Ammonia oxidation (amoA) and nitrogen fixation (nifH) genes along metasandstone and limestone caves of Brazil

Abstract

Many cave-dwelling microorganisms have been studied, but little is known about whether genes related to the nitrogen cycle are present in this environment, particularly in sandstone caves. The profiles of nitrogen fixation (nifH) and archaeal and bacterial ammonia oxidation (amoA) genes were analyzed along a cave of metasandstone and limestone lithologies. Using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis and quantitative PCR to evaluate these genes, we analyzed the variations in the microorganisms and frequency of these genes along the caves. In both caves analyzed for nifH, different community profiles were observed, particularly in entrance samples. Quantitative PCR of the genes showed that samples from the entrance of the caves contained a larger number of copies of amoA. In addition, archaeal amoA was predominant compared to bacterial amoA. The present study demonstrated that the genes for nitrogen fixation and ammonia oxidation are present in both cave lithologies and that their frequency and the organisms present along the caves vary with factors such as the characteristics of the cave itself.     

Evolution of cave living in Hawaiian Schrankia (Lepidoptera: Noctuidae) with description of a remarkable new cave species

Although temperate cave‐adapted fauna may evolve as a result of climatic change, tropical cave dwellers probably colonize caves through adaptive shifts to exploit new resources. The founding populations may have traits that make colonization of underground spaces even more likely. To investigate the process of cave adaptation and the number of times that flightlessness has evolved in a group of reportedly flightless Hawaiian cave moths, we tested the flight ability of 54 Schrankia individuals from seven caves on two islands. Several caves on one island were sampled because separate caves could have been colonized by underground connections after flightlessness had already evolved. A phylogeny based on approximately 1500 bp of mtDNA and nDNA showed that Schrankia howarthi sp. nov. invaded caves on two islands, Maui and Hawaii. Cave‐adapted adults are not consistently flightless but instead are polymorphic for flight ability. Although the new species appears well suited to underground living, some individuals were found living above ground as well. These individuals, which are capable of flight, suggest that this normally cave‐limited species is able to colonize other, geographically separated caves via above‐ground dispersal. This is the first example of an apparently cave‐adapted species that occurs in caves on two separate Hawaiian islands. A revision of the other Hawaiian Schrankia is presented, revealing that Schrankia simplex, Schrankia oxygramma, Schrankia sarothrura, and Schrankia arrhecta are all junior synonyms of Schrankia altivolans. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 114–139.     

Anthropization level of Lascaux Cave microbiome shown by regional‐scale comparisons of pristine and anthropized caves

Limestone areas across the world develop karstic caves, which are populated by a wide range of macro‐ and microorganisms. Many of these caves display Paleolithic art or outstanding speleothems, and in the last century they have been subjected to anthropization due to touristic management and intense human frequentation. Despite their cultural importance and associated conservation issues, the impact of anthropization on cave biodiversity is not known. Here, we show that anthropization is associated with specific cave biota modifications. We compared diversity in four pristine caves, four anthropized show caves, and the iconic Lascaux Cave with even stronger anthropization. The predominant microbial higher taxa were the same in all caves, but the most anthropized cave (Lascaux) was unique as it differed from the eight others by a higher proportion of Bacteroidetes bacteria and the absence of Euryarchaeota and Woesearchaeota archaea. Anthropization resulted in lower diversity and altered community structure for bacteria and archaea on cave walls, especially in Lascaux, but with a more limited effect on microeukaryotes and arthropods. Our findings fill a key gap in our understanding of the response of karstic communities to anthropization, by revealing that tourism‐related anthropization impacts on the prokaryotic microbiome rather than on eukaryotic residents, and that it shapes cave biota irrespective of cave natural features.     

