16 and Karst Environments

Natuschka M. Lee,1 Daniela B. Meisinger,1 Roman Aubrecht,2 Lubomir Kovacik,3 Cesareo Saiz-Jimenez,4 Sushmitha Baskar,5 Ramanathan Baskar,5 Wolfgang Liebl,1 Megan L. Porter6 and Annette Summers Engel7 1Department of Microbiology, Technische Universität München, Freising, ; 2Department of Geology and Palaeontology, Comenius University, Bratislava, ; 3Department of Botany, Comenius University, Bratislava, Slovakia; 4Instituto de Recursos Naturales y Agrobiologia, IRNAS-CSIC, Sevilla, ; 5Department of Environmental Science and Engineering, Guru Jambheshwar University of Science and Technology, Hisar, ; 6Department of Biological Sciences, University of Maryland, Baltimore, USA; 7Department of Geology and Geophysics, Louisiana State University, Baton Rouge, USA; now at: Department of Earth and Planetary Sciences, University of Tennessee, Knoxville, USA

16.1 Introduction a variety of different types of caves and intriguing creatures have been discov- Caves have played a fascinating role through- ered. Therefore, cave-based sciences play out the history of our planet and of our cul- an important role in enhancing our under- ture in different ways. Our first associations standing of the history of our planet and with caves centre on how they served as also form a foundation for exploring novel primitive dwelling sites for animals and concepts about the boundaries of life and the , and as settings for mysterious fan- evolution of extreme dark life ecosystems on tasies and myths (Fig. 16.1). Today, scien- Earth, as well as in other parts of the Universe tific exploration has added another aspect (Krajick, 2001; NOVA, 2002; SPACE/Malik to our connection to caves by revealing a and Writer, 2005; Forti, 2009). plethora of unexpected insights into diverse disciplines, including the natural sciences (geology, palaeontology, climatology, phys- 16.2 Description of Caves: Definition, ics, chemistry, biology and cosmology), medical sciences and engineering, as well Distribution and Biogeochemistry as social disciplines such as archaeology, theology, and the cultural history of man- 16.2.1 Introduction kind (Fig. 16.2). The foundation for this is the extreme nature of caves, characterized Many different definitions have been used by a lack of light and geographic isolation, to describe a cave or a cavern. The most but also by nutrient limitation and a range general way to describe a cave, irrespective of extreme redox conditions. In recent years, of its geological history and location, is to

© CAB International 2012. Life at Extremes: Environments, 320 Organisms and Strategies for Survival (ed. E.M. Bell) Caves and Karst Environments 321

Fig. 16.1. Entrance zone of a cave: Cascade Caverns, Carter Caves State Resort Park, Kentucky, USA. Depending on the entrance morphology, sunlight only penetrates a limited distance into the cave. © Annette S. Engel.

simply define it as a natural cavity in a rocky hundreds of kilometres in length. Only a environment where at least some part of it is few of these are accessible to humans. In fact, in total darkness. The science of exploration many cave openings consist only of micro- of caves and various karst features is termed scopic fractures. Despite numerous (Gunn, 2004). Speleology is a activities all over the world, it is estimated broad interdisciplinary science; a multitude that, even in well explored areas like Europe of parameters and disciplines needs to be and North America, so far only 50% of all considered for a thorough exploration of caves in these regions have been accessed; the development of caves and their various globally, only ~10% of all caves have been impacts on their surroundings worldwide discovered (Eavis, 2009; Engel, 2011). The throughout history. subsurface can be regarded as one of the least Caves are developed in soluble rocks explored environment types on Earth, second and constitute a characteristic feature of only to the deep oceans. Caves often serve as karst (carbonate rock, such as and the only available natural entrances and con- dolomite) and pseudokarst (non-carbonate nections to the subsurface, offering fascinating rock) landscapes that covers roughly windows into this vast and unexplored habitat. 15–20% of the Earth’s ice-free land surface Because of the increasing interest in cave (Ford and Williams, 2007). Although caves sciences and all the promising prospects they are found in various regions of the Earth and offer, plus the development of improved cav- at all latitudes, caves are generally not inter- ing technology that allows access to even the connected over large physiographic prov- most difficult caves, many novel types of inces, limited by the extent of rock type. caves have been discovered during the last Caves come in a wide range of shapes and decade (Eavis, 2009). Son Doong Cave, sizes, from micro-fissures to caverns several , found in 2009, contains the largest thousands of metres deep and high, and discovered cavern to date, measuring up to 322 N.M. Lee et al.

