Fifty million years of beetle evolution along the Antarctic Polar Front.

Global cooling and glacial-interglacial cycles since Antarctica's isolation have been responsible for the diversification of the region's marine fauna. By contrast, these same Earth system processes are thought to have played little role terrestrially, other than driving widespread extinctions. Here, we show that on islands along the Antarctic Polar Front, paleoclimatic processes have been key to diversification of one of the world's most geographically isolated and unique groups of herbivorous beetles-Ectemnorhinini weevils. Combining phylogenomic, phylogenetic, and phylogeographic approaches, we demonstrate that these weevils colonized the sub-Antarctic islands from Africa at least 50 Ma ago and repeatedly dispersed among them. As the climate cooled from the mid-Miocene, diversification of the beetles accelerated, resulting in two species-rich clades. One of these clades specialized to feed on cryptogams, typical of the polar habitats that came to prevail under Miocene conditions yet remarkable as a food source for any beetle. This clade's most unusual representative is a marine weevil currently undergoing further speciation. The other clade retained the more common weevil habit of feeding on angiosperms, which likely survived glaciation in isolated refugia. Diversification of Ectemnorhinini weevils occurred in synchrony with many other Antarctic radiations, including penguins and notothenioid fishes, and coincided with major environmental changes. Our results thus indicate that geo-climatically driven diversification has progressed similarly for Antarctic marine and terrestrial organisms since the Miocene, potentially constituting a general biodiversity paradigm that should be sought broadly for the region's taxa.

Studies on chill coma recovery in the ladybird, Harmonia axyridis: Ontogenetic profile, effect of repeated cold exposures, and capacity to predict winter survival.

The harlequin ladybird, Harmonia axyridis, is one of the most successful invasive insect species worldwide. We investigated whether (i) chill coma recovery time (CCRt) changes during the ontogenetic development of this species, (ii) CCRt varies in response to repeated cold shocks, and (iii) CCRt could be a good predictor of winter survival ability in adults. CCRt decreased during larval development, the lowest CCRt values were observed in teneral adults (one and four days old), and significantly higher values were observed for older adults (16 and 32 days old). Repeated cold shocks (two hours at -3 °C), interrupted by short (30 min) warm periods (22 °C) resulted in decreased CCRt after the second cold shock, probably depicting an acclimation response, but then CCRt increased with additional cold shocks, likely revealing the accumulation of chill injuries. The CCRt of pre-overwintering individuals was not correlated with their winter survival. This indicates that CCRt is not a reliable measure of cold tolerance in H. axyridis. However, this result could be partially affected by the experimental setup - the use of laboratory-reared individuals who experienced standardized conditions and thus the variability in CCRt of tested animals could be much lower than the variability present in nature. The substantial variation observed over the ontogenetic development of H. axyridis poses important methodological implications for future studies, as animals of the same stage/age should be compared with each other. The observed U-shaped response to repeated cold shocks indicates that the expectation of linearity between cold exposure and insect response is an oversimplification of real situations.

Insects in fluctuating thermal environments.

All climate change scenarios predict an increase in both global temperature means and the magnitude of seasonal and diel temperature variation. The nonlinear relationship between temperature and biological processes means that fluctuating temperatures lead to physiological, life history, and ecological consequences for ectothermic insects that diverge from those predicted from constant temperatures. Fluctuating temperatures that remain within permissive temperature ranges generally improve performance. By contrast, those which extend to stressful temperatures may have either positive impacts, allowing repair of damage accrued during exposure to thermal extremes, or negative impacts from cumulative damage during successive exposures. We discuss the mechanisms underlying these differing effects. Fluctuating temperatures could be used to enhance or weaken insects in applied rearing programs, and any prediction of insect performance in the field-including models of climate change or population performance-must account for the effect of fluctuating temperatures.

Variation in amine composition in plant species: how it integrates macroevolutionary and environmental signals.

