Extended Data Fig. 7: Temperature preference behavior and TPN-V innervation are species-specific and consistent across populations.

(a-c) Preference indexes measured from additional strains of D. persimilis, D. melanogaster, and D. mojavensis, demonstrate consistent behavior within populations of the same species (compare to data shown in Fig. 1b–d, based on different strains -see methods). (a) A D. persimilis strain from Santa Rosa, California (strain 24) (N = 8,8,8,7,7,7 groups of 15 flies for test temperatures 15, 20, 25, 30, 35, and 40 °C, respectively). (b) A wild-derived D. melanogaster strain from Egypt (EG16; N = 13,11,12,12 groups of 15 flies for test temperatures 15, 20, 25, 30, and 35 °C, respectively). (c) A D. mojavensis wrigleyi strain from Catalina Island (D. m. wrigleyi is a subspecies of D. mojavensis; N = 20,19,18,20 groups of 15 flies for 25, 30, 35, and 40 °C, respectively) (d-f) 2-photon z-stacks of dye-labelled TPN-V in each of the three strains shows consistent anatomy within species (compare to data shown in main Figs. 4 and 5, based on different strains of the same species -see methods). (g) A distance matrix of reconstructions (see methods) shows additional strains cluster by species (highlighted branches/datapoints represent additional strains generated from 2P images in d-f, whereas greyed out ones are re-plotted from Fig. 5). Abbreviations: Lateral Horn, LH; Calyx, CA. Scale bars = 25 μm. Asterisks: lateral Accessory Calyx (lACA). Arrowheads = ventral anterior LH, the dotted line is for reference. (In a-c: red lines=mean, filled boxes=one standard deviation, empty boxes=95% CI of the mean, empty circles=groups of flies, grey shading = approx. favorite thermal range, and Ø denotes preference indexes not significantly different from zero in two-tail one sample t-test; p < 0.05, p = [6.30e-3, 3.33e-4, 0.747, 2.47e-5, 7.67e-6, 1.55e-7] for a, p = [2.22e-9, 9.38e-6, 0.488, 1.34e-5, 1.76e-10] for b, and p = [0.888, 3.66e-8, 1.58e-7, 1.13e-5] for c).

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