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The 1989 experiment used eight Drosophila pseudoobscura biological populations from one Bryce Canyon population: four maintained on starch medium, four on maltose, in cages at $25{}^{\circ }\mathrm{C}$. Testing began about a year after establishment. ^[ref]

Before testing, flies spent one generation on cornmeal–molasses–agar. Virgins aged 3–6 days entered multiple-choice chambers: twelve females from each population, followed by males from both populations introduced nearly simultaneously without anesthesia. Observers scored matings for 60–90 minutes under bright indirect lighting. One group's right wing tips were clipped for identification, with clipping reversed between replicates. ^[ref]

All sixteen cross-diet comparisons had positive mating assortment contrasts; eleven departed significantly from random mating. Same-diet comparisons did not show significant departures. Common-medium rearing addressed immediate diet conditioning; same-diet controls addressed separation and establishment population bottlenecks. Mate choice was not the selection target; the biological populations were maintained separately. The result supports partial premating isolation accompanying dietary divergence, not completed speciation. ^[ref]

The causal inference remains limited. A negative same-diet hypothesis test does not prove zero reproductive isolation. One generation under common conditions does not exclude every persistent environmental influence. The chamber readout alone cannot identify a particular gene, establish offspring fertility, or predict gene flow after sustained contact in nature. Further experiments would need to target those questions separately.

For observed pairings between two labels $A,B$, let $N_{\mathrm{same}}=N_{AA}+N_{BB}$ and $N_{\mathrm{cross}}=N_{AB}+N_{BA}$, where the first label denotes the female's group and the second the male's. A mating assortment contrast isIt lies between $-1$ and $1$. Positive $I$ describes an excess of same-label pairings over cross-label pairings; whether that is evidence of assortative mating depends on the null mathematical model and the assay. The four counts form a contingency table.

Here is an original scope check. Suppose independently paired females and males have probabilities $p_f,p_m$ of carrying label $A$. ThenWith $p_f=p_m=1/2$, the expected value is zero. With $p_f=p_m=4/5$, it is $9/25=0.36$, despite pairing being independent of label. This counterexample concerns unequal pool composition; it does not replace the group introductions and contingency table tests in the dietary divergence and mating isolation in Drosophila pseudoobscura experiment. Even with balanced pools, finite observed samples fluctuate: a positive $I$ alone is not a hypothesis test, much less a universal species boundary.