PREMISE OF THE STUDY: While plants show lineage-specific differences in metabolite composition, plant metabolites are also known to vary in response to the environment. The extent to which these different determinants of metabolite composition are mutually independent and recognizable is unknown. Moreover, the extent to which the metabolome can reconcile evolutionary constraint with the needs of the plant for rapid environmental response is unknown. We investigated these questions in plant species representing different phylogenetic lineages and growing in different subantarctic island environments. We studied their amines-metabolites involved in plant response to environmental conditions. METHODS: Nine species were sampled under high salinity, water saturation, and altitude on the Kerguelen Islands. Their profiles of free aromatic, aliphatic, and acetyl-conjugated amines were determined by HPLC. We related amine composition to species and environment using generalized discriminant analyses. KEY RESULTS: Amine composition differed significantly between species within the same environment, and the differences reflected phylogenetic positions. Moreover, across all species, amine metabolism differed between environments, and different lineages occupied different absolute positions in amine/environment space. Interestingly, all species had the same relative shifts in amine composition between environments. CONCLUSION: Our results indicate a similar response of amine composition to abiotic environments in distantly related angiosperms, suggesting environmental flexibility of species is maintained despite major differences in amine composition among lineages. These results aid understanding of how in nature the plant metabolome integrates ecology and evolution, thus providing primordial information on adaptive mechanisms of plant metabolism to climate change.

Temperature and parasitism by Asobara tabida (Hymenoptera: Braconidae) influence larval pupation behaviour in two Drosophila species.

In holometabolous insects, pupation site selection behaviour has large consequences for survival. Here, we investigated the combined effects of temperature and parasitism by the parasitoid Asobara tabida on larval pupation behaviour in two of its main Drosophila sp. hosts differing in their climate origin. We found that larvae of Drosophila melanogaster--a species with a (sub)tropical origin--placed at 25°C pupated higher in rearing jars than those placed at 15°C. The opposite pattern was observed for Drosophila subobscura larvae--a species from temperate regions--which pupated lower, i.e. on or near the substrate at 25°C, than those placed at 15°C. When placed at 25°C, parasitized larvae of both species pupated closer to the substrate than unparasitized ones. Moreover, the Drosophila larvae that had been exposed and probably stung by A. tabida, but were not parasitized, pupated lower than the control unparasitized larvae. These results provide new insights of host behaviour manipulation by A. tabida larvae.

Spotlight on the invasion of a carabid beetle on an oceanic island over a 105-year period.

The flightless beetle Merizodus soledadinus, native to the Falkland Islands and southern South America, was introduced to the sub-Antarctic Kerguelen Islands in the early Twentieth Century. Using available literature data, in addition to collecting more than 2000 new survey (presence/absence) records of M. soledadinus over the 1991-2018 period, we confirmed the best estimate of the introduction date of M. soledadinus to the archipelago, and tracked subsequent changes in its abundance and geographical distribution. The range expansion of this flightless insect was initially slow, but has accelerated over the past 2 decades, in parallel with increased local abundance. Human activities may have facilitated further local colonization by M. soledadinus, which is now widespread in the eastern part of the archipelago. This predatory insect is a major threat to the native invertebrate fauna, in particular to the endemic wingless flies Anatalanta aptera and Calycopteryx moseleyi which can be locally eliminated by the beetle. Our distribution data also suggest an accelerating role of climate change in the range expansion of M. soledadinus, with populations now thriving in low altitude habitats. Considering that no control measures, let alone eradication, are practicable, it is essential to limit any further local range expansion of this aggressively invasive insect through human assistance. This study confirms the crucial importance of long term biosurveillance for the detection and monitoring of non-native species and the timely implementation of control measures.

Transdisciplinary Bioblitz: Rapid biotic and abiotic inventory allows studying environmental changes over 60 years at the Biological Field Station of Paimpont (Brittany, France) and opens new interdisciplinary research opportunities.

