An extraordinary colonial spider community in Sulfur Cave (Albania/Greece) sustained by chemoautotrophy

An extraordinary colonial spider community in Sulfur Cave (Albania/ Greece) sustained by chemoautotrophy* István Urák1, Blerina Vrenozi2, Zofia Głąbiak3,4, Ninon Lecoquierre4,5, Cord Eiberger6, Mark Maraun6, Andrei Ştefan7,8, Jean-François Flot4,9,10, Traian Brad8, Luisa Dainelli11, Serban M. Sarbu8,12,13, Raluca I. Băncilă8,12 1 Department of Life Sciences, Sapientia Hungarian University of Transylvania, 520036 Sfântu Gheorghe, Ciucului str. 50, Romania 2Research Center of Flora and Fauna, Faculty of Natural Sciences, University of Tirana, Petro Nini Luarasi, No 80, Tirana, Albania 3Maastricht Science Programme, University of Maastricht, 6200 MD Maastricht, Netherlands 4Research unit “Evolutionary Biology and Ecology”, Department of Organismal Biology, Université libre de Bruxelles (ULB), 1050 Brussels, Belgium 5Department of Biology, Research group of Ecology, Evolution and Genetics, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Ixelles, Belgium 6J.F. Blumenbach Institute of Zoology and Anthropology, University of Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany 7Department of Molecular Biology, “Grigore Antipa” National Museum of Natural History, Şos. Kiseleff 1, Bucharest 011341, Romania 8Romanian Academy Cluj-Napoca Branch, Emil Racoviţă Institute of Speleology, Str. Clinicilor, Nr. 5-7, 400006 Cluj-Napoca, Romania 9Interuniversity Institute of Bioinformatics in Brussels - (IB)², Brussels, Belgium 10Brussels Laboratory of the Universe - BLU, Brussels, Belgium 11Gruppo Speleologico Archeologico Livornese of the Mediterranean Natural History Museum, 57100 Livorno, Italy 12Emil Racoviţă Institute of Speleology, Biospeleology and Karst Edaphobiology Compartment, Calea 13 Septembrie, Nr. 13, 050711 Bucharest, Romania 13Department of Biological Sciences, California State University, Chico, CA, USA Corresponding author: Traian Brad ([email protected]) Academic editor: Stefano Mammola|Received 17 June 2025|Accepted 25 August 2025|Published 17 October 2025 https://zoobank.org/300E3746-CC81-442A-AB83-BE8E6B8D5DD9 Citation: Urák I, Vrenozi B, Głąbiak Z, Lecoquierre N, Eiberger C, Maraun M, Ştefan A, Flot J-F, Brad T, Dainelli L, Sarbu SM, Băncilă RI (2025) An extraordinary colonial spider community in Sulfur Cave (Albania/Greece) sustained by chemoautotrophy. Subterranean Biology 53: 155–177. https://doi.org/10.3897/subtbiol.53.162344 Abstract We report the discovery and detailed analysis of an extraordinary colonial spider assemblage in Sulfur Cave, a chemoautotrophic sulfidic ecosystem located on the Albania-Greece border. The colony, comprising an estimated 69,000 individuals of Tegenaria domestica (Agelenidae) and more than 42,000 of * Topical Collection: "DarCo: Filling knowledge gaps for European subterranean biodiversity towards its effective conservation", edited by Stefano Mammola, Fabio Stoch. Subterranean Biology 53: 155–177 (2025) doi: 10.3897/subtbiol.53.162344 https://subtbiol.pensoft.net Copyright István Urák et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Subterranean Biology Published by The International Society for Subterranean Biology A peer-reviewed open-access journal István Urák et al. / Subterranean Biology 53: 155–177 (2025) 156 Prinerigone vagans (Linyphiidae), spans a surface area of over 100 m²—representing the first documented case of colonial web formation in these species. Stable isotope analyses (δ¹³C and δ¹⁵N) revealed that the trophic web sustaining this assemblage is fueled by in situ primary production from sulfur-oxidizing microbial biofilms then transferred through chironomid larvae and adults to higher trophic levels. Morphological and molecular data confirmed the identity of the two spider species and revealed that their populations in Sulfur Cave are genetically distinct from other populations. Regarding T. domestica, we found a seasonal pattern in fecundity, with significantly larger egg clutches in early summer. Microbiome analysis of this species also revealed a lower Shannon diversity in the cave population compared with a surface individual captured nearby. Our findings unveil a unique case of facultative coloniality in this cosmopolitan spider, likely driven by resource abundance in a chemoautotrophic cave, and provide new insights into the adaptation and trophic integration of surface species in sulfidic subterranean habitats. Keywords food-web analysis, Prinerigone vagans, Sarandaporo Valley, stable isotope ratio analysis, Tegenaria domestica Introduction Sulfidic subterranean ecosystems are sustained by in situ primary