Cave Invertebrates Research Articles

Caves’ environmental stability shaping subterranean biodiversity in the neotropics

Caves, once thought to be isolated ecosystems", are now understood to have intricate connections with surface environments, particularly evident at their entrances. These connections can significantly affect the microclimate within caves, leading to varying degrees of environmental stability. Our research explores the impact of microclimate conditions, specifically related to cave environmental stability, on biodiversity changes. We surveyed subterranean invertebrates during two different seasonal periods in 17 limestone caves in three karst regions in southeastern Brazil. Our analysis aimed to understand how environmental stability influences the overall richness of cave invertebrates and troglobitic species richness (restricted to subterranean habitats). We hypothesized that more stable caves would experience less fluctuation in relative species richness and lower turnover between seasons. Additionally, we anticipated that caves with greater environmental stability would harbor a higher richness of cave-restricted species in the function of their length. Our findings support these hypotheses, revealing a significant positive correlation between cave environmental stability and species richness. Caves with higher environmental stability demonstrated lower species turnover rates between seasons, indicating enhanced community stability. While environmental stability significantly affected species richness, its influence on cave-restricted species was comparatively less pronounced. This suggests a complex interplay of factors shaping the unique fauna of caves. Nonetheless, the implications of climate change underscore the importance of preserving the environmental stability of these ecosystems. Using environmental stability as a guide can help protect cave biodiversity from the adverse effects of climate change, contributing to broader conservation efforts for these unique habitats.

The gut microbiome mediates adaptation to scarce food in Coleoptera.

Beetles are ubiquitous cave invertebrates worldwide that adapted to scarce subterranean resources when they colonized caves. Here, we investigated the potential role of gut microbiota in the adaptation of beetles to caves from different climatic regions of the Carpathians. The beetles' microbiota was host-specific, reflecting phylogenetic and nutritional adaptation. The microbial community structure further resolved conspecific beetles by caves suggesting microbiota-host coevolution and influences by local environmental factors. The detritivore species hosted a variety of bacteria known to decompose and ferment organic matter, suggesting turnover and host cooperative digestion of the sedimentary microbiota and allochthonous-derived nutrients. The cave Carabidae, with strong mandibula, adapted to predation and scavenging of animal and plant remains, had distinct microbiota dominated by symbiotic lineages Spiroplasma or Wolbachia. All beetles had relatively high levels of fermentative Carnobacterium and Vagococcus involved in lipid accumulation and a reduction of metabolic activity, and both features characterize adaptation to caves.

Worldwide distribution of cave-dwelling Chelodesmidae (Diplopoda, Polydesmida)

Chelodesmidae is one of the most species rich families within the Myriapoda. However, little is known regarding their association with caves. We provide a list of all Chelodesmidae taxa reported from caves, map their worldwide distribution, and discuss the troglomorphic features of the group. A total of 25 species and subspecies from 20 genera and 2 subfamilies have been recorded from 59 caves and cave systems in 11 countries. These numbers represent a surprisingly modest proportion (~3%) of the approximately 800 described species in the family. Records of cave-dwelling chelodesmids appear to be geographically biased with most taxa reported from the Neotropics, likely due to the greater diversity of the group in the region. The lack of published records from the Afrotropics can undoubtedly be attributed to low sampling efforts. In addition, many studies focusing on cave invertebrates, especially in the tropics, often fail to identify chelodesmid taxa to the species level, thus precluding their inclusion on checklists. The majority of the Chelodesmidae reported from caves do not display obvious adaptations to a subterranean lifestyle and are not troglomorphic. The five troglobitic taxa indicated in this study may represent relictual lineages suggesting that few, if any, radiations of chelodesmids within caves have occurred in the past. Increased efforts are needed to bridge the gap between our current understanding and the true diversity of the group in these fragile and threatened habitats, which is necessary to allow policy makers to prioritize appropriate and effective conservation efforts, including the protection of environments severely impacted by human activities.