BACKGROUND: The Biological Field Station of Paimpont (Station Biologique de Paimpont, SBP), owned by the University of Rennes and located in the Brocéliande Forest of Brittany (France), has been hosting student scientific research and field trips during the last 60 years. The study area of the SBP is a landscape mosaic of 17 ha composed of gorse moors, forests, prairies, ponds and creeks. Land use has evolved over time. Historical surveys by students and researchers focused on insects and birds. With this study, we aimed to increase the range of taxa observations, document changes in species composition and landscape and provide a basis for interdisciplinary research perspectives. We gathered historical data, implemented an all-taxon biodiversity inventory (ATBI) in different habitats of the SBP study area, measured abiotic factors in the air, water and soil and performed a photographical landscape observation during the BioBlitz held in July 2017. NEW INFORMATION: During the 24 h BioBlitz, organised by the SBP and the EcoBio lab from the University of Rennes and the French National Center of Scientific Research (CNRS), different habitats were individually sampled. Seventy-seven experts, accompanied by 120 citizens and 12 young people participating in the European Volunteer Service, observed, identified and databased 660 species covering 5 kingdoms, 8 phyla, 21 classes, 90 orders and 247 families. In total, there were 1819 occurrences including records identified to higher taxon ranks, thereby adding one more kingdom and four more phyla. Historical data collection resulted in 1176 species and 4270 occurrences databased. We also recorded 13 climatic parameters, 10 soil parameters and 18 water parameters during the BioBlitz. Current habitats were mapped and socio-ecological landscape changes were assessed with a diachronic approach using 32 historical photographs and historical maps. The coupling of historical biodiversity data with new biotic and abiotic data and a photographic comparison of landscape changes allows an integrative understanding of how the SBP changed from agriculturally-used land to a managed natural area within the last 60 years. Hence, this BioBlitz represents an important holistic sampling of biodiversity for studies on trophic webs or on trophic interactions or on very diverse, but connected, habitats. The integration of social, biotic and abiotic data opens innovative research opportunities on the evolution of socio-ecosystems and landscapes.

Influence of temperature acclimation and gut content on the supercooling ability of the land snail Cornu aspersum.

The invasive land snail Cornu aspersum possesses a low ability to supercool (c. -5 degrees C in winter) and survives only minimal ice formation in its body fluids, what may limit its expansion to colder environments. In the present study, we investigated the influence of acclimation and starvation on its supercooling ability. During eight weeks, individuals were maintained at 20 degrees C, fed or starved, or placed at 5 degrees C, directly or with progressive acclimation to cold and shorter photoperiod. Temperature of crystallisation of whole individual (Tc(I)) and hemolymph (Tc(H)), mass data and gut content were recorded every two weeks. Hemolymphatic glucose and glycerol were measured at the end of experiment and occurrence of intestinal ice-nucleating agents (INA) was researched. Acclimation had no effect on Tc(I) but stimulated purging of the gut. Starvation induced a slight decrease of Tc(I) whereas a high quantity of alimentary particles in the digestive tract limited the supercooling ability. Glucose and glycerol were not synthesized in cold conditions. Mean Tc(H) was low (c. -17 degrees C), some INA being present in hemolymph of fed animals. Intestinal content of starved individuals exhibited a mean Tc of c. -6 degrees C, decreasing to c. -12 degrees after heating, suggesting the presence of organic INA.

Antennal structure of male and female Aphidius rhopalosiphi DeStefani-Peres (Hymenoptera:Braconidae): description and morphological alterations after cold storage or heat exposure.

Several species of the genus Aphidius are used in biological control programs against aphid pests throughout the world and their behavior and physiology are well studied. But despite knowing the importance of sensory organs in their behavior, their antennal structure has never been described. We describe here the types and distribution of antennal sensilla in Aphidius rhopalosiphi, a larval parasitoid of several aphid species and observe how this antennal structure is modified after cold storage or heat exposure. Six types of sensilla were found on both male and female antennae. Male and female antennae differed in the total number of antennomeres (16 in males, 14 in females) and in the number and distribution of three of the six types of sensilla. After cold storage or heat exposure, we observed the appearance of a small number of abnormal sensilla.

Evolution of freezing susceptibility and freezing tolerance in terrestrial arthropods.

Arthropods have evolved various adaptations to survive adverse seasons and it has long been discussed why some arthropods are freezing-susceptible and some are freezing-tolerant. However, which mode of frost resistance came first during the course of evolution? A commonly held opinion is that no choice of strategy has been offered in evolution, because each species of arthropod may have its own evolutionary and natural history, leading to cold-hardiness. Freezing tolerance is more frequent in holometabolous insect orders and partially used by certain vertebrates, like some terrestrially hibernating amphibians and reptiles. Supported by phylogenetic, ontogenetic and ecological arguments, we suggest here that freezing tolerance is more recent than freezing susceptibility in the course of arthropods evolution. In addition, we observe that three basic modes of freezing resistance in insect species exist in the field: (i) permanent or year-round freezing-susceptible species, (ii) alternative or seasonal freezing-susceptible/freezing-tolerant species, (iii) permanent or year-round freezing tolerant species.

Effects of a freezing event during hibernation on further survival, reproduction and growth in the partially freezing tolerant land snail Helix aspersa muller (Gastropoda: helicidae).

