Limestone Cave Research Articles

Bacterial communities forming yellow biofilms in different cave types share a common core

The walls of different types of caves under diverse geological settings (limestone, gypsum and volcanic) are colonized by biofilms of different colors: white, yellow, pink, grey, green to dark brown, but only a few colored biofilms such as the white, yellow and grey ones have been extensively studied. However, an assessment among the microbial communities originating these biofilms in different lithologies is lacking. Here we compare the yellow biofilms from two caves, Covadura and C3, in the Gypsum Karst of Sorbas in Spain, with those from two Spanish limestone caves (Pindal and Santian), and four volcanic caves in Spain and Italy (Viento, Honda del Bejenado, Grotta del Santo, Grotta di Monte Corruccio). The structure of yellow biofilms in gypsum caves closely resembles that found in other Spanish and European limestone caves. However, volcanic cave biofilms exhibit greater variability in their microbial community structure and morphologies. Biofilms from gypsum, limestone and volcanic caves were characterized by the abundance of the genera Crossiella and the gammaproteobacterial wb1-P19. The uncultured Euzebyaceae were abundant in gypsum and Spanish volcanic caves, while in the limestone and Italian volcanic caves, they were rare or absent. Nitrospira was also abundant in limestone and volcanic caves, but not in gypsum caves. Due to the abundances of Crossiella, gammaproteobacterial wb1-P19, and uncultured Euzebyaceae, in many different ecosystems, not only in caves, as recently reported, understanding the functional diversity in which these lineages are involved seems critical. Although we have studied a limited number of yellow biofilms from caves in Spain and Italy, data from other caves in USA and Russia also point out the existence of a similarity among the most abundant members composing the structure of yellow biofilms, suggesting that they share a common core.

Language standardization and railway-walking distance. Levenshtein distance and distribution in GAJ

Abstract In this paper, new quantitative methods were applied to elucidate the geographical distribution of Japanese dialects. (1) Regional differences (individual differences) of usage rate of standard language at each locality in GAJ (Grammar Atlas of Japanese Dialects) were dealt with. (2) In addition to the railway distance from Tokyo to each locality, the walking distance from the railway stations to the survey localities was measured and used as the “railway-walking distance”. (3) Using a scatter-plot in what is called a “limestone cave model”, the values on the axis of standard language usage were reversed, and the Japan Islands were divided into east and west according to their direction from Tokyo. It showed that the standard language usage rate and distance show a proportional relationship even at each locality (individual informant) value. (4) Focusing on the prefectures near Tokyo, the distance from Tokyo regulates the usage rate. From this, the rapid spread of standard language from modern Tokyo and influence of railway can be ascertained. It was found that the phenomena pointed out by the average values by prefectures of vocabulary items in LAJ are valid to the data for each locality (individual) of GAJ.

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.

Microhabitat selection and niche overlap: Drivers of spider coexistence in a tropical limestone cave

AbstractStable temperature, humidity, and oligotrophy may be essential in microhabitat selection, shaping niche overlap and species coexistence within cave ecosystems. To explore this hypothesis, we assessed the distribution and microhabitat preferences of four non‐obligate cave‐dwelling spiders: Loxosceles anomala, Scytodes itapevi, Philoponella vittata, and the exotic Oecobius navus. Using 1‐m2 quadrats, we surveyed spider distribution and abundance in a limestone cave, considering environmental conditions and prey availability during wet and dry periods. Significant differences were observed between moist and dry periods regarding light intensity, wind speed, air temperature and moisture, potential prey, and the abundance of L. anomala. The exotic spider O. navus was more specialist, preferred brighter habitats, and showed lower niche overlap. Conversely, the three native species displayed greater niche overlap and preferred darker and more humid environments. Spatial disparities, rather than temporal variation, in species distribution underscored the influence of cave habitat conditions on species segregation. Furthermore, our findings highlighted the limited ability of the non‐native O. navus to occupy more illuminated cave areas, thereby potentially safeguarding these habitats against biological invasion by this species.

