Cold adaptation across the elevation gradient in an alpine butterfly species complex

1. Temperature acts as a major factor on the timing of activity and behaviour in butterflies, and it might represent a key driver of butterfly diversification along elevation gradients. Under this hypothesis, local adaptation should be found along the elevation gradient, with butterflies from high elevation populations able to remain active at lower ambient temperature than those from low elevation.

2. We recorded the warming-up rate and the thoracic temperature at take-off of 123 individuals of the Alpine butterfly species complex Coenonympha arcania - C. macromma - C. gardetta in controlled conditions.

3. Warming-up rate increased with elevation within C. arcania: high elevation males of C. arcania were able to warm-up more quickly, as compared to low elevation ones.

4. High elevation C. gardetta had a darker underwing pattern than low elevation ones. This high-elevation species was significantly smaller (lower weight and wing surface) than the two other species, and had a faster warming up rate.

5. Our results suggest that the ability to warm-up quickly and to take-flight at a high body temperature evolved adaptively in the high-altitude C. gardetta, and that low temperature at high altitude may explain the absence there of C. arcania, while the hybrid nature of C. macromma is probably the explanation of its elevation overlap with both other species, and its local replacement of C. gardetta.

Methods
Individual Coenonympha specimens were collected in the Massif des Ecrins, French Alps (Isère and Hautes-Alpes, Table 1). All captures using entomological nets took place between 3 and 6 July 2017. C. arcania was collected in three localities: Chaillol-Les Combettes (1275 m), Chaillol (1600 m), where it flies together with C. macromma, and Notre Dame de la Salette (1770 m) where it flies with C. gardetta. C. macromma was collected at three elevations in Chaillol (1600 m, 1850 m and 2000 m). C. gardetta was collected in Notre Dame de la Salette (1800 m) and in Ailefroide (1875 m).

Upon capture, butterflies were individually put into glassine envelopes and cooled to ca. 11°C in a portable fridge. The same day, in a room at ambient temperature (22 to 25°C), each specimen was taken out of the fridge and placed on a piece of cotton under a 150 W neodynium daylight lamp, which has a measured light emission spectrum from 100 to 1400 nm, giving an irradiance of ca. 280 W/m² at the butterfly level.

The thoracic temperature of each specimen was monitored with a testo®895 IR thermometer with the emissivity set at epsilon= 0.95, and the temperature was automatically recorded into a computer database, one datum per second. A previous experiment has shown that the temperature inside the thorax was ca. 1.3°C warmer than the one measured on the surface, with a low variance of this difference. The take-off temperature was the last temperature recorded before the butterfly spontaneously took off. A 4-th degree curve was fitted to the raw data, and its slope was used to estimate the warming-up rate at 26°C, in °C s-1