production predominantly by chemoautotrophic sulfur-oxidizing microorganisms. These organisms utilize the hydrogen sulfide (H2S) as electron donor, and atmospheric dioxygen (O2) as terminal electron acceptor (Chen et al. 2009; Kumaresan et al. 2014). The resulting high amounts of autochthonous organic matter support abundant and diverse aquatic and terrestrial communities of invertebrates as well as a few vertebrate species (Engel 2007; Hutchins et al. 2016). Sulfidic cave ecosystems often harbor numerous endemic species (Brad et al. 2021; Sarbu et al. 2024), some of which exhibit physiological and biochemical adaptations allowing them to survive in highly selective environmental conditions such as H2S toxicity, hypoxia, and very low pH values (Flot et al. 2014; Tobler et al. 2016; Borko et al. 2019). The first chemoautotrophy-based groundwater ecosystem was discovered in Movile Cave in 1986 (Sarbu 1990; Sarbu et al. 1996). Subsequent investigations have revealed similar ecosystems in various locations, including the Frasassi caves (Italy; Sarbu et al. 2000), Ayyalon Cave (Israel; Por et al. 2013), Melissotrypa Cave (Greece; Popa et al. 2020), Tashan-Chah Kabootari Cave (Zagros Mountains, Iran; Malek-Hosseini et al. 2023), the Sharo-Argun cave system (Chechen Republic; Chervyatsova et al. 2020; Antić and Turbanov 2022), and more recently, several sulfidic caves in Albania (Benassi 2024; Sarbu et al. 2024; T. Delić, personal communication) within the Sarandaporo Valley, situated on the border between Greece and Albania (Audy et al. 2022; Kovařík et al. 2023; Sarbu et al. 2024). Sulfur Cave is located at the border of two countries: its entrance is in Greece, while its deep sections are in Albania (Fig. 1). Along with Atmos Cave and Turtle Cave, it forms a hypogenic subterranean network of large rooms and passages lodged in a narrow limestone outcrop that was cut by the Sarandaporo River to form the Vromoner Canyon (Vromoner means “Smelly water” in Greek). Springs located in the deep recesses of the cave feed a sulfidic stream which flows through the entire length of the main An extraordinary colonial spider community in Sulfur Cave 157 cave passage and reaches the Sarandaporo River at the cave entrance. Its water displays a constant year-round temperature of 26 °C and a H2S concentration as high as 65 mg l-1 (Audy et al. 2022), and no seasonal or diurnal variations in flow rate were recorded for the cave stream. Occasional flooding events in the Sarandaporo River are only felt in the cave entrance area and appear to have limited effects on the cave ecosystem. The atmosphere in the cave can reach concentrations of up to 14 ppm of H2S (Audy et al. 2022). The sediments in the stream are covered by abundant white biofilms consisting of filamentous sulfur-oxidizing bacteria (e.g., Beggiatoa spp., Thiothrix spp.). The aquatic cave fauna in this stream is very abundant and includes oligochaetes, gastropods, chironomid larvae, coleoptera larvae and adults, and occasionally some fish. The terrestrial fauna is represented by numerous centipedes, terrestrial isopods, pseudoscorpions, mites, scorpions, spiders, springtails, chironomid adults, and beetles (Sarbu et al. 2024). In the stream passage located close to the cave entrance, a dense swarm of adult chironomid flies fills the air in the immediate vicinity of the sulfidic stream, and a large portion of the cave wall is covered by a massive colonial spider web (Figs 1, 2). The European subterranean spider fauna includes 512 species belonging to 20 families, with at least 192 species considered to be obligate troglobionts (Mammola et al. 2022). In Sulfur Cave, a large colonial spider web covering a significant portion of one of the cave walls was first reported by a team of speleologists from the Czech Speleological Society who explored the sulfidic caves of the Vromoner Canyon and collected samples of fauna (Audy et al. 2022). Taxonomic analysis by V. Ružička reported the presence of the following spider taxa in Sulfur Cave: Tegenaria domestica (Fourcroy, 1785) (Agelenidae), which was identified based solely on the examination of a few female specimens, Metellina merianae (Scopoli, 1763) (Tetragnathidae), Kryptonesticus eremita (Simon, 1880) (Nesticidae), and another unidentified nesticid species. Sarbu et al. (2024) subsequently identified the dominant spider species in the colony as Tegenaria domestica (Clerck, 1757) (Agelenidae) (Fig. 3), commonly known as the “Domestic House Spider” or “Barn Funnel Weaver”. A dense population of Prinerigone vagans (Andouin, 1826) (Linyphiidae) (Fig. 4) was also found co-inhabiting the colonial web. Parasocial