Cyrtodactylus panitvongi, a new cave-dwelling Bent-toed Gecko from Lopburi Province, central Thailand (Squamata: Gekkonidae)

We describe Cyrtodactylus panitvongi sp. nov. from Tham Khao Chan (14°58’42.2”N 101°18’40.6”E), a limestone cave in Lopburi Province, central Thailand. The new species is characterized by the following combination of morphological characters and pattern: maximal known snout-vent length of 85 mm, 15–18 interorbital scales, 35–37 interciliary scales, 10–12 supralabials, 9–11 infralabials, 29–33 paravertebral tubercles of which 22–25 between limb insertions, 18 longitudinal rows of dorsal tubercles at midbody, 40–42 ventral scale rows at midbody, poorly defined, non-denticulate ventrolateral folds, a series of 13 or 14 enlarged femoroprecloacal scales including 2 pores on the proximalmost scales on each side separated by a single smaller medial scale in males, or six precloacal pits on a continuous series of 32–34 enlarged femoroprecloacal scales in females, no precloacal groove or depression, two or three postcloacal spurs on each side of the base of the tail in males and females, no interdigital webbing, 16–19 total subdigital lamellae on 4th toe, a row of widened median subcaudal plates, a banded dorsal pattern including an uninterrupted nuchal collar and three dark bands on dorsum, 10 or 11 dark-colored rings on original tail, and a golden iris. This trogloxene species is added to a long list of squamates endemic to the Saraburi Group Limestones.

A study on the diversity of phlebotomine sand flies (Diptera, Psychodidae) in karstic limestone areas in Vientiane Province, Laos, with a description of two new species of Sergentomyia França and & Parrot

BackgroundSoutheast Asia is well known as a hotspot of biodiversity. However, very little is known about cave-dwelling hematophagous insects that are medically important. Taxonomic knowledge and ecology of phlebotomine sand flies are very poorly studied in Laos, as well as in other countries in the region. Herein, we report species diversity data and some notes on the ecology of the detected species from these karstic limestone areas of Laos.MethodsPhlebotomine sand flies were collected using Centers for Disease Control and Prevention (CDC) light traps from limestone cave locations in three districts of Vientiane Province, Laos. Both morphological and molecular techniques were used for sand fly identification. Species diversity and abundance were analyzed according to sites, locations, collection seasons, and trapping positions.ResultsA total of 6564 sand flies, of which 5038 were females and 1526 were males, were morphologically identified into 20 species belonging to five genera (Chinius, Idiophlebotomus, Phlebotomus, Sergentomyia, and Grassomyia). The most abundant species were Chinius eunicegalatiae, Phlebotomus stantoni, Sergentomyia hivernus, Se. siamensis, and Idiophlebotomus longiforceps. Cytochrome b analysis results supported the morphological identification and revealed that Se. siamensis was separated from other members of the Se. barraudi group. Two new species, Se. dvoraki n. sp. and Se. marolii n. sp., were described. Sand fly density was generally high except in a cave in Vangvieng, with species richness ranging from 14 to 18 across different caves. Outside caves had higher species richness (R = 20) and diversity (H = 2.50) than cave entrances (R = 18, H = 2.41) and interiors (R = 16, H = 2.13). Seasonal variations showed high sand fly density in Feung and Hinheup during both dry and rainy seasons, while Vangvieng had a notable decrease in density during the dry season (D = 6.29).ConclusionsThis study revealed that the diversity of phlebotomine sand fly fauna in Laos, particularly in karstic limestone areas, is greater than previously known. However, the taxonomic status of many species in Laos, as well as Southeast Asia, still needs more in-depth study using both morphological characters and molecular methods. Many species could be found from inside, at the entrance, and outside of caves, indicating a wide range of host-seeking behavior or possible natural breeding in the karstic cave areas.Graphical abstract

Biology and ecology of a deep cave nesting spider wasp, Ageniella evansi Townes, (Hymenoptera: Pompilidae), in Arizona

