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Many ant species are known to exhibit foraging tool use, during which ants place various debris items (e.g., pieces of soil, leaves, pine needles, etc.) into liquid food, and then they carry the food-soaked tools back to the nest. In the present study, we compared the tool-using behavior in captive colonies of two closely related myrmicine ants with different feeding preferences: Aphaenogaster subterranea, an omnivorous species, and Messor structor, a mainly granivorous seed-harvester species. We supplied foraging ants with honey-water baits and six types of objects they could use as tools: sand grains, small soil grains, large soil grains, pine needles, leaves, and sponges. We found that the workers of A. subterranea both dropped more tools into honey-water baits and retrieved more of these tools than the workers of M. structor. While A. subterranea preferred smaller tools over larger ones, tool preferences for M. structor did not differ significantly from random. In addition, tool dropping was significantly faster in A. subterranea, and both the dropping and retrieving of tools began significantly earlier than in M. structor. For Aphaenogaster species that regularly utilize and compete for liquid food sources, the ability to efficiently transport liquid food via tools may be more important than it is for seed-harvester ants. Dropping tools into liquids, however, may still be useful for seed-harvester species as a means to supplement diet with liquid food during periods of seed shortage and also to serve as a means of getting rid of unwanted liquids close to the nest.

Climate change is one of the major threats to biodiversity, but its impact varies among the species. Bark beetles (Ips spp.), as well as other wood-boring pests of European forests, show escalating numbers in response to the changes driven by climate change and seriously affect the survival of the forests through the massive killing of trees. Many methods were developed to control these wood-boring beetles, however, their implementation can be detrimental for other forest specialists. Ants are widely used for biological pest-control, so in our study, we aimed to test the effect of Formica polyctena on the control of the wood-boring beetles. The results show that the proportion of infested trees is significantly reduced by the increase of the number of F. polyctena nests, with a strong effect on those infested by Ips species. We also show that the boring beetle community is shaped by different biotic and abiotic factors, including the presence of F. polyctena nests. However, the boring beetle infestation was not related to the latitude, altitude and age of the forests. Based on our results, we assert the effectiveness of the red wood ants as biological pest control and the importance of their conservation to keep the health of the forests.

In the presence of territorial dominant ant species, lower-ranked ants change their foraging strategy by shifting their activity in time and space and by switching to suboptimal food sources. Altho...

Background Interspecific interactions within ecological networks can influence animal fitness and behaviour, including nest-site selection of birds and ants. Previous studies revealed that nesting birds and ants may benefit from cohabitation, with interspecific attraction through their nest-site choice, but mutual interactions have not yet been tested. We explored a previously undescribed ecological link between ground-nesting birds and ants raising their own broods (larvae and pupae) within the birds’ nests in a temperate primeval forest of lowland Europe. We tested whether the occurrence of ant broods within bird nests resulted from a mutual or one-sided interspecific attraction that operated through nest-site choice and was modified by weather conditions. Results We found a non-random occupation of bird nests by ants raising their own broods within them, which indicated interspecific attraction driven solely by the ants. The birds’ preference to nest near tussocks of vegetation showed little overlap with the most frequent placement of ant colonies among fallen deciduous tree-leaves, dead wood and moss. Additionally, birds did not appear to select forest localities with high densities of ant colonies. The occurrence of ant broods within bird nests was also unrelated to bird nest placement near to specific habitat features. The attractiveness of bird nests to ants appeared to increase with the thermal activity of the birds warming their nests, and also during cool and wet weather when the occurrence of ant broods within bird nests was most frequent. Ants often remained in the nests after the birds had vacated them, with only a slight reduction in the probability of ant brood occurrence over time. Conclusions The natural patterns of bird nest colonisation by ants support the hypothesis of ants’ attraction to warm nests of birds to raise their broods under advantageous thermal conditions. Similar relationships may occur between other warm-blooded, nest-building vertebrates and nest-dwelling invertebrates, which depend on ambient temperatures. The findings advance our understanding of these poorly recognised interspecific interactions, and can inform future studies of ecological networks.