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Fig. 16.2. (a) Grotta dei Cervi, (discovered in February 1970), with black wall paintings, made with bat guano. © Cesareo Saiz-Jimenez. (b) Castle in front of cave, Predjamski Grad, . © Annette S. Engel.

140 by 140 m and 4.5 km in length. The cave known, and was discovered in deepest cave, Krubera Cave, , near in 2004 at an altitude of nearly 5000 m. the Black Sea, was discovered in 2001 and Several fascinating underwater caves have extends to a depth of −2191 m into the sub- also been described, such as those in the surface. Qaqa Mach’ay Cave is the highest Yucatan Peninsula, , and the Nullarbor Caves and Karst Environments 323

Plain, ; each area contains hun- However, it is not only the size and dreds of kilometres of submerged passage- depth of caves that are impressive (Figs 16.3 ways. However, even caves that have been and 16.4); many of their contents undisput- known about and explored for many years, edly continue to capture our imagination e.g. Mammoth Cave, Kentucky, USA, also and curiosity, e.g. their spectacular spele- continue to reveal astonishing insights. othems (cave rock and mineral formations), Due to natural karstification as well as such as stalactites, stalagmites and giant continued exploration, Mammoth Cave is crystals that may be over 70 m in length, now estimated to cover a distance of around and fascinating creatures belonging to 600 km, and as such represents the longest unique, extreme ecosystems (Taylor, 1999; cave known on Earth (for a summary of Krajick, 2001; Culver and Pipan, 2009; Eavis, spectacular cave types, see NSS GEO2 com- 2009, http://www.canyonsworldwide.com/ mittee, 2011). crystals/mainframe3.html).

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Fig. 16.3. Examples of spectacular constructions in different types of caves. (a) The large room of Škocjan Caves Regional Park, Slovenia, a UNESCO site. This photo highlights the massive size that speleothems can reach (people for scale in the lower left). © Annette S. Engel. (b) Unique formations of unknown nature (possibly biospeleothems) inside a sandstone cave in Venezuela (Aubrecht et al., 2008). © Jan Schloegl and Roman Aubrecht. (c) Entrances to caves can be very small and embedded in water; Cascade Caverns, Carter Caves State Resort Park, Kentucky, USA. © Annette S. Engel. (d) Example of different colour formations due to various microbiological redox processes. Sulfidic spring in a cave with toxic hydrogen sulfidic gases, Lower Kane Cave, Wyoming, USA. © Annette S. Engel. 324 N.M. Lee et al.

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Fig. 16.4. Different types of speleothems and biospeleothems in a sandstone cave in Venezuela. (a–c) sandstone cave in Venezuela, © Roman Aubrecht. (d) Driny Cave in Slovakia, © Lubomir Kovacik.

16.2.2 Classification and formation of caves Although new caves are constantly being formed somewhere on Earth, the Caves are classified using several criteria majority of the caves so far described and (Northup and Lavoie, 2001; Gunn, 2004; explored have most likely persisted for Engel, 2011): the solid rock/bedrock type, longer periods, from thousands to millions the proximity to the groundwater table, the of years. Irrespective of their speleogenetic overall passage morphology and organiza- histories, all caves are constantly changing tion (i.e. size, length, depth, routes of fluid over time, either by natural karstification flow, etc.) and the speleogenetic history processes or simply due to continued explo- (related to the origin and development of ration. Depending on the proximity to the caves). So far, at least 250 different minerals groundwater table, two different general have been described from karst and pseu- modes of cave formation can be discerned: dokarst settings (Hill and Forti, 1997). (i) epigenic caves, as the most commonly However, the most common rock types are described caves, are related to surface pro- calcareous rocks (e.g. limestone, which cesses by formation at or proximal to the underlies about 15% of Earth’s surface) and groundwater table, and are critically depend- basaltic rock (e.g. lava tubes). Other less ent on the hydrological conditions of the common rock types include other volcanic region; and (ii) hypogenic caves, which are deposits, gypsum, granite, quartzite, sand- related to subsurface processes because they stone, salt and even ice. form by the action of rising fluids, such as Caves and Karst Environments 325