Tolerance of ectothermic animals to freezing is often estimated by assessing survival a few days after the treatment. However, in the long term, ice formation in the body tissues can affect survival, as well as reproductive capability and growth. The land snail Helix aspersa survives only short durations with ice in its tissues, to a lethal limit of 40 to 60 % of its body water frozen. Adult and immature snails were treated during their winter dormancy period to a freezing event above this limit; their survival was observed both in the short and long term, as well as their ability to reproduce (adults) and grow (immature snails). Treated snails were compared with a control group, which was not frozen. No difference appeared in the survival, reproduction and growth of control and frozen snails. This study confirms partial freezing tolerance in this population of Helix aspersa.

Well wrapped eggs: effects of egg shell structure on heat resistance and hatchling mass in the invasive land snail Cornu aspersum.

In ectotherms, the temperature changes that accompany climate shifts, microhabitat changes, and species range extensions can have profound effects on the performance of organisms. The aim of this laboratory study on the terrestrial invasive gastropod Cornu aspersum was to investigate the effect of dietary calcium source on egg shell structure and heat resistance of eggs in two populations from different climatic regions of France (Western Atlantic and Mediterranean). To date no literature is known about heat stress in calcified ectothermic eggs while exposed to heat peaks using fluctuating thermal regimes and optimal humidity. In snails from the Mediterranean population fed exclusively with CaCO(3) from limestone we found the thinnest egg shells and the smallest hatchlings. Limestone represents the most accessible calcium source for snails, but is, however, responsible for thinner and more mineralized egg shells (higher ash content). Hence thicker egg shells result from a low mineralized mucopolysaccharide-glycoprotein matrix that could play a nutritional role for hatchlings. Exposed to heat peaks, eggs in both populations had lower incubation time variability at the detriment of hatching rate. This study highlights the need for functional studies in this invasive species to understand the effects on population dynamics of interacting biotic and abiotic environmental factors under climate and anthropic habitat changes.

Evolution of metabolic rate in a parasitic wasp: the role of limitation in intrinsic resources.

Metabolic rate, a physiological trait closely related to fitness traits, is expected to evolve in response to two main environmental variables: (1) climate, low metabolic rates being found in dry and hot regions when comparing populations originating from different climates in a common garden experiment and (2) resource limitations, low metabolic rates being selected when resources are limited. The main goal of this study was to investigate if differences in intrinsic resource limitations may have disrupted the expected evolution of metabolic rate in response to climate in a parasitic wasp. We compared CO(2) production of females from 4 populations of a Drosophila parasitoid, Leptopilina boulardi, as an estimate of their metabolic rate. Two populations from a hot and dry area able to synthesise lipids de novo at adult stage were compared with two populations originating from a mild and humid climate where no lipid accumulation during adult life was observed. These last females are thus more limited in lipids than the first ones. We observed that a high metabolic rate has been selected in hot and dry environments, contrarily to the results of a great majority of studies. We suggest that lipogenesis occurring there may have allowed the selection of a higher metabolic rate, as females are less limited in energetic resources than females from the mild environment. A high metabolic rate may have been selected there as it partly compensates for the long distances that females have to cross to find laying opportunities in distant orchards. We suggest that intrinsic resources should be integrated when investigating geographical variations in metabolism as this factor may disrupt evolution in response to climate.

Overwintering strategy of two weevils infesting three gorse species: when cold hardiness meets plant-insect interactions.

The cold hardiness of two closely related weevil species, Exapion ulicis and E. lemovicinum was studied in relation to their life cycles. These two seed-eating weevils reproduce on Ulex plant species with different fruiting phenologies. E. ulicis lays eggs in spring and overwinters as an adult while E. lemovicinum lays eggs in autumn and overwinters as a larva. Adult weevils were collected in natural populations of Brittany (Western France) and characterized with morphological and molecular tools before experiments. We showed that both weevil species exhibited low supercooling points (SCPs) with mean seasonal values below -17 degrees C. Fresh mass, moisture content and sex were not correlated to supercooling ability. Weevils died upon freezing and the lower lethal temperatures (LLT) were within the range of SCP, indicating that both species are freezing intolerant. Comparison between species for SCP, LLT and survival to exposure at -8 degrees C in winter showed a higher cold resistance for E. ulicis than for E. lemovicinum. In addition, the seasonal evolution of cold hardiness differed depending on the species. These features suggest that response to cold of weevils is linked to their life cycles, and thus to the life history of their host plants.