Abstract Climate change and the subsequent increase of global temperature are the most current and important threats to biodiversity. Despite the importance of temperature, our knowledge about the level of behavioural and physiological adaptations of ant species from temperate regions to cope with high temperatures is limited compared to the broad knowledge of typical thermal specialists from warmer regions. In the current study, we investigated the temperature-related foraging risk of xerothermic ant species from the temperate climate in Europe, Formica cinerea. Our aims were to check how an increase in external soil temperature affects the foraging activity of workers and how the temperature during development and worker age affects foraging activity in high temperatures. Based on our results, we can draw the following conclusions: (1) the majority of workers utilize a risk-aversive strategy in relation to foraging in high surface temperatures; (2) pupal development temperature affects the risk taken by adult workers: workers that developed in a higher temperature forage more often but for shorter intervals compared to workers that developed in a lower temperature; (3) age is an important factor in temperature-related foraging activity, as with increasing age, workers forage significantly longer at the highest temperatures. Our study is one of the first to assess the potential factors that can affect the foraging risk of ants from a temperate climate in high ambient temperatures. Significance statement Our study is the first direct test of workers' age and the development temperature of pupae on the thermal-related foraging strategy of adult F. cinerea workers. It shows that worker age and the development temperature of pupae interact to promote tolerance of thermal stress. We found that with increasing age, workers are prone to forage significantly longer at the highest and riskiest temperatures. Workers that developed in the high temperature (28°C) foraged more often but for shorter intervals compared to workers that developed in the lower temperature (20°C). Interestingly, the factor of age is more significant for ants that developed in the higher temperature of 28°C; the foraging time of these ants significantly increased with their age.

Climate change is one of the major threats to biodiversity, but its impact varies among the species. Bark beetles (Ips spp.), as well as other wood-boring pests of European forests, show escalating numbers in response to the changes driven by climate change and seriously affect the survival of the forests through the massive killing of trees. Many methods were developed to control these wood-boring beetles, however, their implementation can be detrimental for other forest specialists. Ants are widely used for biological pest-control, so in our study, we aimed to test the effect of F. polyctena on the control of the wood-boring beetles. The results show that the proportion of infested trees is significantly reduced by the increase of the number of F. polyctena nests, with a strong effect on Ips species. We also show that the boring beetle community is shaped by different biotic and abiotic factors, including the presence of F. polyctena nests. However, the boring beetle infestation was not related to the latitude, altitude and age of the forests. Based on our results, we assert the effectiveness of the red wood ants as biological pest control and the importance of their conservation to keep the health of the forests.

Nesting birds can act as thermal ecosystem engineers by providing warm habitats that may attract arthropods to colonise the nest structure. This cohabitation of birds and nest-dwelling invertebrates may foster symbiotic relationships between them, but evidence is lacking. We investigated whether ants are attracted to bird nests by the heat generated by the hosts, and/or the nests’ structural insulation properties, to raise their broods (larvae and/or pupae) in advantageous thermal conditions. We found that the endothermic activity of birds within their nests created ‘heat islands’, with thermal conditions potentially promoting the survival and development of ant larvae in cool environments. We experimentally confirmed that the presence of heat within bird nests, and not the structure itself, attracted the ants to colonise the nests. As ants might benefit from exploiting warm bird nests, this may be a previously overlooked commensal, mutualistic or parasitic relationship which may be ecologically significant and globally widespread among various nesting birds and reproducing ants. Similar interspecific interactions may exist with other arthropods that reproduce in avian and mammalian nests. Further research is needed to reveal the nature of these relationships between such taxa, and to understand the role of warm-blooded animals as thermal ecosystem engineers.