water or gases, at or below the water table 3. Volcanism leading to lava flows that (Engel, 2011). The most commonly described produce lava tube caves (Halliday, 2004), hypogenic caves to date are sulfidic caves, which first may result in sterilized surfaces which are formed by a combination of abi- after a volcanic eruption that may then be otically and microbially produced sulfuric colonized through time. Examples of exten- acid (Engel, 2007). Irrespective of the cave’s sive lava tube caves have been described for relation to surface or subsurface processes, the Kilauea Volcano, , USA. the character of the void spaces and the 4. Physical weathering by water, which fluid flow patterns are crucial to the con- results in the formation of sea (littoral) caves tinuous evolution of the cave, as these pro- due to the constant pounding of waves and duce the foundation for all flow systems digging out of seashore cliffs (Bunnell, 2004). and circulations relevant for the various 5. Anchialine caves along the coast are speleogenetic processes. formed by the constant solubilization of Depending on rock type, and geochemi- host rock at a freshwater–saline water cal and geophysical (including climatic) interface. conditions, at least six different speleoge- 6. Ice (glacier) caves are formed by streams netic pro cesses may take place (Northup and that erode tunnels under and through gla- Lavoie, 2001; Gunn, 2004; Engel, 2011). ciers (Gunn, 2004; Fig. 16.5). 1. Solubilization of the host rock and the precipitation of minerals, which may even- (a) tually initiate the formation of speleothems. Speleothems are secondary mineral deposits and may contain a number of different minerals or unconsolidated materials such as clays and organic matter. So far, at least 38 different types of speleothems have been described (e.g. stalactites, stalagmites, helic- tites, cave pearls, curtains of dripstone, flow- stone, rimstone, pool fingers, etc.), based on the formation mechanism and dominant (bio)-geochemical reactions (Hill and Forti, 1997; Boston et al., 2001; Self and Hill, 2003; Melim et al., 2009; Lavoie et al., 2010; see the website of the NOAA Paleoclimatology Speleothem (2011) for information about the (b) value of speleothems for paleoclimatology research; Fig. 16.4). Examples of caves con- taining a variety of different speleothem types include Mammoth Cave, Kentucky, USA, and Castañar de Ibor Cave, Spain. 2. Solubilization of the host rock from sulfuric acid-driven speleogenesis, which may dissolve calcareous rocks to generate significantly large caverns and often are associated with active microbiological colo- nization if reactive solutions are still present (Engel, 2007). Examples of caves formed from sulfuric acid speleogenesis include Carlsbad Cavern, New Mexico and Lower Fig. 16.5. (a) Isfjellelva Cave, Vestre Torellbreen, Kane Cave, Wyoming, USA, as well as Svalbard, © Stanislav Rehak. (b) Tone Cave, Movile Cave, . Tonebreen Glacier, Svalbard, © Stanislav Rehak. 326 N.M. Lee et al.

16.2.3 The cave environment In contrast to surface habitats, condi- tions in the deeper parts of caves are gener- Several parameters contribute to the forma- ally more stable (i.e. stable temperature tion of the cave environment (Northup and throughout the year), but however stable, Lavoie, 2001; Gunn, 2004; Barton and the conditions can also be considered more Northup, 2007; Engel, 2011). Intergranular extreme simply because it is constantly dark spaces, pores, joints, fractures and fissures, in this zone, thereby making photosynthesis and dissolutionally enlarged conduits and impossible. As such, deep cave environments cave passages form a porosity and permea- are generally considered to be extremely bility continuum that can be colonized by oligotrophic (nutrient poor) because many organisms. An opening connected to the of the resources needed for surface-based surface can have three major zones based on ecosystems, such as light and organic mat- light penetration and intensity: (i) the ter, are limited. Some systems with direct entrance zone, which is exposed to full sun- hydrological connections to the surface may light and experiences the daily light cycle; occasionally be subject to catastrophic events, (ii) the twilight zone; and (iii) the dark zone, such as floods (Fig. 16.7), whereas caves in where no light penetrates (Fig. 16.6). Different deserts may undergo long periods of types of life, and subsequently adaptations drought, and lava tube systems may experi- expressed by that life, are generally corre- ence renewed volcanic activity. For hypo- lated to these zones because of specific genic caves, there may also be a range of physico-chemical and geochemical condi- hazardous conditions, including toxic con- tions related to photic gradients. Entrance centrations of inorganic compounds such as and twilight zone conditions are tolerable sulfur, heavy metals, lethal gases, or radio- to a wide variety of organisms, from insects activity (Fig. 16.8). Depending on the rock to vertebrates, with little modification to type, caves can also have extreme pH and their overall lifestyle. redox gradients, such as the interface between

How Cave Biology Works @2009 HowStuffWorks

Cave Zones

Entrance Twilight Dark Zone Zone Zone Sunlight Less light No light Variable Minor temperature Constant Temperature changes temperature Green vegetation Minimal plant life

Fig. 16.6. The three zones (entrance, twilight, dark) of a cave (graph from www.howstuffworks.com). Caves and Karst Environments 327

Fig. 16.7. Some cave systems with direct hydrological connections to the surface may occasionally be subject to catastrophic events, such as floods. Here, scientists wade through a flooded cave to reach their sampling site. © Annette S. Engel.