Controversies and hidden risks in biodiversity offsets in critically threatened Canga (ironstone) ecosystems in Brazil

AbstractCanga, or ironstone, ecosystems are hotspots of old-growth plant diversity and highly specialized cave invertebrates. These ancient metalliferous habitats are amongst the most threatened ecosystems because of the destruction caused by large-scale iron ore mining. International debate on biodiversity offsets is increasing because these mechanisms are seen as tools for potentially balancing economic development with conservation biodiversity. Leading mining companies worldwide, including some of the largest iron ore producers in Brazil, are signatories to offset principles and best practices that aim to achieve no net loss of habitats, species or ecosystem functions. We aimed to analyse whether Brazilian legal requirements for biodiversity offsets result in the achievement of conservation outcomes or in elevated threat of extinction in canga ecosystems. We evaluated technical reports that support decision-making related to environmental licensing for iron ore mining and specific offset proposals linked to the Atlantic Forest Act. We found a relevant net loss in canga ecosystems and observed shortcomings related to the equivalency and transparency of offset principles. These deficiencies are mainly related to lax norms and regulations and the absence of an integrated database for accessing information on environmental licensing processes. We argue that both policy flaws and low engagement by the Brazilian mining industry in implementing offset principles have increased the threat of extinction in canga ecosystems.

From light to darkness: the duality of influence of habitat heterogeneity on Neotropical terrestrial cave invertebrate communities

ABSTRACT The theory of habitat heterogeneity, which treats one of the global drivers of biodiversity, is valid for several taxonomic groups and distinct ecosystems, including subterranean environments. However, knowledge about the factors that influence the structure of subterranean communities remains limited, especially in Neotropical regions. We sought to understand the main drivers of invertebrate species richness and composition. For that, we compared the substrate elements of twilight and deep-cave zones and their associated invertebrate communities. Variations in habitat heterogeneity were significantly related to richness in the deep cave zone, but had no effect on the fauna associated with the twilight zone. The local heterogeneity enhanced the richness in the deep cave zone due to the increase in the number of available habitats and the effect on decreasing niche overlap. For communities’ composition, the geographic distribution of caves explained the similarity between the twilight zone of different caves. Therefore, nearby caves have a more similar fauna in the entrance regions but conversely, exhibit a higher dissimilarity between communities associated with the deep-cave zone. This study highlights the influence of local habitat heterogeneity on the invertebrate communities associated with different zones of the cave.

The pleiotropic effects of melanin loss in cave snails Physella sp.

A traditional explanation for the loss of pigmentation in cave dwellers is the absence of negative selection acting on surface species to remove all albinos from the population. Recently, however, evidence has been accumulating that albinism in the cavefish Astyanax mexicanus has several beneficial pleiotropic effects. Albino cavefish have higher catecholamine levels due to a loss-of-function mutation in the oca2 gene, higher resistance to anesthesia, and sleep less than their pigmented, surface-dwelling conspecifics. All of these traits are beneficial to cavefish, given the scarcity of food and the availability of mates in caves. To understand whether pleiotropy might be a general mechanism for the loss of melanin pigmentation in cave animals, we use Physella sp., a freshwater snail from SW Illinois, USA. Within one cave Physella sp. occurs in several different morphs, ranging from completely albino to fully pigmented individuals. Using melanogenic substrate assay, we demonstrated that the first step of melanin synthesis is blocked in Physella sp. To investigate the pleiotropic effects of this blockade, we tested anesthesia resistance (AR). Similar to A. mexicanus, albino morphs of Physella sp. exhibited higher AR levels compared to pigmented morphs, with AR being controlled by the noradrenergic system in both morphs. AR and the noradrenergic system are linked to melanin synthesis via a common precursor L-tyrosine, which can be redirected between melanin and catecholamine pathways. Using RNA sequencing of albino and pigmented morphs of Physella sp. we aim to gain further insight into other pleiotropic effects of melanin loss. Our results suggest that pleiotropy may be involved in the evolution of albinism in both cave vertebrates and invertebrates.

Study of the Invertebrate diversity in Prometheus Show Cave (Georgia, Caucasus)

Prometheus Cave is one of the largest caves in Georgia among the local six show caves. Before opening the cave as a tourist attraction, no research was conducted on the cave to study the invertebrate community living there, despite the cave's status as a natural monument. Before our study, only 22 species of invertebrates were known from Prometheus Cave, while none of the invertebrate species have been reported from adjacent non-touristic Datvi and Melouri caves. Cave invertebrate fauna was monitored monthly from 2018 to 2021 in the Prometheus cave and adjacent non-touristic Datvi and Melouri caves. The sampling was conducted on at least ten sites per cave each month. The abundance and incidence of the invertebrates were recorded on each site during field surveys. We ordinated the invertebrate diversity of the studied caves using Principal Component Analysis (PCA). After intensive investigations for three years, the number of species in Prometheus Cave increased from 22 to 47. Besides, ten species in Datvi and 11 species in Melouri caves were found for the first time. Our results suggest the existence of a significant difference in the diversity of the cave invertebrate fauna between the touristic part of Prometheus show Cave and its non-touristic part - Alpinist's hall, Datvi, and Melouri caves. The primary factor differentiating Prometheus show Cave from the others is the lower abundance of the species. Hurlbert's PIE and rarefaction analysis of the diversity explains that the diversity is also most unevenly distributed in this cave.