Supercooling ability is surprisingly invariable in eggs of the land snail Cantareus aspersus.

From an ontogenetic point of view, invertebrate eggs are generally the most freezing intolerant stage of a species. Development state, water content and acclimation may affect their supercooling ability. In this study, we measured fresh mass, water content and temperature of crystallisation (T(c)) of eggs of the edible land snail Cantareus aspersus, depending on its form ("aspersa"vs. "maxima"), incubation temperature (20, 12 and 7 degrees C) and physiological age (as part of the complete development). We also tested their tolerance to freezing. Despite a high number of individual observations (n=759) and significant differences of fresh mass and water content between both subspecies, no effect of origin, incubation temperature or development state has been found in this study. T(c) remained constant whatever the condition, with an overall mean of -5.40+/-0.24 degrees C (mean+/-SD). We suggest that fresh mass is important, a high water content and a constantly wet surface confer to land snail eggs a poor ability to supercool. Moreover, the presence of ice nucleating agents at the egg surface (microorganisms present in the soil, calcium carbonate crystals of the egg shell) might induce freezing. Thus, considering the present results, to delay hatching by cryopreservation of eggs does not seem possible.

Impact of extreme temperatures on parasitoids in a climate change perspective.

Parasitoids depend on a series of adaptations to the ecology and physiology of their hosts and host plants for survival and are thus likely highly susceptible to changes in environmental conditions. We analyze the effects of global warming and extreme temperatures on the life-history traits of parasitoids and interactions with their hosts. Adaptations of parasitoids to low temperatures are similar to those of most ectotherms, but these adaptations are constrained by the responses of their hosts. Life-history traits are affected by cold exposure, and extreme temperatures can reduce endosymbiont populations inside a parasitoid, eventually eliminating populations of endosymbionts that are susceptible to high temperatures. In several cases, divergences between the thermal preferences of the host and those of the parasitoid lead to a disruption of the temporal or geographical synchronization, increasing the risk of host outbreaks. A careful analysis on how host-parasitoid systems react to changes in temperature is needed so that researchers may predict and manage the consequences of global change at the ecosystem level.

Does fluctuating thermal regime trigger free amino acid production in the parasitic wasp Aphidius colemani (Hymenoptera: Aphidiinae)?

When stressful cold-exposure is interrupted by short warm intervals, physiological recovery is possible, and this improves markedly the survival of insects. Fluctuating thermal regime (FTR) may act as a cue triggering the initiation of a metabolic response involving synthesis of cryoprotective compounds, such as free amino acids (FAA). Since specific changes in FAA levels can provide a good indication of the overall response of an organism to stressful conditions, we investigated temporal changes in FAA body contents of the parasitoid Aphidius colemani Viereck during exposure to FTR (4 degrees C: 20 degrees C for 22 h: 2 h per day) versus constant low temperature (4 degrees C). Physiological response during cold-exposure was clearly dissimilar between thermal treatments. Under constant cold-exposure FAA pool increased, whereas it decreased with cold-exposure duration in FTR. No single FAA accumulation could explain the higher survival under FTR. We propose that instead of considering FAA as a part of cryoprotective arsenal, FAA accumulation should rather be regarded as a symptom of a cold-induced physiological response. This is much less manifest under FTR, as the warm intervals likely allow a periodic reactivation of normal metabolic activities and a recovery of developmental processes.

Freezing or supercooling: how does an aquatic subterranean crustacean survive exposures at subzero temperatures?

Crystallization temperature (T(c)), resistance to inoculative freezing (IF), ice contents, bound water, protein and glycogen body contents were measured in the aquatic subterranean crustacean Niphargus rhenorhodanensis and in the morphologically close surface-dwelling aquatic crustacean Gammarus fossarum, both acclimated at 12 degrees C, 3 degrees C and -2 degrees C. Cold acclimation induced an increase in the T(c) values in both species but no survival was observed after thawing. However, after inoculation at high sub-zero temperatures, cold-acclimated N. rhenorhodanensis survived whereas all others, including the 3 degrees C and -2 degrees C acclimated G. fossarum died. In its aquatic environment, N. rhenorhodanensis is likely to encounter inoculative freezing before reaching the T(c) and IF tolerance appears as a highly adaptive trait in this species. Bound water and glycogen were found to increase in the 3 degrees C and -2 degrees C acclimated N. rhenorhodanensis, whereas no variation was observed in G. fossarum. Considering the hydrophilic properties of glycogen, such a rise may be correlated with the increased bound water measured in cold-acclimated N. rhenorhodanensis, and may be linked to the survival of this species when it was inoculated. The ecological significance of the survival of the aquatic subterranean crustacean to inoculative freezing is paradoxical, as temperature is currently highly buffered in its habitat. However, we assume that past geographical distribution and resulting life history traits of N. rhenorhodanensis are key parameters in the current cold-hardiness of the species.

