Species delimitation infers the boundaries and number of species from evidence. No single arbitrary percentage of nucleotide sequence difference is a universal boundary. Divergence within an old, widespread species can exceed divergence between recently separated species; geography and multiple kinds of evidence matter. [ref]
Species Concepts and Species Delimitation by Kevin de Queiroz. pp. 884–885, “Alternatives to the Traditional Properties”: divergence cutoffs and geographic differentiation
An original counterexample to a universal percentage rule: an assay could examine a strongly conserved gene in one comparison and a rapidly changing nucleotide sequence in another. Equal percentages then answer different historical questions. Even the denominator needs specifying: all sites, aligned sites, or only a filtered region? A threshold is an operational rule for a stated assay and group of organisms, whose errors need evaluating. It is not a definition that automatically transfers to every species.
Contrasting operational evidence for species delimitation
. Original hypothetical comparison of morphology, mating and ancestry evidence. Each observation tests a different claim; the combinations are thought experiments, not a dataset or verdicts about named species. “Not tested” does not mean absent. Details.
The diagram's hypothetical pair B has similar morphology but reduced mating and distinct ancestry evidence. A morphological species concept could miss that distinction. Pair C instead has different morphology but compatible mating and ancestry that fails to separate the samples. That combination calls for checking variation in phenotype within a species. Pair A has only a structural difference observed: the other criteria remain untested. These examples show why missing evidence, disagreeing evidence and agreeing evidence require different interpretations.
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