Ganxiao Dong: A Hotspot of Cave Biodiversity in Northern Guangxi, China

Located in the core zone of Mulun National Nature Reserve in northern Guangxi, the limestone cave Ganxiao Dong harbours the richest cave fauna currently known in China. In total, 26 species of cave invertebrates have been recognized so far, in spite of limited sampling efforts. Of them, 20 are troglobionts or stygobionts, including one snail, four millipedes, three spiders, one harvestman, three isopods, two springtails, two crickets, one non-glowing sticky worm, and three trechine beetles. Six other species are troglophiles. The most remarkable characteristic of this fauna is its high number of troglomorphic species, especially among millipedes, crickets and beetles.

Do different relevance attributes indicate the same conservation priorities? A case study in caves of southeastern Brazil

In the last decade, the scientific community brought to the debate gaps that slow down the advance of knowledge regarding global biodiversity. More recently, this discussion has reached subterranean environments, where these gaps are even more dramatic due to the relict and vulnerable nature of their species. In this context, we tested ecological metrics related to some of these gaps, checking if the biological relevance of the caves would change depending on ecological attributes related to each metric. The study was carried out in caves from southeastern Brazil, located in a region presenting a high richness of troglobitic species restricted to a narrow geographical extent. Thus, we verified: (a) the cave invertebrate communities’ vulnerability with the Vulnerability Index and the Importance Value for Cave Conservation; (b) the distribution and endemicity of the troglobitic species with the Endemicity Index; (c) the phylogenetic diversity of the troglobitic species considering the average taxonomic distinction (∆+), their richness and evenness. We observed a considerable change in the ordering of the caves’ biological relevance according to each tested attribute (index). We discussed how each of these metrics and their attributes indirectly relate to: (a) the preservation and maintenance of the phylogenetic diversity of subterranean communities; (b) the spatial restrictions of different groups, where the greater their restrictions, the greater their vulnerability; (c) the preservation of caves with high biological relevance considering these different attributes together. Thus, we recommend the use of different metrics so that different ecological attributes can be considered, supporting actions that aim to preserve caves in highly altered regions. Finally, we find that the most biologically important cave in the region is not protected (Gruta da Morena cave). We warn that this cave needs to be contemplated by a conservation unit in the region urgently.

Planarians, a Neglected Component of Biodiversity in Groundwaters

Underground waters are still one of the most important sources of drinking water for the planet. Moreover, the fauna that inhabits these waters is still little known, even if it could be used as an effective bioindicator. Among cave invertebrates, planarians are strongly suited to be used as a study model to understand adaptations and trophic web features. Here, we show a systematic literature review that aims to investigate the studies done so far on groundwater-dwelling planarians. The research was done using Google Scholar and Web of Science databases. Using the key words “Planarian cave” and “Flatworm Cave” we found 2273 papers that our selection reduced to only 48, providing 113 usable observations on 107 different species of planarians from both groundwaters and springs. Among the most interesting results, it emerged that planarians are at the top of the food chain in two thirds of the reported caves, and in both groundwaters and springs they show a high variability of morphological adaptations to subterranean environments. This is a first attempt to review the phylogeny of the groundwater-dwelling planarias, focusing on the online literature. The paucity of information underlines that scarce attention has been dedicated to these animals. Further revisions, including old papers and books, not available online will be necessary.

Does allochthonous leaf litter structure terrestrial cave invertebrate assemblages?