Cannibalistic necrophagy is rarely observed in social hymenopterans, although a lack of food could easily favour such behaviour. One of the main supposed reasons for the rarity of necrophagy is that eating of nestmate corpses carries the risk of rapid spread of pathogens or parasites. Here we present an experimental laboratory study on behaviour indicating consumption of nestmate corpses in the ant Formica polyctena. We examined whether starvation and the fungal infection level of the corpses affects the occurrence of cannibalistic necrophagy. Our results showed that the ants distinguished between corpses of different types and with different levels of infection risk, adjusting their behaviour accordingly. The frequency of behaviours indicating cannibalistic necrophagy increased during starvation, although these behaviours seem to be fairly common in F. polyctena even in the presence of other food sources. The occurrence and significance of cannibalistic necrophagy deserve further research because, in addition to providing additional food, it may be part of the hygienic behaviour repertoire. The ability to detect infections and handle pathogens are important behavioural adaptations for social insects, crucial for the fitness of both individual workers and the entire colony.

Simple Summary The red wood ants are considered to be one of the main ecosystem engineers and keystone species of the habitats in which they exist. Most of the species from this species group inhabit coniferous forests, which, due to the consequences of anthropogenic climate change, are continuously cut down. Therefore, the main habitat of these important species is rapidly disappearing. We investigated the consequences of the absence of conifers (clear-cut area and deciduous forest) on one of the members of this species group, namely Formica polyctena. We have found that compared with the mixed-coniferous forest stand, the absence of coniferous species resulted in significant changes in the colony and nest structure of F. polyctena. In addition, the colony size was also smaller in these sites. These changes suggest that although F. polyctena is able to survive in suboptimal habitats, still their population decline is expected and urges conservation managers to apply necessary action plans for their protection. Abstract The consequences of anthropogenic climate change are one of the major concerns of conservation biology. A cascade of negative effects is expected to affect various ecosystems, one of which is Central European coniferous forests and their unique biota. These coniferous forests are the primary habitat of many forest specialist species such as red wood ants. Climate change-induced rising of temperature allows trees to skip winter hibernation, making them more vulnerable to storms that cause wind felling, and in turn, promotes bark beetle infestations that results in unscheduled clear-cuttings. Red wood ants can also be exposed to such habitat changes. We investigated the effects of bark beetle-induced clear-cutting and the absence of coniferous trees on colonies of Formica polyctena, including a mixed-coniferous forest as a reference. Our aim was to investigate how these habitat features affect the nest characteristics and nesting habits of F. polyctena. Our results indicate that, in the absence of conifers, F. polyctena tend to use different alternatives for nest material, colony structure, and food sources. However, the vitality of F. polyctena colonies significantly decreased (smaller nest mound volumes). Our study highlights the ecological flexibility of this forest specialist and its potential to survive under extreme conditions.

Red wood ants are keystone species of forest ecosystems in Europe. Environmental factors and habitat characteristics affect the size of their nest mounds, an important trait being in concordance with a colony&rsquo, s well-being and impact on its surroundings. In this study, we investigated the effect of large-scale (latitude and altitude) and small-scale environmental factors (e.g., characteristics of the forest) on the size of nest mounds of Formica polyctena in Central Europe. We predicted that the change in nest size is in accordance with Bergmann&rsquo, s rule that states that the body size of endotherm animals increases with the higher latitude and/or altitude. We found that the size of nests increased along the latitudinal gradient in accordance with Bergmann&rsquo, s rule. The irradiation was the most important factor responsible for the changes in nest size, but temperature and local factors, like the perimeter of the trees and their distance from the nest, were also involved. Considering our results, we can better understand the long-term effects and consequences of the fast-changing environmental factors on this ecologically important group. This knowledge can contribute to the planning of forest management tactics in concordance with the assurance of the long-term survival of red wood ants.