ECOMORPHOLOGICAL CONVERGENCE OF CAVE COMMUNITIES

Extreme selective environments are commonly believed to funnel evolution toward a few predictable outcomes. Caves are well‐known extreme environments with characteristically adapted faunas that are similar in appearance, physiology, and behavior all over the world, even if not closely related. Morphological diversity between closely related cave species has been explained by difference in time since colonization and different ecological influence from the surface. Here, we tested a more classical hypothesis: morphological diversity is niche‐based, and different morphologies reflect properties of microhabitats within caves. We analyzed seven communities with altogether 30 species of the subterranean amphipod (crustacean) genus Niphargus using multivariate morphometrics, multinomial logit models cross‐validation, and phylogenetic reconstruction. Species clustered into four distinct ecomorph classes—small pore, cave stream, cave lake, and lake giants—associated with specific cave microhabitats and of multiple independent phylogenetic origins. Traits commonly regarded as adaptations to caves, such as antenna length, were shown to be related to microhabitat parameters, such as flow velocity. These results demonstrate that under the selection pressure of extreme environment, the ecomorphological structure of communities can converge. Thus, morphological diversity does not result from adaptive response to temporal and ecological gradients, but from fine‐level niche partitioning.     

Big family,warm home,and lots of friends: Pteronotus large colonies affect species richness and occupation inside caves

Roosts are essential for the survival of most animals. Due to homothermic requirements, mammals are particularly dependent on roost quality and availability. Bats select their roosts in a species-specific way, likely related to species´ different physiological and adaptive needs. Unlike species whose individuals roost solitarily, roost selection is critical for bats forming large colonies due to the requirements for maintaining thousands of individuals in a single shelter. This is the case of Pteronotus (Mormoopidae), whose colonies reach hundreds of thousands of bats. Using captures, bioacoustics, and automated censuses, we evaluated how cave size, ceiling characteristics, environmental stability, temperature, and humidity influence the formation of exceptionally large colonies, species richness and composition in caves in north-eastern Brazil. We expected that colonies would be positively related to cave size and stability, internal cave selection would be species-specific, but larger and more environmentally stable caves would have higher richness. Pteronotus colonies were positively related to cave size, stability, and ceiling characteristics, and their presence strongly influenced cave temperature variation. Species richness was positively correlated to a cave stability index. Species other than Pteronotus preferred different climatic and ceiling characteristics. We detected an indirect influence of the large colonies of Pteronotus on the species richness and occupation inside caves. On the other hand, such caves favor species coexistence, as they offer a range of microenvironments, reducing niche overlap in their interior. Pteronotus gymnonotus and Pteronotus personatus are both key- and umbrella-species for cave ecosystems, stressing the need for specific conservation strategies in Brazil. Abstract in Portuguese is available with online material.     

Diversity and ecology of the bryophytes in the cave environment: a study on the volcanic and karstic caves of Sicily

The results of a long investigation (20 years) on bryophyte diversity of 28 Sicilian caves are reported; the study regarded both karstic and volcanic caves, located at altitudes ranging from 15 to 2030 m a.s.l. The bryophyte diversity consists of a total of 136 taxa, 20 liverworts, 3 hornworts and 113 mosses. The ecological analysis was performed using the Ellenberg indicator values for moisture, light and soil reaction to the substrate. On the whole, mesophytic and meso-hygrophytic, sciophytic and photo-sciophytic species strongly prevail independently from the geological nature of the caves; the values of the soil reaction to the substrate well reflect the characteristics of the substrate. The phytogeographical analysis shows the prevalence of the Mediterranean species, followed by the temperate and southern-temperate species. It is remarkable the occurrence of boreo-arctic-montane species, which find refuge for their survival in some high-altitude caves. Some typical species well adapted to the cave environment are reported, including Amphidium mougeotii, Isopterygiopsis pulchella, Thamnobryum alopecurum and Rhynchostegiella tenella, showing a troglophilous character. The occurrence of species of phytogeographical significance, as well as of conservation interest, e.g. Ptychostomum cernuum, Tortula bolanderi, Rhabdoweisia fugax, Grimmia torquata, Brachytheciastrum collinum, very rare in Italy, is emphasized too.     

Energy and speleogenesis: Key determinants of terrestrial species richness in caves