ABSTRACT Allochthonous materials are the main sources of nutrients for most cave invertebrate communities. However, scant research exists about the differences in quality of various cave nutrient sources. We investigated whether different types of leaf litter would attract different terrestrial invertebrates within cave ecosystems. We placed six leaf types: (1) Maple (Acer saccharum) (2) Dogwood (Cornus florida) (3) Sweetgum (Liquidambar styraciflua) (4) Pine (Pinus taeda) (5) Swamp White Oak (Quercus bicolor) (6) Southern Red Oak (Quercus falcata) in three caves in north Alabama. Leaf sets were placed within the twilight and dark zones within each cave and gathered at 30, 60, and 100-day intervals. Leaves were dried, weighed, and C/N analyses of leaves were determined. Pine had significantly higher C/N ratios whereas dogwood and red oak had the lowest. However, abundances of the most commonly collected invertebrates (e.g. Collembola and Acari) were not affected by the leaf type despite some leaves (e.g. pine) being a lower quality food source. Additionally, no differences in overall invertebrate abundance or taxa richness were found neither among the leaf types nor between the twilight and dark zones. Our data suggests that terrestrial invertebrate abundance is not solely driven by food quality and could be driven by multiple factors.

Microhabitat selection of a Sicilian subterranean woodlouse and its implications for cave management

Human activities in subterranean environments can affect different ecosystem components, including the resident fauna. Subterranean terrestrial invertebrates are particularly sensitive to environmental change, especially microclimatic variations. For instance, microclimate modifications caused by the visitors may directly affect local fauna in caves opened to the public. However, since numerous factors act synergistically in modulating the distribution and abundance of subterranean species, it remains challenging to differentiate the impact of human intervention from that of other factors. Therefore, evidence of the impact of tourism on cave invertebrate fauna remains scarce. Over a year and with approximately two visits a month, we investigated the effects of the presence of visitors on the subterranean endemic woodlouse Armadillidium lagrecai in the strict natural reserve of Monello Cave (Sicily, Italy). We found that natural microclimatic fluctuations, and not direct human disturbance, were the main factors driving the distribution of A. lagrecai. Specifically, A. lagrecai select for more climatically stable areas of the cave, where the temperature was constantly warm and the relative humidity close to saturation. We also observed a significant temporal effect, with a greater abundance of A. lagrecai in summer and a gradual decrease during the winter months. The number of visitors in the Monello Cave had no effect on the abundance and distribution of A. lagrecai. However, considering the high sensitivity of the species to microclimatic variations, it seems likely that a significant increase in the number of visitors to the cave could indirectly affect this species by altering local microclimate. Constant monitoring of the environmental parameters within the cave is therefore recommended.

Epigean and hypogean drivers of Neotropical subterranean communities

AbstractAimIn addition to cave size and other subterranean habitat characteristics, cave entrances are important structurers of neotropical cave communities. However, little is known about the epigean ecosystems factors that might dictate the influence of entrances and the relationship among surface and cave communities, as entrance regions are ecotonal zones between the epigean and hypogean ecosystems. We aimed to assess the influence of epigean and hypogean factors as potential drivers of invertebrate species richness in Neotropical caves as well as the influence of the regional pool of invertebrate diversity on cave invertebrate diversity.LocationLimestone caves in south‐eastern Brazil.TaxonInvertebratesMethodsData were collected in 48 caves within the Cerrado biome (Brazilian Savanna). The fauna sampling was performed by a direct intuitive search (DIS). Landscape characteristics and climatic parameters were accessed using geographic information systems. The characterization of the caves occurred during each sampling event. Regional diversity was based on the Taxonomic Catalog of Brazilian Fauna. To clarify the factors that significantly influence invertebrate species richness of the caves, we used generalized linear models (GLM). The relationship between regional diversity and cave diversity was evaluated based on linear models (LM).ResultsOverall, 1,173 species were found, of which 72 were obligate subterranean dwellers. The cave total species richness was influenced by hypogean factors and epigean factors. The species richness of obligate cave dwellers was significantly explained only by cave size. The regional pool of invertebrate species influenced the levels of biodiversity in the studied caves. The taxa with troglobitic species tended to be more diverse in caves than expected.Main conclusionsIn addition to the influences of intrinsic cave variables, we highlight the importance of epigean factors on subterranean diversity in the Neotropics. The study shows the significant influence of the regional species pool on cave faunal composition. These findings reinforce the importance of considering the surrounding areas in actions regarding cave biodiversity conservation.