Territorial strategy in animals is characterized by the monopoly of resources inside a protected area, the territory. The presence of territorial species considerably alters the behavior of co-occurring submissives, as it is known in several submissive ant species living on the territories of red wood ants in temperate regions. On the other hand, as a rule, territorial species cannot share the same territory and usually exclude each other. However, this ‘rule’ is inferred from the almost complete lack of data on the coexistence of rival territorials, and not from observations regarding the effective exclusion or behavioral inhibition of one territorial species by other. In the frame of this study, we investigated the foraging strategy of the territorial red wood ant Formica pratensis that occurred inside a large polydomous system of another territorial ant species, F. exsecta. Formica pratensis colonies outside the supercolony served as control. Within the F. exsecta supercolony, F. pratensis showed the characteristics of a subordinate species characterized by low discovery and exploitation success of artificial food sources. On the contrary, control colonies outside the polydomous system clearly behaved like typical territorials as they successfully monopolized the majority of the baits. In addition, submissive species were more successful around the F. pratensis nests within the supercolony, than outside of it. As suggested by our results, territorial species could co-occur with other territorials given certain plasticity in their behavior and small colony size. Nevertheless, exclusion also happens as proven by our field observations.

Ant richness and abundance are negatively affected by the invasion of alien goldenrods (Solidago sp.). However, little is known about the mechanisms standing behind the impact of the invaders on ant life history, such as colony investments in growth and reproduction. We examined this problem of the investments of Myrmica rubra ant colonies living in different grasslands invaded and non-invaded by goldenrods. Altogether, 47 colonies were analysed; and for each colony, we calculated the number of queens, workers and the production of young workers, gynes, and males. We found that colonies from invaded meadows are smaller in size, but have a similar number of adult queens compared to colonies from non-invaded sites. We also found different brood investments among colonies from invaded and non-invaded meadows-colonies from non-invaded meadows produce more young workers and invest more in growth, whereas colonies from invaded meadows invest more in reproduction through higher gyne production. Male production was at a similar level in colonies from both habitat types. The observed patterns may be explained by the effect of various environmental factors occurring in both grassland types, such as stress in changed habitats, higher competition among gynes in non-invaded grasslands, or finally, by the adaptive colony-level response of ants to stress. The higher production of gynes observed in the invaded grasslands may support dispersal and enhance the probability of establishing a colony in a more favourable location.

This work was supported by European Commission (FP7-MC-ERG-2009-256524 to PdE). Tool use by non-human animals has received much research attention in the last couple of decades. Nonetheless, research has focused mostly on vertebrates, particularly primates and corvids, even though tool use has also been documented in insects. One of the best documented examples involves ants using debris (e.g., sand grains, mud, leaf fragments) to collect and transport liquid food to their nest. However, little is known about the factors that determine the selection of materials to be used as tool. We investigated tool selection in two species of Aphaenogaster ants by giving them the choice between different kinds of potential tools (natural and artificial objects). Ant workers showed a clear preference for certain materials to be used as tool objects. Tool selection was also shaped by familiarity with the material as ants developed a preference for artificial tools with a good soaking capacity that cannot be found in their natural environment. Our results indicate that ants of this genus evolved unique foraging strategies and show plasticity in their behaviour. Postprint

Successful evacuation of a peculiar ‘colony’ of the wood ant Formica polyctena Först., for years trapped within an old bunker previously used for storing nuclear weapons (see Czechowski et al. 2016), is reported. Using an experimentally installed boardwalk, the imprisoned ants managed to get through the ventilation pipe to their maternal nest on the top of the bunker. In our previous report, we left open the question of how the ‘colony’ could survive seemingly without food. Here we show that the ‘colony’ in the bunker survived and grew thanks to an influx of workers from the source nest above the bunker and mass consumption of corpses of the imprisoned nestmates.