The aim of this study was to unravel the relative role played by speleogenesis (i.e., the process in which a cave is formed), landscape‐scale variables, and geophysical factors in the determination of species richness in caves. Biological inventories from 21 caves located in the southeastern Iberian Peninsula along with partial least square (PLS) regression analysis were used to assess the relative importance of the different explanatory variables. The caves were grouped according to the similarity in their species composition; the effect that spatial distance could have on similarity was also studied using correlation between matrices. The energy and speleogenesis of caves accounted for 44.3% of the variation in species richness. The trophic level of each cave was the most significant factor in PLS regression analysis, and epigenic caves (i.e., those formed by the action of percolating water) had significantly more species than hypogenic ones (i.e., those formed by the action of upward flows in confined aquifers). Dissimilarity among the caves was very high (multiple‐site β_sim = 0.92). Two main groups of caves were revealed through the cluster analysis, one formed by the western caves and the other by the eastern ones. The significant—but low—correlation found between faunistic dissimilarity and geographical distance (r = .16) disappeared once the caves were split into the two groups. The extreme beta‐diversity suggests a very low connection among the caves and/or a very low dispersal capacity of the species. In the region under study, two main factors are intimately related to the richness of terrestrial subterranean species in caves: the amount of organic material (trophic level) and the formation process (genesis). This is the first time that the history of a cave genesis has been quantitatively considered to assess its importance in explaining richness patterns in comparison with other factors more widely recognized.     

Cave invertebrate assemblages differ between native and exotic leaf litter

Abstract Allochtonous leaf litter is an important source of energy and nutrients for invertebrates in cave ecosystems. A change to the quality or quantity of litter entering caves has the potential to disrupt the structure and function of cave communities. In this study, we adopted an experimental approach to examine rates of leaf litter decomposition and the invertebrate assemblages colonizing native and exotic leaf litter in limestone caves in the Jenolan Caves Karst Conservation Reserve, New South Wales, Australia. We deployed traps containing leaf litter from exotic sycamore (Acer pseudoplatanus) and radiata pine (Pinus radiata) trees and native eucalypts (Eucalyptus spp.) in twilight zones (near the cave entrance) and areas deep within the caves for 3 months. Thirty‐two invertebrate morphospecies were recorded from the litter traps, with greater richness and abundance evident in the samples from the twilight zone compared with areas deep within the cave. Sycamore litter had significantly greater richness and abundance of invertebrates compared with eucalypt and pine litter in samples from the twilight zone, but there was no difference in richness or abundance among litter samples placed deep within the cave. Relative rates of decay of the three litters were sycamore > eucalypt > pine. We discuss the potential for the higher decomposition rates and specific leaf area in sycamores to explain their higher invertebrate diversity and abundance. Our findings have important implications for the management of exotic plants and the contribution of their leaf litter to subterranean ecosystems.     

Sampling adequacy in an extreme environment: species richness patterns in Slovenian caves

Caves harbor a rich fauna unique to subterranean environments. Although extensive records of cave animals are available, only a small fraction of known caves in any region have been biologically assessed. We investigated the impact of incomplete sampling using one of the richest, best documented cave faunas in the world – that of the Dinaric karst of Slovenia. We utilized time snapshots (1940, 1970, and 2000) of the caves and cave fauna to analyze stability of hotspots, spatial pattern, and relationship between number of species and number of caves. Using data aggregated into 100km² hexagons, the location of hotspots, black–white joins, Moran's I, and spatial autocorrelation all remained constant, at least from 1970 on. The linear regression coefficient of the relationship between number of caves and number of species declined with time. Most hexagons had been sampled, but there was no indication that any hexagon had been sampled intensively enough for the accumulation curve of number of caves versus number of species within a hexagon to reach an asymptote. This appeared to be the result of a highly skewed distribution of species richness among caves. Number and position of hotspots can be predicted from information on these few high diversity caves.     

Bacterial and Fungal Diversity of Quaternary Cave Sediment Deposits

The bacterial and fungal assemblages of clastic sediments collected from two caves located in north-western Romania were investigated by assessing ITS and 16S rRNA gene diversity. Bacterial members belonging to Chloroflexi, Nitrospirae, Proteobacteria, Firmicutes, Acidobacteria, Gemmatimonadetes, and fungal members of Ascomycota were identified. Except for Bacillus sp., all bacteria were related to uncultured or unknown species and the majority (86%) of the bacterial sequences from one of the caves had no close GenBank relatives. The bacterial sequences obtained clustered with species found in extreme environments. Half of the bacterial operational taxonomic units were clustered with clones isolated from deep subsurface sediments of a radioactively contaminated site in the USA. The present study represents the first attempt to identify microorganisms in Quaternary cave sediments.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Phenotypic plasticity in the greater mouse-eared bat in extremely different roost conditions

