There is a clear historical instance of a new species being created by natural processes. See https://en.wikipedia.org/wiki/Spartina_anglica
Species with extended geographical ranges can have infertility between the populations at the ends of the distribution most clearly demonstrated by ring species where the population extends round some obstructive feature and where the two "ends" have met up and do not interbreed. Such populations could be split into two species by exterminating it in the middle part of the range. The obvious ethical considerations prevent this as a deliberate experiment but it could happen as a consequence of human interference with the environment for economic purposes.