Tourism effects on the subterranean fauna in a Central American cave

Abstract Tourism activity affects the cave's physical environment in several ways, such as damaging speleothems, altering the cave structure for ease of access, and the shifting of cave microclimate. Nevertheless, it is less clear how the pressure of touristic activities affects the cave fauna. This study aimed to investigate the effects from tourism activities on the cave invertebrate fauna from Gruta de Lanquín Cave (Alta Verapaz, Guatemala). For this, quadrats were placed throughout the cave, where invertebrates were sampled and substrate features were measured. Results showed that the community structure differed among touristic and non‐touristic areas of the cave and the troglobitic species are structured differently than the rest of the invertebrate community in the cave. It was detected that the pathways designated for touristic use have different substrate features than their adjacent areas. In order to maintain the conservation of the invertebrate species, several management recommendations are provided, such as determining a well‐established walking path that avoids cave microhabitats and the maintenance of a supervised guided tour.

Database of Romanian cave invertebrates with a Red List of cave species and a list of hotspot/coldspot caves.

BackgroundThe increasing human impact in Romanian caves raises the urgency of publishing a correct database of the strictly-adapted cave fauna. Previous attempts at indexing cave fauna and classifying caves by using their fauna opened many questions regarding the use of an incomplete list of cave species and mixed lists of troglobionts/stygobionts with troglophiles/stygophiles for ranking caves with priority for protection. It has also become obvious that there is a need to publish a list of Romanian cave species that are under threat. Cave species in Romania (and elsewhere) are endemic on small ranges, are unique and must be considered as important units for conservation. A cave must be equally protected if it has one or more rare and strictly endemic cave species. Although not exhaustive, we here provide the first checklist of Romanian troglobionts/stygobionts developed in the framework of the DARKFOOD and GROUNDWATERISK projects, coordinated by the “Emil Racovita” Institute of Speleology, Cluj-Napoca, Romania. The GIS application was used to complement the checklist of cave species with data on caves and surface environments above the caves. Until complete data on species diversity and population sizes are made available for each cave, measures of conservation can be implemented, based on the presence/absence of cave species, while classifications of caves for protection, based on the number of species, must be avoided. We also propose a list of Romanian caves with fauna that are under threat and a tentative Red List of Romanian troglobiont/stygobionts.New informationThis is the first database with identified troglobiont and stygobiont species of Romania, with a critical analysis of their distribution inside the country. A list of caves that need protection for their rare and unique species and a tentative Red List of Romanian cave fauna are also added. A total of 173 species were identified, of which 77 troglobionts and 96 stygobionts are currently registered in 366 caves. The database is divided into two parts, one part with a list of troglobionts, their revised systematic position, cave name, cave code and geographic region; and the second part with the same information on stygobionts. The database represents the contribution of many active researchers, who are the authors of this paper and of review publications of many other authors of the "Emil Racoviță" Institute of Speleology.

The role of microhabitats in structuring cave invertebrate communities in Guatemala

Several studies have tried to elucidate the main environmental features driving invertebrate community structure in cave environments. They found that many factors influence the community structure, but rarely focused on how substrate types and heterogeneity might shape these communities. Therefore, the objective of this study was to assess which substrate features and whether or not substrate heterogeneity determines the invertebrate community structure (species richness and composition) in a set of limestone caves in Guatemala. We hypothesized that the troglobitic fauna responds differently to habitat structure regarding species richness and composition than non-troglobitic fauna because they are more specialized to live in subterranean habitats. Using 30 m2 transects, the invertebrate fauna was collected and the substrate features were measured. The results showed that community responded to the presence of guano, cobbles, boulders, and substrate heterogeneity. The positive relationship between non-troglobitic species composition with the presence of guano reinforces the importance of food resources for structuring invertebrate cave communities in Guatemalan caves. Furthermore, the troglobitic species responded to different substrate features when compared to non-troglobitic species. For them, instead of the presence of organic matter, a higher variety of abiotic microhabitats seem to be the main driver for species diversity within a cave. The high specialization level of troglobitic organisms might be the reason why they respond differently to environmental conditions. The findings of this study highlight the importance of biological surveys for understanding cave biodiversity and give insights on how this biodiversity might be distributed within a cave. Conservation measures should keep in mind the target organisms and if such measures aim to protect a broad variety of organisms, then one should aim to preserve as many microhabitats and trophic resources as possible.

Eukoenenia ibitipoca: the first troglobitic Palpigradi (Arachnida) from a quartzitic cave.