Bats use various roost types with a wide spectrum of ecological features. The greater mouse-eared bat Myotis myotis (Borkhausen, 1797), creates nurseries in attics and caves in Central Europe. The stable low temperature and high humidity cave microclimate contrasts that of attics, which may alter species adaptations and life strategies. We analysed population characteristics (composition, body condition, parasite load, and immune response) and genetic relatedness of two proximal M. myotis populations. Age, sexual and parasite species composition were similar between the cave and attic sites. However, a significantly higher parasite load and body condition was detected in the post-partum females and juveniles of the cave colony (n = 263 bats from the cave, 231 from the attic), with the cave colony females having a significantly stronger immune response (n = 2 caves and 2 attics, 20 females per site). There was no evidence for genetic divergence between cave and attic populations (n = 3 caves and 3 attics, 24 females per site), indicating that different population characteristics are not genetically based and that M. myotis is an example of a species with rather unique phenotypic plasticity.     

Local genetic structure in the critically endangered,cave‐associated perennial herb Primulina tabacum (Gesneriaceae)

The local spatial genetic structures of cave‐associated plants are seldom studied. Given that these plants are mainly confined to small areas in and around the entrances of caves, we hypothesized that they might lack genetic structures at local scales. To test this hypothesis, we sampled two large populations (named D and T) of a critically endangered perennial herb, Primulina tabacum, which is endemic to karst caves in southern China. We analysed nine microsatellite loci and sequenced four chloroplast DNA (cpDNA) intergenic spacer regions to study the genetic diversity and structure within and between both populations. Both populations have distinct genetic characteristics. Samples from two subpopulations in population D showed considerable genetic divergence. This is not consistent with the hypothesis that P. tabacum has a weak genetic structure at a local scale. However, 94% of the individuals in population T shared the same multilocus genotype, which indicates little genetic structure within this population. The contributions of seed flow, pollen flow and (sub)population history to the genetic diversity and structure in each and both populations are discussed. Our study is the first to investigate local genetic diversity and structure in a cave‐associated plant, and provides valuable information for the sustainable conservation of such species. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 747–756.     

Cave lithology determining the structure of the invertebrate communities in the Brazilian Atlantic Rain Forest

In Brazil, only limestone caves and a few caves in sandstone, iron ore and granite rocks had their invertebrate communities evaluated. Being such, the present study aimed to promote a comparative analysis of the structure of the invertebrate communities in caves associated to carbonatic, magmatic, siliciclastic and ferruginous rocks of the Brazilian Atlantic forest. Significant differences in the relative richness, abundance and diversity were observed between lithologies. The average relative richness was higher in the ferruginous caves (0.53 spp). The total number of troglomorphic species was significantly different among caves and the highest average richness occurred at ferruginous caves (5.79 spp/cave). Siliciclastic, carbonatic and magmatic caves presented a higher quantitative similarity of the fauna. Ferruginous caves revealed communities with a fauna composition different from the other lithologies. The total richness of invertebrates correlated significantly and positively with the linear development in the siliciclastic caves (R_s = 0.67, P < 0.05), carbonatic (R_s = 0.71, P < 0.05) and ferruginous (R_s = 0.74, P < 0.05). The rock type in which the cave is inserted can determine differences in the richness of invertebrate troglophyles and troglobites. Therefore, on creating value attributes, the size of the caves should always come related to their lithology by the fact that same sized caves associated to different lithologies, possess communities with quite diverse structures.     

The distribution of cave twilight‐zone spiders depends on microclimatic features and trophic supply

Caves are not closed systems. Trophic dynamics in these habitats are driven by resource availability, and species that move between cave and outdoor environments may play a major role in resource availability. Spiders are among the most abundant invertebrates in caves; however, very few studies have tested factors hypothesized to affect the distribution of spiders among caves, and it is not known whether the trophic features of caves play a role in determining the occurrence, abundance, or breeding success of spiders. We assessed the distribution of the cave‐dwelling orb‐weaver spider Meta menardi in Italy, in a Mediterranean and in a Pre‐alpine area during summer and winter. We analyzed the relationships between spider distribution and multiple cave features, describing both the abiotic and the biotic environment. Using visual encounter surveys, the detection probability of this species was high, indicating that this technique provides reliable information on spider distribution. In Mediterranean caves, spider presence was more likely in cold and wet caves with abundant dipterans. In Pre‐alpine caves, spider presence was more likely in deep caves with abundant dipterans. Dipteran abundance was the variable best explaining spider distribution when pooling all sampled caves. This study shows that adults of M. menardi do not occur randomly among caves, but select caves with specific features. Prey availability and abiotic features are major determinants of habitat suitability for cave spiders. The strong relationship between spider distribution and prey availability suggests that the distribution of these spiders might be an indicator of the resources available in the twilight zones of caves.     

