Yes, if you want to have 16 lanes for pci 1 gpu, you can only put a second m2 ssd in slot 3 or 4 without any loss.

There are 2 more slots that run via the chipset of the mainboard ☺️ it is best to check the manual (online)

Yes, with the ASUS ROG Crosshair X870E Hero motherboard, it is recommended to install an M.2 SSD in the M.2_1 slot to utilise the full PCIe 5.0 x4 bandwidth without compromising the performance of the graphics card. This slot does not share any lanes with the graphics card and therefore offers optimum performance for an M.2 SSD.

The other M.2 slots, especially M.2_3, share PCIe lanes with the graphics card. If you install an M.2 SSD in these slots, this can reduce the bandwidth of the graphics card, which could affect performance.

Therefore, it is best to install the M.2 SSD in the M.2_1 slot to ensure maximum performance for both the SSD and the graphics card.

M.2_1 and M.2_2 should be possible without losses, if I read the manual correctly.

You only have PCIe x8 (instead of x16) if M.2_2 and M.2_3 have an SSD at the same time or M.2_3 alone.