ABSTRACT Cavernicolous populations of the spider wasp Ageniella evansi Townes, 1957 (Hymenoptera: Pompilidae) nest deep within limestone caves at Colossal Cave Mountain Park near Tucson, Arizona, USA. These remarkable populations are the only truly cavernicolous pompilid wasps currently known. As many as 79 females were documented nesting simultaneously within Arkenstone Cave. The facts that the wasps do not nest near cave entrances but only deep within the interior of the caves, are philopatric to each occupied cave and have been present each year for decades confirm their cavernicolous behaviour as subtroglophiles. Studies conducted for over 30 years revealed many insights into the behaviour of these wasps in addition to their nesting deep within limestone caves, including: intraspecific and interspecific competition, plasticity in host selection, precision solar navigation, evidence that some form of communal nesting may be present and behaviour suggestive of rudimentary cognition in the species. This study demonstrates the importance of assessing all animals found in caves as potential ecological elements and not focusing solely on exotic troglobiotic species.

Narrative cave art in Indonesia by 51,200 years ago.

Previous dating research indicated that the Indonesian island of Sulawesi is host to some of the oldest known rock art1-3. That work was based on solution uranium-series (U-series) analysis of calcite deposits overlying rock art in the limestone caves of Maros-Pangkep, South Sulawesi1-3. Here we use a novelapplication of this approach-laser-ablation U-series imaging-to re-date some of the earliest cave art in this karst area and to determine the age of stylistically similar motifs at other Maros-Pangkep sites. This method provides enhanced spatial accuracy, resulting in older minimum ages for previously dated art. We show that a hunting scene from Leang Bulu' Sipong 4, which was originally dated using the previous approach to a minimum of 43,900 thousand years ago (ka)3, has a minimum age of50.2 ± 2.2 ka, and so is at least 4,040 years older than thought. Using the imaging approach, we also assign a minimum age of 53.5 ± 2.3 ka to a newly described cave art scene at Leang Karampuang. Painted at least 51,200 years ago, this narrative composition, which depicts human-like figures interacting with a pig, is now the earliest known surviving example of representational art, and visual storytelling, in the world3. Our findings show that figurative portrayals of anthropomorphic figures and animals have a deeper origin in the history of modern human (Homo sapiens) image-making than recognized to date, as does their representation in composed scenes.

Paleoclimatic and environmental conditions of the Lower-Middle Ordovician carbonate reservoir in the middle of Tarim Basin (NW China): Constrained by petrographic, geochemical, and carbon–oxygen isotopic characteristics

Breakthroughs have been made recently in the exploration of hydrocarbon in the middle of Tarim Basin, NW China. The complexity of the Lower-Middle Ordovician carbonate reservoir remains enigmatic and hinders the source, developmental mechanism of the calcite fills, and paleo-environmental conditions of the Yingshan and Yijianfang Formations have been unclear. Here we present the related paleoenvironmental settings and diagenetic history, using petrographic, geochemical, and carbon (13C) and oxygen (18O) isotopic characteristics of calcite fracture, cave, and fissure fillings of a Lower-Middle Ordovician carbonate reservoir. The results indicate that the limestone caves dominantly consist of CaO, FeO, and MgO, and minor quantities of TiO2 and MnO. Among the trace elements, Ni reported the highest values (with an average of 1162.16 ppm) and other trace elements such as Ba, V, and Ga occur in minor values (with an average of 71.03, 19.18, and 12.92 ppm) respectively. The enrichment of light rare earth elements (LREEs) over the heavy rare earth elements (HREEs), with positive and negative Ce, and negative Eu anomalies indicate the contexts of oxic and anoxic conditions in seawater and sediments interface. The variation in δ18OPDB values ranges from −4.72 to −14.48‰ (with an average of −10.12‰), indicating a setting of paleo-karstification by atmospheric freshwater dissolution. While the variation in δ13CPDB indicates relatively significant values, ranging between 0.82 to −3.80‰ (with an average of −1.07‰), suggests that the paleo-karstification in this region has experienced episodes of supergene and burial diagenesis. The paleo-temperature average value of 59.1 °C, and the paleo-salinity average (Z-value) of 120.07, indicating a relatively warm paleoclimatic environment during the development of calcite-filling deposits. The results suggest several paleo-environmental conditions when paleokarst occurred: a marine deposition environment, an atmospheric freshwater karstification environment, a shallow burial paleo-karstification environment, and a high-temperature deep-burial environment in Yingshan and Yijianfang Formation in the Shunbei area. This study serves as an analog for similar carbonate systems worldwide, providing valuable insights for understanding paleo-karstification processes in different tectonic and climatic settings. Comparative studies between the Shunbei area, Tarim Basin, and other carbonate platforms contribute to our knowledge of karst evolution and its implications for reservoir development and groundwater resources.