中华菊头蝠的栖息生态特征及种群保护

中华菊头蝠(Rhinolophus sinicus)主要分布于我国境内,属于典型的洞栖食虫性蝙蝠,具有重要的研究意义与保护价值。近年,在湘西州境内的16个溶洞中对其栖息生态特征进行了观察,共记录到其个体12942只次,数据分析结果显示：该蝠是该区域溶洞中的居留型优势蝠种,具有集群冬眠的习性。每年的9月中下旬开始逐步聚群冬眠,翌年的3月底开始苏醒,其夏季的繁育场所非常隐蔽,通常位于人无法进入的洞段或人迹罕至的洞穴。总体上,其栖点主要集中于距洞口150—200 m(91.60%)的洞段,离地高度6—10 m(91.75%),多以“双足悬挂”的栖姿将身体悬空倒挂于洞道的顶壁。其体温与栖点温度之间不存在显著性差异(P>0.05),但具有明显的线性正相关关系(R~2=0.8886)。其栖点安全性春夏季高,秋冬季中等偏低。中华菊头蝠作为该区域洞栖性蝙蝠的优势种,在维持洞穴生态系统的稳定中发挥着至关重要的作用,加强对洞穴生态系统中蝙蝠伞护性的研究有助于洞穴生态保护教育及对蝙蝠种群的保护实践。     

The Occurrence of Fungi in the Recently Discovered Jarkowicka Cave in the Karkonosze Mts. (Poland)

Our study is one of the very few cases of speleomycological research in recently discovered caves. The aim of this research was to assess the population size of fungal colonies and their species composition in the Jarkowicka cave, discovered in 2012. The air samples were taken from one location outside the cave and from two locations inside of it. Mycological evaluation of the rocks inside the cave was performed usingswab sampling procedure. In the Jarkowicka cave we found 22 species of fungi, including 13 isolated from air at the entrance and from the walls, and 8 species from air inside the cave. Cladosporium spp. were the fungi most frequently isolated from internal atmosphere of the Jarkowicka cave, and from the external air. On the other hand, the fungi most frequently isolated from the rocks were Mucor spp. We found several species not yet described as cave inhabitants: Hypocrea pachybasioides, Cladosporium uredinicola, and Embellisia abundans. Our study may provide a basis for comparison to other similar studies conducted in frequently visited caves by tourists.     

Identification of roots in lava tube caves using molecular techniques: implications for conservation of cave arthropod faunas

Lava tube cave ecosystems on the volcanic islands of Hawai‘i support communities of rare and highly specialized cave arthropods. In these cave ecosystems, plant roots, both living and dead, provide the main energy source for cave animals. Loss of deep-rooted plants over caves will affect populations of cave-adapted animals living below. Furthermore, the loss of native plant species will likely eliminate host specific cave animals. Thus, identification of plant roots currently found in caves is necessary for the development of effective management actions that encourage the growth of appropriate deep-rooted plant species, thereby protecting the underlying cave ecosystem. We used molecular techniques to identify plant roots found within cave ecosystems on the islands of Maui and Hawai‘i. Sequences of the internal transcribed spacer (ITS) regions and the 5.8S gene of nuclear ribosomal DNA from cave roots were compared to sequences of known plant species either collected on the surface over the footprint of each cave or to sequences accessioned in GenBank. Roots in the cave ecosystem studied on Maui belonged to two alien tree species: Eucalyptus tereticornis and Grevillea robusta. Within the Hawai‘i cave ecosystem, roots of two plant species were identified: the alien tree G. robusta and the native vine Cocculus orbiculatus. The Maui cave ecosystem supports populations of at least 28 species of arthropods, including eight that are blind obligate cave inhabitants. The Hawai‘i cave ecosystem supports 18 arthropod species, of which three are cave-adapted. Creating protected reserves around biologically significant caves, controlling, and preventing the introduction of harmful invasive plant species within the cave footprint, and encouraging the establishment of deep-rooted native plant species is essential for the continued survival of the unique ecosystems found within Hawaiian lava tube cave systems.     