or colonial behavior (i.e., cooperation in web building by sharing the same spatial framework) has often been reported in subtropical and tropical arachnid communities. However, to date the only documented case of sub-social behavior in a cave-dwelling spider is that of Goeldia sp., observed by Almeida-Silva et al. (2009) in a Brazilian cave. This involved limited cooperative behavior and does not meet the criteria for true coloniality. Therefore, no confirmed cases of coloniality—defined by stable, large-scale cooperative web structures—have been reported among cave spiders (Mammola and Isaia 2017). Spider colonies, sometimes exceeding 100,000 individuals, are often correlated with locally high abundance of food resources (Rypstra 1979; Uetz 1983; Uetz and Hodge 1990; Uetz and Hieber 1997). Within Central Europe, the araneid spider Larinioides sclopetarius (Clerck 1757) is one of the few species known to form colonies with aggregations of 60 to 200 individuals across multiple generations building interconnected orb-webs (Schmitt 2004). Colonial behavior has never been observed in any agelenid or linyphiid spiders. István Urák et al. / Subterranean Biology 53: 155–177 (2025) 158 Food webs in caves are generally little studied since the trophic relationships between invertebrates and also between invertebrates and microorganisms are difficult to observe. One method to overcome those problems is the use of stable isotopes for understanding trophic levels in cave ecosystems. To analyze the trophic structure of cave animals and their food resources in Sulfur Cave, we therefore used stable isotope ratios of carbon (13C/12C) and nitrogen (15N/14N). These ratios are often used to analyze trophic levels in invertebrate communities (Maraun et al. 2023) as they integrate animal dietary information over longer periods of time, which allows to reveal general characteristics of the trophic structure of food webs (Tiunov 2007; Nielsen et al. 2018). As 13C is little enriched across trophic levels, it serves as an indicator for the base of the food web (Peterson and Fry 1987; Potapov et al. 2019a). In contrast, 15N is enriched by about three delta units per trophic level thereby reflecting the trophic position of consumers (Post 2002; Pollierer et al. 2009; Potapov et al. 2019b). This study presents a detailed characterization of the remarkable spider colony discovered within Sulfur Cave, focusing on its spatial distribution and dimensions, species composition and population density, and the trophic resources sustaining this unique assemblage. We also documented aspects of reproductive biology (egg number and size) of the dominant species within the colonial web, and examined the presence and distribution of other spider species within Sulfur Cave to understand habitat partitioning and potentially distinct food web dynamics. Figure 1. Plan of Sulfur Cave, with depiction of the main sulfidic springs (blue areas), and the large spider web in the vicinity of the cave entrance (brown areas) (modified from Audy et al. 2022 and Sarbu et al. 2024). An extraordinary colonial spider community in Sulfur Cave 159 Materials and methods Spider specimens were manually collected from both large and small webs throughout Sulfur Cave. Voucher specimens were preserved in 70% ethanol and deposited in the collection of the Museum of Natural Sciences, University of Tirana, Albania, and the zoological collection of the Department of Life Sciences, Sapientia Hungarian University of Transylvania, Romania. For stable isotope analysis samples of invertebrates and of microbial biofilms covering the aquatic sediments in the cave stream were collected manually and stored at –80 °C prior to analysis. Figure 2. The colonial spider web in Sulfur Cave, is home to a mixed colony of Tegenaria domestica and Prinerigone vagans. A. Side view; B. frontal view. The swarm of adult chironomids is visible near the cave stream. Photo A: Marek Audy. István Urák et al. / Subterranean Biology 53: 155–177 (2025) 160 Morphological identification of spiders from Sulfur Cave Spider external morphology, with a specific focus on male and female genitalia was examined using a ZEISS Stemi 2000-C and a Nikon SMZ1270 stereomicroscope. The taxonomic identification was based on the resources available at Spiders of Europe (Nentwig et al. 2025). Images of the female epigyne and male pedipalp of T. domestica were taken using a Nikon D5600 DSLR camera mounted on a Nikon SMZ1270 stereomicroscope (Fig. 3B, C). The specimens of P. vagans were photographed using a Kern ODC 825 microscope camera attached to a Kern OZL 464 trinocular stereomicroscope. Images of the