Tribasodes kamedai Nomura is not a strictly cavernicolous species (Coleoptera: Staphylinidae: Pselaphinae)

Tribasodes kamedai Nomura, 2012 was described based on specimens collected in a limestone cave in Okinawa-jima Island. We demonstrate that this species is not a strict troglobite, but presumably a trogloxene, as it was recently collected in new localities from forest leaf litter and hollow trees. Tribasodes kamedai is redescribed in detail, with the first illustrations of female genitalia, notes on interspecific variability, and new distributional records.

Potential for limestone caves and their sediments as an onshore archive : A key to reading paleo-environment and human history

Potential for limestone caves and their sediments as an onshore archive : A key to reading paleo-environment and human history

Boea cavernarum Zich & B.Gray (Gesneriaceae), a remarkable new species from north Queensland, Australia

Boea cavernarum, restricted to near limestone crevices and caves in NE Queensland, is described. Images of whole plants, flowers, fruit and leaf indumentum, a distribution map, and a key to all species of Boea in Australia are provided. Thirteen species are now recognised in Boea.

Mechanical Properties, Tissue Structure, and Elemental Composition of the Walking Leg Tips of Coconut Crabs

The coconut crab, Birgus latro, has black protrusions on the tops of its walking legs and claw fingers. In addition, there are regularly aligned small black protrusions on parts of the exoskeleton surface of the claws and leg. In this study, the elemental composition, crystal structure, tissue structure, and mechanical properties of these protrusions were studied using a materials science approach, and the results were compared with those of mineralized cuticle. These leg tips were found to be a non-calcified fibrous tissue of α-chitin connected to the mineralized cuticle. The tip of the second walking leg was elongated and had a pointed shape with an oval cavity at its center that was more than 1000 times larger than the pore tubes (100–350 nm) of the mineralized cuticle. It was very soft, with a hardness of 0.4 GPa, corresponding to 11–12% of the hardness of the hard exocuticle and 55–57% of the hardness of the soft endocuticle. The elastic modulus of 8.0 GPa obtained by means of nanoindentation testing was consistent with that of α-chitin fibers of shrimp shells obtained by means of tensile testing. These soft protrusions provide a secure grip on the surfaces of trees or rocks and protect the claw fingertips. It was concluded that the black protrusions are related to a unique ecological (engaging in vertical movements, entering and exiting limestone caves, and escape behavior) aspect of the coconut crab, the largest terrestrial crustacean.

Detection of Lactic Acid Bacteria in Metasandstone and Limestone Caves of Chapada Diamantina, Brazil

Detection of Lactic Acid Bacteria in Metasandstone and Limestone Caves of Chapada Diamantina, Brazil

Finger Fluting in Prehistoric Caves: A Critical Analysis of the Evidence for Children, Sexing and Tracing of Individuals

