DOI: 10.5061/dryad.sqv9s4n16
title: Contribution of genetic versus plastic responses to adaptive patterns in a widespread butterfly along a latitudinal cline

description: Understanding how organisms adapt to complex environments is a central
 goal of evolutionary biology and ecology. This issue is of special
 interest in the current era of rapidly changing climatic conditions. Here,
 we investigate clinal variation and plastic responses in life history,
 morphology, and physiology in the butterfly Pieris napi along a
 pan-European gradient by exposing butterflies raised in captivity to
 different temperatures. We found clinal variation in body size, growth
 rates and concomitant development time, wing aspect ratio, wing
 melanisation, and heat tolerance. Individuals from warmer environments
 were more heat-tolerant, had less melanised wings and a shorter
 development but still they were larger than individuals from cooler
 environments. These findings suggest selection for rapid growth in the
 warmth and for wing melanisation in the cold, and thus fine-tuned genetic
 adaptation to local climates. Irrespective of the origin of butterflies,
 the effects of higher developmental temperature were largely as expected,
 speeding up development, reducing body size, potential metabolic activity,
 and wing melanisation, while increasing heat tolerance. At least in part,
 these patterns likely reflect adaptive phenotypic plasticity. In summary,
 our study revealed pronounced plastic and genetic responses, which may
 indicate high adaptive capacities in our study organism. Whether this may
 help such species though to deal with current climate change needs further
 investigation, as clinal patterns have typically evolved over long
 periods.
description: For this study, we collected
 freshly-eclosed, spring generation females from a total of nine
 populations along a latitudinal gradient from northern Italy to Sweden
 (Fig. 1). We sampled three replicate populations each in northern Italy
 (I: Torino 45.11°N / 7.48°E, Pavia 45.21°N / 9.27°E, Mantova 45.21°N /
 10.75°E), northern Germany (G: Wahrenholz 52.64°N / 10.61°E, Rathenow
 52.65°N / 12.44°E, Strausberg 52.60°N / 13.86°E), and Sweden (S: Örebro
 59.29°N / 15.01°E, Eskilstuna 59.36°N / 16.54°E, Stockholm 58.95°N /
 17.58°E). Mean annual (Italy: 13ºC, Germany, 9ºC, Sweden: 6ºC) and the
 mean temperature during the vegetation period (May to September; Italy:
 21ºC, Germany: 16ºC, Sweden: 14ºC) followed a temperature gradient across
 latitudes while precipitation is higher in Italy than in Germany and
 Sweden (Supplementary Table S1). The minimal straight distance between two
 populations was 73 km, the total latitudinal gradient spanned ca. 1660 km.
 We collected a total of 74 females from Italy, 94 from Germany, and 76
 from Sweden between 19<sup>th</sup> of April and
 14<sup>th</sup> of June 2016. All females were afterwards
 transferred to Greifswald University for egg-laying. <b>Experimental
 design</b> Field-caught females were kept
 individually in translucent 1 litre plastic pots covered with gauze, which
 were placed into a climate chamber set at a constant temperature of 25°C,
 65 % relative humidity, and a photoperiod of L18:D6. Females where fed
 <i>ad libitum</i> with water, a 10% sugar solution, and
 additionally flowers (e.g. <i>Sambucus nigra</i>,
 <i>Taraxacum </i>spec., <i>Senecio </i>spec.). For
 oviposition, they were provided daily with a fresh cutting of <i>A.
 petiolata</i>. The resulting eggs were collected daily, counted, and
 kept separated by oviposition day and female under the above conditions
 until hatching. Eggs were collected until the death of the females. After
 hatching, all larvae were transferred individually to translucent plastic
 pots (250 mL) lined with moist tissue and cuttings of <i>A.
 petiolata</i> <i>ad libitum</i>. Host-plants were
 replaced on a daily basis. On day 3 after hatching, larvae were randomly
 divided among two rearing temperatures (18 and 25°C), using a split-brood
 design. These temperatures were chosen to mimic summer conditions (July)
 in Germany / Sweden and Italy (Supplementary Table S1). Larval rearing
 took place in climate cabinets (Sanyo MLR-351H; Bad Nenndorf, Germany) at
 60% relative humidity and L18:D6. For each individual, we recorded larval
 development time, pupal development time, and pupal mass (one day after
 pupation, KERN ABJ-120-4M; 0.1 mg accuracy). Larval growth rate was
 calculated as the natural logarithm of pupal mass / larval time. One day
 after adult eclosion, butterflies were subjected to a heat knock-down
 assay. Therefore, they were individually placed into translucent plastic
 pots (100 ml) in a randomized block design, and exposed to a constant
 temperature of 43°C (climate cabinet Sanyo MIR-553; Bad Nenndorf,
 Germany). The time until physical knock down, characterized by an
 inability to move in a coordinated manner, was recorded. Butterflies were
 afterwards frozen at -80°C for later analyses. Frozen butterflies were later
 thawed and their adult fresh mass was measured with a digital scale (KERN
 ABJ-120-4M). Then, the head, wings and legs were removed, and the thorax
 and abdomen were separated before being weighed. The thorax-abdomen ratio
 was calculated as the thorax mass divided by the abdomen mass. Butterfly
 wings were used to measure wing morphology and melanisation. Therefore, we
 took a photograph of one dorsal fore- and one ventral hindwing per
 individual under standardized illumination with a PC microscope camera
 (Veho MS-004 Discovery Deluxe USB Microscope). The ventral hindwing was
 used as its melanisation is known to influence heat gain during lateral
 basking (Heinrich, 1996), whilst dorsal forewing melanisation influences
 the heat gain during dorsal basking (Kingsolver, 1987). To score wing
 area, we used the “lasso” function in Adobe Photoshop CS6. Wing
 melanisation was defined as the percentage of black wing area
 (Supplementary Figure S2). We used a threshold approach with a fixed value
 of 128 (on a scale of 0 to 255), turning each pixel on the butterfly wing
 into either black or white (Adobe Photoshop CS6). We additionally
 calculated fore- to hindwing ratio (forewing area divided by hindwing
 area), wing loading (total body mass divided by forewing area), and wing
 aspect ratio to examine wing shape (4 x forewing
 length<sup>2</sup> divided by forewing area; Berwaerts
 <i>et al.</i>, 2002). <b>Physiological
 parameters</b> We measured the following
 parameters related to oxidative stress: (1) potential metabolic activity
 (PMA), (2) two markers of antioxidant defences: total hemolymph
 antioxidant capacity (OXY) and glutathione (a non-enzymatic antioxidant;
 GSH), and (3) three markers of oxidative damage: hydroperoxide
 concentration (a Reactive Oxygen Metabolite; ROM), malondialdehyde
 concentration (a marker of lipid peroxidation; MDA), and DNA damage
 (8OHdG). To keep the size of the experiments manageable, PMA, OXY and ROM
 were only measured in males. Frozen abdomen were cut into two
 similar-sized parts and weighed (± 0.1 mg). The first half was used to
 measure PMA, while the second half was used to measure OXY and ROM. For
 measuring GSH, MDA, and 8OHdG, we used thoraces and abdomen from other
 males and females. For details on measuring physiological parameters, see
 Supplementary Material S3.