female epigyne and male pedipalp were taken using a Kern ODC 825 microscope camera mounted on a Kern OBN 132 trinocular optical microscope (Fig. 3). Multifocal images were compiled using HeliconFocus software, digital images were processed using Photoshop software. Molecular identification of spiders from Sulfur Cave and haplotype analysis The morphological characterization of the spiders was complemented by DNA barcoding. DNA was isolated from the prosoma or legs of specimens collected in absolute ethanol and kept at 4 °C using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. For Tegenaria spiders, we amplified and sequenced Folmer’s fragment (COI) of the cytochrome c oxidase mitochondrial gene of 26 cave-collected individuals and 1 surface-collected individual (caught near the entrance of nearby Pixaria Cave) using the protocol described in Collard et al. (2025); Nanopore reads were subsequently assembled using amplicon_sorter (Vierstraete and Braeckman 2022). For the other spiders (namely, two specimens of Kryptonesticus, three specimens of Prinerigone, and two specimens of Metellina), a slightly shorter fragment of COI mitochondrial gene was amplified using the LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’; Folmer 1994) and chelicerate_reverse_2 (5’-GATGGCCAAAAAATCAAAATAAATG-3’; Barrett and Hebert 2005) primer pair. Those PCRs were performed in a 40 μL volume containing 1X Green GoTaq® Flexi Buffer (Promega, Madison, WI, USA), 2.5 mM MgCl2, 1X BSA, 0.1 mM dNTP, 1 U/μL GoTaq® Flexi DNA polymerase, 0.1 μM each primer and up to 10 ng/μL DNA. The cycling conditions consisted of an initial denaturation at 95 °C for 2 minutes followed by 5 cycles of denaturation at 94 °C for 40 seconds, annealing at 45 °C for 40 seconds, extension at 72 °C for 1 minute, 35 cycles of denaturation at 94 °C for 40 seconds, annealing at 51 °C for 40 seconds, extension at 72 °C for 1 minute and a final extension step at 72 °C for 5 minutes. Sanger sequencing was performed by Macrogen (Amsterdam, The Netherlands) and the resulting trace files were visually inspected and curated in Chromas v.2.6.6 (Technelysium Ltd., South Brisbane, Australia) and CodonCode Aligner v.3.7.1 (CodonCode Corporation, MA, USA). The resulting sequences were compared to the publicly available databases GenBank (Sayers et al. 2022) and Barcode of Life Data System (Ratnasingham and Hebert 2024). An extraordinary colonial spider community in Sulfur Cave 161 Sequences belonging to the same taxon or related taxa were downloaded and aligned on the MAFFT web server (Katoh et al. 2019; https://mafft.cbrc.jp/align-ment/server/ index.html, accessed on the 29th of March 2025), sequence statistics and haplotype diversity (Hd) were estimated in DnaSP v.6 (Rozas et al. 2017) and haplotype networks were drawn in HaplowebMaker (Spöri and Flot 2020) using the median-joining algorithm (Bandelt et al. 1999). Figure 3. Tegenaria domestica in Sulfur Cave. A Female next to a funnel shaped hole in the colonial spider web. B Female epigyne. C Male pedipalp with retrolateral view. István Urák et al. / Subterranean Biology 53: 155–177 (2025) 162 16S metabarcoding analyses As T. domestica is the dominant species in the communal web, our preliminary microbiome analysis has focused on this species. It included six individuals caught at the cave entrance, 13 individuals caught in the first section of the spider wall encountered when walking from the entrance into the cave, and 13 individuals caught in the second section of the spider wall (both on the left bank of the stream); as well as one surface individual caught near the entrance of Pixaria Cave, a few kilometers away, as a comparison. Whereas the first section of the Sulfur Cave spider wall is characterized by numerous egg sacs visible on the web, the second section is almost devoid of egg sacs, suggesting that reproduction chiefly occurs on the first section. Whole opisthosomas Figure 4. Prinerigone vagans in Sulfur Cave. Male and male pedipalp (right). Female and female epigyne (left). An extraordinary colonial spider community in Sulfur Cave 163 were used for DNA extraction, because the gut of spiders is not easy to separate from surrounding tissues. Although there are several organs located in the abdomen that contain microbial communities, the midgut is the largest one and it has been suggested in the literature that microbes associated with other organs do not influence the results significantly (see Kennedy et al. 2020 and references therein). DNA was extracted using the DNeasy PowerSoil Pro kit (Qiagen, UK), then 16S amplification and Nanopore sequencing was performed using the protocol