Several factors can influence individual and group behavioral variation that can have important fitness consequences. In this study, we tested how two habitat types (seminatural meadows and meadows invaded by Solidago plants) and factors like colony and worker size and nest density influence behavioral (activity, meanderness, exploration, aggression, and nest displacement) variation on different levels of the social organization of Myrmica rubra ants and how these might affect the colony productivity. We assumed that the factors within the two habitat types exert different selective pressures on individual and colony behavioral variation that affects colony productivity. Our results showed individual-/colony-specific expression of both mean and residual behavioral variation of the studied behavioral traits. Although habitat type did not have any direct effect, habitat-dependent factors, like colony size and nest density influenced the individual mean and residual variation of several traits. We also found personality at the individual-level and at the colony level. Exploration positively influenced the total- and worker production in both habitats. Worker aggression influenced all the productivity parameters in seminatural meadows, whereas activity had a positive effect on the worker and total production in invaded meadows. Our results suggest that habitat type, through its environmental characteristics, can affect different behavioral traits both at the individual and colony level and that those with the strongest effect on colony productivity primarily shape the personality of individuals. Our results highlight the need for complex environmental manipulations to fully understand the effects shaping behavior and reproduction in colony-living species., The behavior of your family can depend on the place you live and may affect the amount of resources you gain. We studied this relationship in ants living in two environmentally different meadows. Ants living in big families and in more crowded places have to be aggressive to produce more brood. Ants living in less crowded but food-limited places have to move more to reproduce successfully. Exploration skills are beneficial in both habitats.

The first observation of alate gyne of Dolichoderus quadripunctatus (L.) visiting aphids is described. A gyne walked along a foraging trail to the aphid Panaphis juglandis Goeze colony where it imbibed honeydew excreted on the leaf by the aphids, after which it returned to the trail. This recurred during two more days, always a single alate gyne at a time; hence the total number of gynes, one or more, remained open. The phenomenon, hitherto practically unknown in ants, is presented against the background of the biology of the species and discussed in the context of specific environmental circumstances and the colony dynamics.

The hierarchical organization of multi-species ant communities can be determined by the colony size and worker density-dependent differences in behaviour, recruitment efficiency, and aggressiveness between colonies of different species. In this study, we compared the competitive effects of two territorial wood ant species, Formica rufa and F. polyctena, on the ant species subordinated to them (encounterers and submissives) that nest close-by their mounds. The nests of subordinates were mapped in 10 m2 plots around the mounds of each of the two wood ant species, situated at a distance of 10, 20 and 30 m in four directions. The most abundant subordinate species were two Myrmica species: M. rubra and M. ruginodis. Interestingly, the nests of subordinates had an even diversity, density and distribution along with the distance from the wood ant mounds. The wood ant presence had a species-specific negative effect on some of the subordinate species, which can be in relation with the smaller mound sizes in F. rufa compared to F. polyctena. Based on our results it seems that subordinate species, due to their adaptability, might tolerate the circumstances close-by the wood ant mounds by changes in their strategies, foraging behaviour, and switching to the use of alternative food sources, like corpses of wood ants present in high abundance. Although these might not allow reaching normal colony size and reproduction, they may allow the colonies to survive while maintaining relatively large nest densities.

Ant corpses, besides representing threat of infection by pathogens and parasites, can also be used during interspecific conflicts to inhibit the activity of the attacked colony, or they can be consumed as food. In the view of the former, the signal properties of corpses can be manifold. Besides discriminating nestmates and foes, the corpses of different ant species may act as cues for foragers, signaling the presence of other rival species, and triggering appropriate responses (e.g., alarm, retreat or foraging). In our study, we examined the responses of the facultative slave-maker Formica sanguinea and those of the obligate Polyergus rufescens towards corpses of nestmates, non-nestmate conspecifics, heterospecific slave-makers and their slaves, and corpses of non-enslaved host species under laboratory conditions. Both dulotic species responded differently to corpses of different origin. In F. sanguinea, the most intensive response was elicited by the corpses of P. rufescens and its slave, but also the corpses of non-nestmate conspecifics and their slaves elicited many adverse responses. In P. rufescens, the corpses of non-nestmate conspecifics and their slaves elicited the most adverse response. Both dulotic species distinguished corpses of their slaves from corpses of non-enslaved hosts. Based on our results, ant corpses are not meaningless objects scattered in the field, but cues carrying information that trigger different behavioral responses, and in F. sanguinea they can even represent an important food source.

