Punctuation mark unknown in settlement (6)
I believe the answer is:
colony
'settlement' is the definition.
('colony' can be a synonym of 'settlement')
'punctuation mark unknown' is the wordplay.
'punctuation mark' becomes 'colon' (colon is a kind of punctuation mark).
'unknown' becomes 'y' (algebra).
'colon'+'y'='COLONY'
'in' is the link.
(Other definitions for colony that I've seen before include "Country under the political control of another and occupied by settlers" , "Group of animals of the same type living together" , "Group of organisms of the same type living together" , "Country controlled by another's settlers" , "Community" .)