The largest European lion Panthera leo spelaea (Goldfuss 1810) population from the Zoolithen Cave,Germany: specialised cave bear predators of Europe

Remains of 13 individuals with 3/1 male/female ratio of the extinct Upper Pleistocene lion Panthera leo spelaea (Goldfuss, 1810) from the Zoolithen Cave near Burggeilenreuth (Bavaria, Germany) include the holotype skull and all paratype material. The highest mortality rate for the Zoolithen Cave lions is in their reproductive adult ages. Bite marks on lion bones or skulls are results of hyena activities, or rare cannibalism of lions under stress situations. Lions were possibly also killed in battles with cave bears during predation on hibernating bears in winter times. This cave bear hunt specialisation in caves overlaps with the ecological behaviour of cave bear feeding by Ice Age-spotted hyenas. Both largest Ice Age predators, lions and hyenas, had to specialise on feeding herbivorous cave bears in boreal forest mountainous cave rich regions, where the mammoth steppe megafauna prey was absent. This cave bear hunt by felids, and scavenging by hyenas and other large carnivores such as leopards and wolves explains why cave bears hibernated deep in to the European caves, for protection reasons against predators. Within such lion–cave bear and even lion–hyena conflicts in the caves lions must have been killed sometimes, explaining mainly the skeleton occurrences in different European caves.     

Cryogenic Minerals in Hawaiian Lava Tubes: A Geochemical and Microbiological Exploration

The Mauna Loa volcano, on the Island of Hawaii, has numerous young lava tubes. Among them, two at high altitudes are known to contain ice year-round: Mauna Loa Icecave (MLIC) and the Arsia Cave. These unusual caves harbor cold, humid, dark, and biologically restricted environments. Secondary minerals and ice were sampled from both caves to explore their geochemical and microbiological characteristics. The minerals sampled from the deep parts of the caves, where near freezing temperatures prevail, are all multi-phase and consist mainly of secondary amorphous silica SiO₂, cryptocrystalline calcite CaCO₃, and gypsum CaSO₄·2H₂O. Based on carbon and oxygen stable isotope ratios, all sampled calcite is cryogenic. The isotopic composition of falls on the global meteoric line, indicating that little evaporation has occurred. The microbial diversity of a silica and calcite deposit in the MLIC and from ice pond water in the Arsia Cave was explored by analysis of ～50,000 small subunit ribosomal RNA gene fragments via amplicon sequencing. Analyses reveal that the Hawaiian ice caves harbor unique microbial diversity distinct from other environments, including cave environments, in Hawaii and worldwide. Actinobacteria and Proteobacteria were the most abundant microbial phyla detected, which is largely consistent with studies of other oligotrophic cave environments. The cold, isolated, oligotrophic basaltic lava cave environment in Hawaii provides a unique opportunity to understand microbial biogeography not only on Earth but also on other planets.     

Ecological characteristics of a cave‐dwelling moth species,Triphosa dubitata (Lepidoptera: Geometridae), in Baram and Ssang caves in Gangwon Province,Korea

This study was performed to clarify the ecological characteristics of a cave‐dwelling moth species, Triphosa dubitata Linnaeus (Lepidoptera: Geometridae), at Baram cave and at Ssang cave within Pyeongchang‐gun of Gangwon province in south Korea. We investigated T. dubitata population for 3 days from March 2013 to October 2013 (except September) and in January 2014. We used a section investigation method in the caves every month to determine their ecological characteristic. A nocturnal investigation using a high intensity discharge lamp adjacent to the entrance of the cave was conducted in parallel with a diurnal investigation using butterfly traps to investigate the movement of T. dubitata. In order to investigate the range of habitat temperature of moths, we used a dual laser targeting infrared thermometer to measure the cave temperatures. We caught individuals of T. dubitata L.: 8,574 individuals in Baram cave and 828 individuals in Ssang cave. As 9,095 individuals (97 %) appeared at a temperature of ?3°C ~ 8°C, it was verified that the adults were living at specific temperatures. In addition, T. dubitata adults were not active outside the caves; they entered the cave as soon as they developed from pupa into adults. These results showed that T. dubitata depended on food plants outside of the cave during the egg–larva–pupa stages, and they used the cave as a habitat for long‐term survival when they develop into adults.