manufacturer:
x16
x16/x16
x16/x8/x8 or 8x/8x/16x
x8/x8/x8/x8
I dont have this board and have never used it so I am just going off basic PCI-E knowledge and what the manufacturer says. But if you put a card in slot 2 it might run at 8x speed. It depends on how exactly the lanes can be configured by the board. It might be slots 1 and 3 can be 16x and slot 2 can only be a max of 8x. Or it might allow any slot to be 16x if there is only 1 or 2 cards installed and then split lanes as more cards get installed.
EDIT: according to a picture at techpowerup showing a closeup of the PCI-E slots, it looks like all contain full 16x electrical pins. So if only 2 cards are installed, they can be in any of the slots and still run at 16x speeds. That was where my confusion came from before, many boards do not have the full 16x electrical pins in the secondary PCI-E slots that get lanes split to them with multiple cards installed.
If you put a card in slot #3, it would run at 16x while the first 2 cards run at 8x or opposite depending on what the cards are. For example if you had an audio card and raid card in slots 1 and 2 and a graphics in slot 3 then the 3rd slot would run at 16x. But if you plugged them all in the opposite way then the graphics in slot 1 would run at 16x while the others had a max of 8x. If you then also put a card in the #4 slot, all slots run at 8x speeds.
In addition to those, it looks like the actual PCI-E 4x slot will continue to work at 4x speeds regardless of the other slot configurations.
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