Finger flutings are channels drawn in soft sediments covering walls, floors and ceilings of some limestone caves in Europe and Australia and in some cases date as far back as 50,000 years ago. Initial research focused on why they were made, but more recently, as part of a growing interest in the individual in the past, researchers began asking questions about who made them. This shift in direction has led to claims that by measuring the width of flutings made with the three middle fingers of either hand, archaeologists can infer the ordinal age, sex and individuality of the ‘fluter’. These claims rest on a single dataset created in 2006. In this paper, we undertake the first critical analysis of that dataset and its concomitant methodologies. We argue that sample size, uneven distribution of sex and age within the sample, non-standardised medium, human variability, the lack of comparability between an experimental context and real cave environments and assumptions about demographic modelling effectively negate all previous claims. To sum, we find no substantial evidence for the claims that an age, sex and individual tracing can be revealed by measuring finger flutings as described by Sharpe and Van Gelder (Antiquity 80: 937-947, 2006a; Cambridge Archaeological Journal 16: 281–95, 2006b; Rock Art Research 23: 179–98, 2006c). As a case study, we discuss Koonalda Cave in southern Australia. Koonalda has the largest and most intact display of finger flutings in the world and is also part of a cultural landscape maintained and curated by Mirning people.

Uncovering what lies within: The first scientific account of cave-dwelling ground beetles (Coleoptera: Carabidae) in the Philippines with the description of a new genus, new subgenus, and four new species from Mindanao

For the first time in the Philippines, cave surveys were carried out in search of cave beetles. In this paper we describe a new troglobite genus, Kweba gen. nov., with two new species K. magbabaya sp. nov. and K. diwata sp. nov. We describe a new subgenus: Trogloabacetus subgen. nov. of the genus Metabacetus Bates, 1892, with the addition of two new species, M. (T.) baltazarae sp. nov. and M. (T.) desideratus sp. nov. from various limestone caves of Mindanao Island. Anaulacus (s.str.) fasciatus (Schmidt-Goebel, 1846) is also reported from the cave habitats. A key to the new and related genera, images of habitus and genitalia, and distribution maps are provided.

The geomicrobiology of limestone, sulfuric acid speleogenetic, and volcanic caves: basic concepts and future perspectives.

Caves are ubiquitous subterranean voids, accounting for a still largely unexplored surface of the Earth underground. Due to the absence of sunlight and physical segregation, caves are naturally colonized by microorganisms that have developed distinctive capabilities to thrive under extreme conditions of darkness and oligotrophy. Here, the microbiomes colonizing three frequently studied cave types, i.e., limestone, sulfuric acid speleogenetic (SAS), and lava tubes among volcanic caves, have comparatively been reviewed. Geological configurations, nutrient availability, and energy flows in caves are key ecological drivers shaping cave microbiomes through photic, twilight, transient, and deep cave zones. Chemoheterotrophic microbial communities, whose sustenance depends on nutrients supplied from outside, are prevalent in limestone and volcanic caves, while elevated inorganic chemical energy is available in SAS caves, enabling primary production through chemolithoautotrophy. The 16S rRNA-based metataxonomic profiles of cave microbiomes were retrieved from previous studies employing the Illumina platform for sequencing the prokaryotic V3-V4 hypervariable region to compare the microbial community structures from different cave systems and environmental samples. Limestone caves and lava tubes are colonized by largely overlapping bacterial phyla, with the prevalence of Pseudomonadota and Actinomycetota, whereas the co-dominance of Pseudomonadota and Campylobacterota members characterizes SAS caves. Most of the metataxonomic profiling data have so far been collected from the twilight and transient zones, while deep cave zones remain elusive, deserving further exploration. Integrative approaches for future geomicrobiology studies are suggested to gain comprehensive insights into the different cave types and zones. This review also poses novel research questions for unveiling the metabolic and genomic capabilities of cave microorganisms, paving the way for their potential biotechnological applications.

Amphichorda monjolensissp. nov., a new fungal species isolated from a Brazilian limestone cave, with an update on acremonium-like species in Bionectriaceae.