Although the arachnids of the order Palpigradi are very common in subterranean habitats, no species has yet been recorded from siliciclastic caves. During a survey of cave invertebrates conducted in quartizitic caves in southern Minas Gerais state (Brazil), the occurrence of a new troglobitic palpigrade was revealed. Accordingly, in this paper we describe Eukoenenia ibitipoca sp. nov. which is readily distinguished from most species of this genus by the presence of eight pairs of setae on propeltidium.

Species-area model predicting diversity loss in an artificially flooded cave in Brazil

Subterranean environments are poorly known regarding many ecological aspects, such as community structure and its response to different disturbances. To estimate the effects of ground area lost in a limestone cave community in Southeastern Brazil, the invertebrate fauna was sampled before 76% of the cave floor was submerged by the filling of a hydroeletric power plant reservoir. Then, a 2-year monitoring was conducted. A species-area curve based on empiric data was constructed and the z-value of the species-area equation was calculated, what allowed estimating the expected cave richness after flooding comparing with data obtained during the monitoring. The results support the species-area relationship hypothesis; the cave community showed a drastic reduction of richness after losing area. Furthermore, it was also possible to estimate the species richness using the species-area equation. Moreover, the cave community showed a high temporal beta diversity when comparing the community sampled before and after the inundation; this pattern becomed less pronunciated over time. A high z-value (z = 0.58) was found for the cave species-area equation, indicating that subterranean communities are even more damaged by area loss than other environments probably due to the reduced mobility of cave invertebrates and the physical isolation of this environment. The present study highlighted that area loss resulted in a drastic reduction of cave richness. Additionally, it became evident that whenever possible studies should consider the original condition of a cave community and their responses after disturbances. Such strategy is critically important for conservation purposes.

Richness and taxonomic distinctness of cave invertebrates from the northeastern state of Goiás, central Brazil: a vulnerable and singular area

The karst area of the northeastern state of Goiás comprises two main municipalities: São Domingos and Posse. São Domingos is inside the limits of a Full Protection Conservation Unit known as Parque Estadual de Terra Ronca (PETeR), where a high number of caves occurs, some of them surpassing 10 km in length. Despite their protection by law, uncontrolled tourism has been threatening the integrity of the unique and fragile cave systems of São Domingos. On the other hand, the caves of Posse are much less visited, with its subterranean fauna poorly unknown and are strongly threatened by the urban areas and mining activities in the vicinity. We conducted six systematic surveys of invertebrates in 12 caves, seven of which are located in São Domingos and five in Posse, between 2010 and 2012. Immediately outside the caves, we sampled several microhabitats for comparison. We estimated richness, abundance, and taxonomic distinctness of the communities. We recorded 1,941 individuals of 344 morphospecies. Caves from São Domingos showed a higher species richness and were more taxonomic distinct than caves from Posse. Most morphospecies were considered troglophiles (188). We also found one trogloxene and one troglobite, as well as nine troglomorphic taxa that possibly are troglobitic as well. Both regions have markedly singular caves regarding its subterranean fauna, with high values of taxonomic distinctness. However, the richer caves were not necessarily the most taxonomic distinct. Conservation measures are especially necessary in the region of Posse, where caves are not legally protected and are within an area of significant urbanization. Cave entrances of Posse are near deforested vicinities, in some cases with domestic wastes and cement plants nearby.

The hypogean invertebrate fauna of Georgia (Caucasus)

Information about the cave invertebrates of Georgia, Caucasus, is summarised, resulting in 43 troglo- and 43 stygobiont taxa reported from 64 caves. Species distribution analyses were conducted for 61 caves harbouring 58 invertebrate taxa, with the majority of caves (39) located in Apkhazeti (north-western Georgia). In 22 caves from central-west Georgia (Samegrelo, Imereti and Racha-Lechkhumi regions of west Georgia) 31 taxa are reported. Composition of cave fauna differed strongly between the caves in Apkhazeti and the central-west of Georgia. Only two taxa of the total 86 were shared, resulting in negligible similarity (Sørensen-Dice coefficient Ss=4.8%). Rarefaction indicated an increase in number of species with additional sampling could increase species richness from 58 to 76 for caves in Apkhazeti and from 31 to 69 for caves in central-west Georgia. These findings suggest that the low invertebrate species richness observed in caves of western Georgia is the result of insufficient sampling. A pairwise approach to analysing species co-occurrence showed ten positive spatial associations in 7 out of 86 cave species, all from Kveda Shakurani and Tsebelda caves. The species co-occurring in the same microhabitat require further study to understand their relationships.