Example. If the root store contains CA1 and CA2, you can configure Website 1 with no IIS CTL and both CA1 and CA2 are trusted. If for WebSite 2 you create IIS CTL and add CA1, then only CA1 will be trusted for Site 2.
Note: Using CTL's you cannot limit the list of CA's sent back to the client during the TLS handshake. I.e. you can't use CTL's to limit the list of certificates that Internet Explorer is showing.