Competition is one of the basic mechanisms shaping ant assemblages. Dominant territorial species are known to restrictively influence the traits of subordinates in various ways. However, there could be differences in the effects of dominants due to differences in their colony structure, lifestyle and also behaviour. We tested this hypothesis in natural circumstances in an area where a Formica exsecta Nyl. supercolony neighbours a strong population of the European slave-maker F. sanguinea Latr. For the purpose of our study three different sites were selected: one dominated by F. exsecta, a neighbouring site dominated by F. sanguinea, and a third site where both species co-occurred. We analyzed the structure of subordinate ant communities based on nest counts, and by recording the activity of ants at baits. Based on our findings the structure of ant communities differed significantly among the three sites. The F. exsecta site was characterized by the lowest nest density of subordinates, especially in the case of aggressive species as Lasius platythorax Seifert. At baits even the simple presence of the dominant limited the abundance and occurrence of subordinates. In contrast, no such negative effect could be revealed at the F. sanguinea site. The community of the mixed site showed intermediate features in many respects. Generally, the supercolonial F. exsecta had a bigger influence on the ant community than the slave-making F. sanguinea. On the other hand, our findings revealed a slight protective role of the territorial F. exsecta for potential slave species to F. sanguinea. The current study underlines the importance of differences between dominant ant species in shaping differentially ant communities even within the same restricted area.

Intensive management causes significant changes to the habitat structure of forest stands and threatens forest specialist insect species. To assess and counteract the effect of periodic intensive forestry interventions, such as clear-cutting and reforestation, it is important to adequately quantify the recovery rate and composition of the native biota. We aimed to characterize the above parameters for ant assemblages in forests with different structure and age. Epigeic ant assemblages were studied using pitfall traps in young (6–10 years old), middle-aged (23–26 years old) and mature (35–37 years old) poplar forest plantations in the Kiskunsag region of Hungary. Species richness of ant assemblages and the abundance of nonforest ants did not differ among the age classes. The abundance of forest specialist ants was, however, higher in middle-aged and mature stands compared with young plantations. The assemblage composition of ants significantly correlated with habitat structure and forest age. We conclude that nonforest ant species can persist throughout the entire forestry cycle in poplar plantations with moderate tree density. The present study suggests that forest age and habitat structure together shape the ant assemblages of lowland poplar plantations.

The management of natural and seminatural systems often leads to disturbance associated with the appearance of non-native species. The spread of these species is increasing due to global environmental changes combined with local management interventions. These non-native species may establish self-sustaining populations influencing ecosystem functions, including the habitat use of native species. Here we explore the response of diplopods, spider and ant assemblages and the activity-density of individual species to the establishment of the non-native plant species, Asclepias syriaca in a disturbed poplar forest in Hungary. The relationship between the species richness of spiders and ants and the structural features of A. syriaca was weak. We found a significant relationship between the structural features of A. syriaca stands and the density and activity of the diplopod Megaphyllum unilineatum. We explain this relationship by the modified microclimate and litter quality of the habitats invaded by A. syriaca. The species composition of ant and spider assemblages was sensitive to A. syriaca. Asclepias syriaca had a negative local effect on the abundance of two spider species which were common in the studied forest. However, A. syriaca positively influenced the abundance of two ant species, most probably via indirect trophic relationships, as they feed on aphids living on A. syriaca. Our study shows that invasive plants can have mixed effects on local invertebrate assemblages. It is therefore crucial to understand how native assemblages respond to these changes in order to better manage these novel ecosystems and maximize their biodiversity benefits.