described in Collard et al. (2025). The obtained reads were then analyzed in Emu (Curry et al. 2022) using its default database (Stoddard et al. 2015; O’Leary et al. 2016; Schoch et al. 2020). Population density estimation of colony forming spiders and associated species The density of the T. domestica population was estimated by counting individual funnel-shaped webs and then extrapolating these counts to the surface area occupied by the colony. A 15 × 15 cm quadrant was placed near the cave wall where the colony was present, at 30 randomly selected locations. At each location two highresolution pictures were taken of each quadrat. The number of individual webs per quadrat, identified by the central funnel structure, including those overlapping the center of the web, was counted from the pictures. Note that this methodology is likely to slightly overestimate the total individual count in the colony, as some funnel webs may be abandoned/unoccupied. This survey was carried out in October 2023, April 2024 and March 2025. To calculate the surface area occupied by the colony, the length (L) and width (W) of the web-covered section of the wall were measured, and the area was computed using the formula: Area = L × W, assuming the colony forms an approximately rectangular patch. The P. vagans population density was estimated in March 2025 by counting individual spiders visible within 15 × 10 cm quadrants photographed at 30 random locations. The total estimated population size was extrapolated to half of the surface area determined for T. domestica, reflecting the observed spatial distribution pattern of P. vagans. The density of flies associated with the colony was estimated using a similar approach: individuals were counted within quadrats based on photographs, and the mean density was extrapolated to the total area occupied by the colony. Reproductive biology of Tegenaria domestica - egg number and size analysis A total of 86 egg sacs of T. domestica were collected from the surface of the colonial web during three sampling periods: June 2024 (n = 15), October 2024 (n = 40), and March 2025 (n = 31). Each egg sac was carefully opened using fine-tipped tweezers, and the number of eggs per clutch was recorded. Photographs were taken for further documentation. To assess egg size, 20 egg clutches were randomly selected, and three eggs from each clutch were measured using a Stage Micrometer Microscope calibrated to a precision of 0.01 mm. István Urák et al. / Subterranean Biology 53: 155–177 (2025) 170 In the deep recesses of the cave, far from the cave entrance and the colonial web, two additional web-building spider species were also observed: Kryptonesticus eremita (Simon, 1880) (Nesticidae), previously reported from Sulfur Cave by Audy et al. (2022), and a very small, blind, and depigmented species of Cataleptoneta (Leptonetidae). Both K. eremita and Cataleptoneta sp. were present in large numbers but were spatially limited to areas where the cave walls were moist and free of gypsum crusts. Genetic and geographic patterns of spider species in Sulfur Cave Haplotype networks revealed both similarities and contrasts among the spider species found in Sulfur Cave. For T. domestica, K. eremita, and P. vagans, the cave individuals shared distinct haplotypes not found in broader regional datasets, though all were closely related, i.e. within three mutations, to widespread haplotypes from Europe, the Middle East, or Asia. In contrast, M. merianae showed a more complex and reticulated haplotype network, with two individuals from Sulfur Cave belonging to different haplotypes, one shared with German samples. Despite the geographical proximity of some samples to Sulfur Cave (e.g. from Slovenia, Bulgaria, North Macedonia), they were quite distant in the network. This suggests that M. merianae is both more geographically mobile and more genetically diverse than the other spider species found in Sulfur Cave. Figure 9. Metellina merianae. Female (left) and male (right) in individual webs on the cave wall. An extraordinary colonial spider community in Sulfur Cave 171 Reproductive biology of T. domestica The reproductive effort, measured as the number of eggs per sac, differed from values reported in the literature for surface-dwelling conspecifics. Females of T. domestica are known to lay six to eight egg sacs at an interval of 20–25 days, with the first cocoon containing up to 100 eggs, followed by smaller successive clutches (Trabalon et al. 1992). Numerous egg-sacks were observed in the large spider web, but their number was not estimated as the multi-layered web made it impossible to count the egg clutches. The