More complex than expected: Cold hardiness and the concentration of cryoprotectants in overwintering larvae of five Erebia butterflies (Lepidoptera: Nymphalidae)

Understanding the factors restricting the distribution of some insect species to high altitudes is hindered by poor knowledge of temporal changes in their cold hardiness during overwintering. We studied overwintering larvae of five species of Erebia butterflies (Lepidoptera: Nymphalidae: Satyrinae) differing in altitudinal distribution: lowland E. medusa, submountain E. aethiops, subalpine E. pronoe, alpine E. cassioides, and subnivean E. pluto. We subjected them to three treatments, AutumnWarm (13/8°C), imitating conditions prior to overwintering; AutumnCold (5/0°C), imitating late autumn conditions; and WinterCold (5/0°C), differing from AutumnCold by a shorter photoperiod and longer exposure to zero temperatures. Supercooling points (SCP) did not differ between species in the AutumnWarm treatment, despite large differences in the concentrations of cryoprotectants (CrPC; lowest in E. medusa and E. aethiops). Lowland E. medusa was freeze-tolerant, the subalpine, alpine and subnivean species were freeze-avoidant, whereas submountain E. aethiops displayed a mixed strategy. SCPs diverged in the AutumnCold treatment: it increased in the lowland E. medusa (from -16.5 to -10.8°C) and reached the lowest value in E. cassioides (-21.7°C). In WinterCold, SCP increased in subalpine E. pronoe (from -16.1°C in AutumnWarm and -18.7°C in AutumnCold to -12.6°C). E. medusa decreased and E. aethiops increased their CrPCs between autumn and winter; the highest CrPC was recorded in subnivean E. pluto. CrPC did not correlate with SCP across species and treatments. Cryoprotectant profiles corroborated the difference between lowland and freeze-tolerant E. medusa and the three high altitude freeze-avoidant species, with E. aethiops in an intermediate position. Glycerol was surprisingly rare, trehalose was important in all species, and such rare compounds as monopalmitin and monostearin were abundantly present in E. pronoe, E. cassioides and E. pluto.