Forest management has highly modified the structure of the European forests. Harvesting and post-harvest regeneration leads to a simplified forest structure. Our main objective was to detect the effects of habitat structure and forest age on the ground-dwelling spider diversity and assemblage composition of poplar forests at the Hungarian Great Plain. Our results demonstrate that the rarefaction diversity and the number of forest specialists closely correlated with the structural parameters of the forest floor, however, the age and canopy closure did not influence these parameters. According to redundancy analysis, the composition of spider assemblages was determined solely by habitat structure, with habitat structure having a major effect on the species composition and diversity of spider assemblages. A direct effect of forest age on the spider assemblages was not detected, due to the presence of different habitat types in the surrounding landscape, which may serve as suitable habitats for source-populations of spiders with different habitat requirements. Our results highlight the importance structural complexity of forests for maintaining forest spider diversity and preserving the regional species pool of spiders.

Group living involves major health risks, since it facilitates the spread of pathogens and parasites among members. To counteract this negative effect, social insects, such as ants, adopt several social defensive strategies, one of the most widespread being the collective disposal of corpses often outside the nest, which represent potential sources of infection. Corpses might even be used during interspecific conflicts to inhibit the activity of the attacked nest or they could be consumed as food. As intra- and interspecific relationships are manifold and often vary in terms of fitness consequences for the interacting species, it can be hypothesized that responses to corpses of different origin can vary. Scattered or piled up corpses of different ant species could act as cues for foragers, signalling the presence of other species, and triggering appropriate responses: alarm, retreat or foraging. We examined the reactions of the ant Formica cinerea under natural conditions to corpses of co-occurring Formica species. Formica cinerea responded differentially to corpses of different origin. Those of the territorial Formica polyctena and the slave-maker Formica sanguinea elicited more aggression and their corpses were removed sooner than other dead bodies. The majority of corpses were carried inside the nest, with the exception of Formica fusca corpses, which may signify the lack of specific interest in this species. The removal of nestmate and foreign conspecific corpses could be explained through social prophylaxis, and that of superior competitors additionally by conflict avoidance and/or by food supplementation. Based on our results, for ants corpses are not meaningless objects scattered around ant nests, but cues that carry information that trigger different behavioural reactions.

Background and aims: Dolines are small- to large-sized bowl-shaped depressions of karst surfaces. They may constitute important microrefugia, as thermal inversion often maintains cooler conditions within them. This study aimed to identify the effects of large- (macroclimate) and small-scale (slope aspect and vegetation type) environmental factors on cool-adapted plants in karst dolines of East-Central Europe. We also evaluated the potential of these dolines to be microrefugia that mitigate the effects of climate change on cool-adapted plants in both forest and grassland ecosystems. • Methods: We compared surveys of plant species composition that were made between 2007 and 2015 in 21 dolines distributed across four mountain ranges (sites) in Hungary and Romania. We examined the effects of environmental factors on the distribution and number of cool-adapted plants on three scales: (1) regional (all sites); (2) within sites and; (3) within dolines. Generalized linear models and non-parametric tests were used for the analyses. • Key Results: Macroclimate, vegetation type and aspect were all significant predictors of the diversity of cool-adapted plants. More cool-adapted plants were recorded in the coolest site, with only few found in the warmest site. At the warmest site, the distribution of cool-adapted plants was restricted to the deepest parts of dolines. Within sites of intermediate temperature and humidity, the effect of vegetation type and aspect on the diversity of cool-adapted plants was often significant, with more taxa being found in grasslands (versus forests) and on north-facing slopes (versus south-facing slopes). • Conclusions: There is large variation in the number and spatial distribution of cool-adapted plants in karst dolines, which is related to large- and small-scale environmental factors. Both macro- and microrefugia are therefore likely to play important roles in facilitating the persistence of cool-adapted plants under global warming. Refereed/Peer-reviewed