Phenotypic plasticity in response to temperature is expected to play a key role in how organisms cope with climate change. Evolved differences in plastic responses are often linked to historical differences in average temperatures, yet we know little about how behavioral plasticity is affected by prevailing thermal environments. In this study, we used a common-garden design to test whether historical differences in average temperatures caused evolutionary divergence in the plasticity of mating behavior of Western mosquitofish (Gambusia affinis) inhabiting geothermal springs with average source temperatures spanning from 18.8 to 33.3 C. We found population differences in the thermal plasticity of courtship displays, copulation attempts, copulations, and mating efficiency, but these differences could not be explained by average source temperatures. We also tested for differences in thermal optima and maximum performance in mating behavior among populations. We found that only the maximum number of displays differed among populations, although these differences were also unrelated to source temperature. While temperature may have predictable evolutionary consequences for some thermally sensitive traits, our findings are inconsistent with theoretical predictions of evolutionary responses to divergent average temperatures, highlighting the need for greater synergy between empirical and theoretical work to understand thermal adaptation.

These data were collected to investigate the effect of differences in average source population temperature on the evolution of behavioral plasticity in mating behavior in Western mosquitofish. Mosquitofish from 5 geothermal springs in Bishop, CA with source temperatures ranging from 18.9 to 33.3 C were collected and reared for two generations in a common thermal environment. Mating behavior of each F2 male was observed at 5 ecologically relevant ambient temperatures (15, 20, 26, 32, and 37 ºC) that encompassed the thermal gradient of source populations. The order of observation temperatures was randomized for individual males, although all males were first observed at intermediate temperatures (20, 26, 32) due to possible mortality at extreme temperatures (Otto 1973; Wilson 2005). Males were assayed with two standardized female fish from pond separate from the focal populations. Behavioral assays occurred in blue-felt-lined observation chambers (40 x 21 x 12 cm deep). A camera (Olympus Stylus TG-4, Bethlehem, PA) was placed on a shelf above each tank to record behavior, with two 18 W lights affixed beneath the shelf to illuminate the field of view. 20-minute behavioral interactions were recorded after a 20 minute acclimation period. After each male's first assay, males were removed from the tank and photographed.

Video recordings of mosquitofish interactions were analyzed using CowLog 3.0 behavioral coding software (Hänninen and Pastell 2009). Courtship displays occurred when males arched their body into an sigmoid-shape characteristic of poecilid courtship behavior (Bisazza 1993). Copulation attempts were counted when a male approached a female from behind and oriented beneath the female caudal peduncle, a position required for copulation in livebearing fish (Bisazza et al. 2001). Copulations occurred after a subset of attempts and were identified by the rapid twisting motion that accompanied the male removal of the gonopodium from the female gonoduct (Wilson 2005). Video observers were trained to identify behaviors on a standardized set of videos before collecting data to ensure consistency among observations. Each video observer was blind to treatment and source population identity when identifying and counting behaviors. 

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