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Reproductive isolation consists of barriers that reduce gene flow through sexual reproduction. Distinguish premating isolation from barriers after mating. Barriers before fertilization also include incompatible gametes; barriers after fertilization can reduce offspring survival or fertility. ^[ref]

An assay should state which step it measures. Observing fewer cross-group matings does not measure offspring fertility; observing fertilization does not demonstrate that the offspring will reproduce. These distinctions make a partial barrier informative without declaring completed speciation.

Speciation is the formation of distinct species through evolution. Detecting a difference between biological populations is a starting observation; its interpretation depends on the stated species concept and the evidence for lasting separation. Reproductive isolation is one important route to limiting gene flow in sexual reproduction.

A species concept states what should make organisms members of the same species; an operational criterion states what observations will test that claim. Reproductive isolation, distinguishable morphology and ancestry inferred through phylogenetics answer different questions. In a lineage-based interpretation, they are evidence for separation of evolving biological populations, and need not appear together. ^[ref]