underlying factors contributing to a potential reduction in clutch size in the Sulfur Cave population of this species are yet to be determined, although environmental factors such as perpetual darkness and sulfidic conditions have been shown to be associated with reduced fecundity and increased offspring size (Riesch et al. 2010). Microbiomes of caveand surface-dwelling T. domestica At the microbiome level, we found that, by contrast to one surface-dwelling individual of T. domestica, collected in the vicinity of the cave, in which approximately 30 different bacterial genera were detected, the cave-dwelling individuals investigated exhibited markedly lower bacterial diversity. In these individuals a large proportion of the reads originated from intracellular bacterial symbionts, such as Mycoplasmopsis, Mycoplasma and Wolbachia. Although the results are quite preliminary and do not allow to distinguish the relative contributions of the various parts of the opisthosoma to the measured diversity, it suggests that the population of T. domestica from Sulfur Cave presents a much-reduced microbial diversity than surface ones. Alternate food web in Sulfur Cave Preliminary observations indicate that in the deep cave sections, the food web is based on chemosynthetic carbon fixation in terrestrial microbial biofilms that cover the moist cave walls. These biofilms consist of sulfur-oxidizing microorganisms (unpublished). Numerous specimens of Graeconiscus sp. (Isopoda, Trichoniscidae) and dense populations of collembola appear to feed on this cave-wall microbiome, forming the base of a food web that supports two species of spiders and other small terrestrial predators such as centipedes, pseudoscorpions, mites, and beetles (Sarbu et al. 2024). Conclusions Similarly to other sulfidic subterranean ecosystems based on autochthonous food production by chemoautotrophic microorganisms, Sulfur Cave in the Vromoner Canyon located on the border between Greece and Albania contains exceptionally abundant and diverse invertebrate communities that thrive in total darkness. The most impressive occurrence in this cave is a large colonial spider web that covers an estimated István Urák et al. / Subterranean Biology 53: 155–177 (2025) 172 100 m2 of cave wall and hosts ~69,000 specimens of T. domestica and ~42,000 specimens of P. vagans. These are both surface species that have never been reported to form colonies, and molecular evidence suggests that the Sulfur Cave population does not exchange individuals with the surface. Preliminary analyses of the microbiome of T. domestica also suggest that the cave population of this species is isolated from surface ones and presents a depauperate microbial diversity. Acknowledgements The authors are grateful to Mihai Hristescu, Ruxandra Nițescu, Marius Kenesz, Norm Rosene, Alexandru Crînguș, Geza Zakarias, Claire Chauveau, Olivier Collard, Sarah Flot, Alice Salussolia, Maria Fotiadi, Andreea-Rebeka Zsigmond, Andrei Sarbu, and many others who helped with the field work. The taxonomic identifications were performed by Elisabeth Stur and Torbjørn Ekrem (Chironomidae), Bernhard Klausnitzer (Scirtidae), George Popovici (Pseudoscorpiones), and Victor Fet (Scorpiones). Special thanks to Susanne Boening for help during sample preparation for stable isotope measurements and to Svenja Meyer for the animal drawings in Fig. 7. Special thanks to Ingmar Weiss for his useful insights and for his help during the preparation of the manuscript. This research is a vital part of the initiative: “Conservation of the Unique Cave Ecosystems of Aoos-Vjosa River Basin” and the project received EuroSpeleo protection label status and was supported by FSE for 2025. This research was funded by Biodiversa+, the European Biodiversity Partnership under the 2021–2022 BiodivProtect joint call for research proposals, co-funded by the European Commission (GA N°101052342) and with the funding organizations Ministry of Universities and Research (Italy), Agencia Estatal de Investigación – Fundación Biodiversidad (Spain), Fundo Regional para a Ciência e Tecnologia (Portugal), Suomen Akatemia – Ministry of the Environment (Finland), Belgian Science Policy Office (Belgium), Agence Nationale de la Recherche (France), Deutsche Forschungsgemeinschaft e.V. (Germany), Schweizerischer Nationalfonds (Grant N° 31BD30_209583, Switzerland), Fonds zur Förderung der Wissenschaftlichen Forschung (Austria), Ministry of Higher Education, Science and Innovation (Slovenia), and the Executive Agency for Higher Education, Research, Development and Innovation Funding (Romania). 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