Caves are unique environments characterized by spatial limitations, partial or total absence of direct light, and scarcity of organic carbon and nutrients. Caves are shelters for a variety of adapted animals and microorganisms such as fungi, many of which are still unknown. Amphichorda is a fungal genus belonging to the family Bionectriaceae, which includes cave-dwelling and entomopathogenic species with biotechnological applications. In this study, a new fungal species was identified using morphological and multi-locus phylogenetic analyses of the ITS, LSU, and TEF loci, in the Gruta Velha Nova limestone cave located in the Southern Espinhaço Range, Monjolos, Minas Gerais, Brazil. During the exposure of potato dextrose agar plates to the cave environment, an insect from the family Rhaphidophoridae passed by and fed on the culture medium, resulting in three fungal isolates. Phylogenetic analyses showed that these isolates formed a clade distinct from all known species, leading us to introduce a new species, Amphichorda monjolensis, which may be associated with this insect. Here, we also proposed two new combinations for species of acremonium-like fungi in the Bionectriaceae: Bulbithecium globosisporum (synonym: Acremonium globosisporum) and Hapsidospora curva (synonym: Acremonium curvum). The discovery of A. monjolensis highlights the potential of caves as shelters for new species with significant biotechnological importance.

Ancient Genomes From Bronze Age Remains Reveal Deep Diversity and Recent Adaptive Episodes for Human Oral Pathobionts.

Ancient microbial genomes can illuminate pathobiont evolution across millenia, with teeth providing a rich substrate. However, the characterization of prehistoric oral pathobiont diversity is limited. In Europe, only preagricultural genomes have been subject to phylogenetic analysis, with none compared to more recent archaeological periods. Here, we report well-preserved microbiomes from two 4,000-year-old teeth from an Irish limestone cave. These contained bacteria implicated in periodontitis, as well as Streptococcus mutans, the major cause of caries and rare in the ancient genomic record. Despite deriving from the same individual, these teeth produced divergent Tannerella forsythia genomes, indicating higher levels of strain diversity in prehistoric populations. We find evidence of microbiome dysbiosis, with a disproportionate quantity of S. mutans sequences relative to other oral streptococci. This high abundance allowed for metagenomic assembly, resulting in its first reported ancient genome. Phylogenetic analysis indicates major postmedieval population expansions for both species, highlighting the inordinate impact of recent dietary changes. In T. forsythia, this expansion is associated with the replacement of older lineages, possibly reflecting a genome-wide selective sweep. Accordingly, we see dramatic changes in T. forsythia's virulence repertoire across this period. S. mutans shows a contrasting pattern, with deeply divergent lineages persisting in modern populations. This may be due to its highly recombining nature, allowing for maintenance of diversity through selective episodes. Nonetheless, an explosion in recent coalescences and significantly shorter branch lengths separating bacteriocin-carrying strains indicate major changes in S. mutans demography and function coinciding with sugar popularization during the industrial period.

Discovery of Web-Building Spiders in Gua Kelam, Perlis State Park, Malaysia.

A cave represents a subterranean ecosystem that harbours a myriad of unique, peculiar, and secluded flora and fauna. These biotas have evolved with a wide range of ecological adaptations that allow them to thrive in harsh environments with limited light. Gua Kelam 1 constitutes part of the Gua Kelam limestone caves system in the Nakawan Range of Perlis State Park, Malaysia. Previous observations indicated that it harbours a plethora of spider species; however, their existence is still elusive as speleobiological studies remain unexplored. Herein, we identified the cavernicolous spiders found in the dark zone areas of Gua Kelam 1 through a complementary approach based on morphology and DNA barcoding. From the morphological analysis, we described three web-building spiders of JTKK2 and JTKK3 groups down to the species-level to belong to Nephilengys malabarensis, and Orsinome vethi except for Pholcus sp. from JTKK4 individuals. The molecular analysis of the cytochrome oxidase-I (COI) genes of JTKK2 and JTKK3 individuals showed that they exhibited a high degree similarity with N. malabarensis (98.3%), and O. vethi (100.0%), respectively except for JTKK4 individuals with only 91.4% homology with P. kuhapimuk. Phylogenetic analysis also generated a congruent tree, in which the identified species are well nested within the family Araneidae, Tetragnathidae, and Pholcidae. By this integral approach, the three spiders were determined as N. malabarensis, O. vethi, and Pholcus sp. These spiders are originally epigean in their habitat but uniquely thrive in Gua Kelam 1.