Supercooling ability in two populations of the land snail Helix pomatia (Gastropoda: Helicidae) and ice-nucleating activity of gut bacteria.

The land snail Helix pomatia (Gastropoda: Helicidae) is widely distributed in Northern and Central Europe where it may experience subzero temperatures during winter months. Its supercooling ability was studied in two populations of H. pomatia. One population originated from Southern Sweden (Gotaland) and the other from Central France (Auvergne). In the experimental design, they were acclimated, over 2 weeks, to artificial winter conditions (hibernation, T=5 degrees C). The Swedish snails showed a rather limited supercooling ability (temperature of crystallization, T(c)=-6.4+/-0.8 degrees C), significantly greater, however, than the supercooling capacity of the population from France (T(c)=-4.6+/-1.4 degrees C). In artificial spring conditions (3 months of hibernation followed by a progressive acclimation, over 2 weeks, to activity at T=20 degrees C), both populations exhibited a similar high T(c) (-2.0+/-1.0 degrees C). The lower T(c) of hibernating Swedish snails could be due to a greater loss of body water, accompanied by a higher concentration of solutes in the hemolymph. In both populations, the variation in hemolymph osmolality measured between hibernating (250-270 mOsm kg(-1)) and active (165-215 mOsm kg(-1)) snails may be explained by the variation in body water mass and did not suggest the production of colligative cryoprotectants. Moreover, the three bacterial strains, Buttiauxella sp., Kluyvera sp., and Tatumella sp. (Enterobacteriaceae) which were isolated from fed snails, but absent in starved snails, did not show any ice-nucleating activity at temperatures higher than -9 degrees C. Only the strain Kluyvera sp. initiated nucleation at -9 degrees C. This strain, therefore, is a weak, also termed a Type III or Class C ice-nucleating active bacterium, but with no influence on the supercooling ability of individual snails. In summary, fluctuations in body water mass of hibernating snail populations, triggering changes in osmolyte concentration, rather than the presence of endogenous ice-nucleating-active bacteria, accounts for fluctuations in their T(c).

Metabolic responses to cold in subterranean crustaceans.

Changes in polyol, sugar and free amino acid (FAA) body contents were investigated in the aquatic, subterranean (i.e. hypogean) crustaceans Niphargus rhenorhodanensis and Niphargus virei and in a morphologically close aquatic, surface-dwelling (i.e. epigean) crustacean Gammarus fossarum acclimated to 12 degrees C, 3 degrees C and -2 degrees C. With decreasing temperature, G. fossarum significantly increased its alanine and glutamine levels, while trehalose body content was found to increase above control levels only at -2 degrees C. N. virei showed moderate increases of alanine and glycine, and no change in trehalose level was observed in this species. By contrast, N. rhenorhodanensis was the only species showing a significant rise in its total FAA pool, mainly explained by alanine, glycine, arginine and glutamine accumulations. This species also gradually increased its trehalose body content with decreasing temperature. Several cold-hardy ectotherms show metabolic responses to cold that are identical to those observed in N. rhenorhodanensis. A previous comparative study showed that the hypogean N. rhenorhodanensis exhibited a survival time (Lt50) at -2 degrees C that was 26.3 times and 2.6 times higher than the hypogean N. virei and the epigean G. fossarum, respectively. Thus, crustacean levels of FAA and trehalose were correlated with their respective cold tolerances. Such differences in metabolic responses to cold in both hypogean organisms were unexpected since they both live in thermally buffered biotopes. Considering the current distribution areas of the two subterranean crustaceans studied, we assume that the cold hardiness found in the hypogean N. rhenorhodanensis could be correlated with its biogeography history during the quaternary glaciations.