Fig. 16.8. Castañar de Ibor, Spain, is a cave with extremely high radon (222Rn) concentrations reaching up to 50,462 Bq m−3. Cigna (2005) studied 220 caves around the world and calculated an annual average value of 2500 Bq m−3, well below the range of Castañar de Ibor Cave. © Sergio Sanchez-Moral. 328 N.M. Lee et al.

the host rock and cave passage atmosphere, also Gómez et al., Chapter 26, this volume). or rock and water. The rock itself may con- Therefore, cave research has been used to tain various reduced compounds that create enhance our understanding of the mecha- significantly different redox gradients within nisms of biological adaptation to extreme short distances. These redox-variable environ- conditions, the interactions between organ- ments play a crucial role in the development of isms and minerals, the role of inorganic complex micro- and macro-biological com- matter in different dark environments, the munities that each have differing impacts on evolution and speciation of biological sys- the speleogenetic history of a cave system tems under extreme conditions, and also (Northup and Lavoie, 2001; Gunn, 2004; led to various biotechnological applications Engel, 2011). (i.e. screening of novel enzymes or pharma- ceutical research).

16.3 The Biology of Caves 16.3.2 Life in caves 16.3.1 History of biological cave research All kinds of life forms (i.e. viruses, bacteria The first biological studies in caves were (including cyanobacteria), fungi, algae, pro- initiated in the Middle Ages in Europe and tists, plants, animals) have been found in . In the centuries that followed, the caves (Culver and Pipan, 2009; Romero, biology of caves only evoked interest among 2009), in either an active or fossilized state a limited group of specialists (Culver and and in a range of different types of habitats Pipan, 2009; Romero, 2009; Engel, 2010). (i.e. rocks, springs, pools, cave walls (Fig. 16.9), In the late 1970s to early 1980s dark life or even dispersed in the air). Depending on ecosystems were discovered at hydrother- the environmental conditions, cave organ- mal vent systems in the Atlantic Ocean (see isms can be either motile or sessile, and can also Lutz, Chapter 13, this volume), provid- be directly or indirectly associated with ing insights into the evolution of life adapted other organisms in symbiotic associations to extreme and dark conditions. Shortly (e.g. parasitic or mutualistic). In general, after this marine discovery the extreme ter- ecological interactions profoundly influ- restrial dark life ecosystem in the Movile ence the development and maintenance of Cave in Romania was described (Sarbu et al., cave ecosystem dynamics and food webs. 1996). Both habitats (vents and caves) Such interactions can include how chemo- revealed astonishingly rich ecosystems of lithoautotrophic microorganisms form the various faunal species, geochemically fuelled base for some subsurface ecosystems, whereby by novel species of microorganisms. From rich and diverse higher level organisms are this time forward, the modern era of extreme sustained, but also can include how bat environment research in caves began. With guano in some caves provides a significant the further development of caving technol- energy and nutrient source for many other ogy and molecular biological tools that organisms (Figs 16.10 and 16.11b). enabled the exploration of previously inac- Depending on the location and the con- cessible caves and of unculturable, novel ditions in the cave, organisms may be either species, the number of discoveries of non-residents or permanent residents (Culver extreme ecosystems in different types of and Pipan, 2009; Romero, 2009). Non- caves has consistently increased. Based on resident organisms, referred to as acciden- the investigations conducted to date, it has tals, enter a cave occasionally via wind, become obvious that caves may serve as excel- water (groundwater, sea spray, rain water), lent model systems for several fundamental or air, as sediment or spores, or can even be biological disciplines, in particular geobiol- carried into a cave by other animals. ogy and astrobiology (Taylor, 1999; Boston Depending on their actions and length of et al., 2001; Engel and Northup, 2008; see stay in the cave, they may have a profound Caves and Karst Environments 329

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Fig. 16.9. Microbial growth on walls in caves: (a) colonies of white and gold actinomycetes and other bacteria on a cave wall, Slovenia, © Annette S. Engel; (b) fungal snottites from Sharps Cave, West Virginia, USA, © Megan Porter; (c) cyanobacterial growth, Cave Bojnice, Slovakia, © Lubomir Kovacik; (d) biovermiculations on the cave wall in the Frasassi Caves, Italy. These features, composed of clays and organic matter, are thought to be formed by microbial and nematode activity, © Annette S. Engel. influence on the resident populations. The and groundwater, such as metals, sulfur and mechanism for the origin of permanent methane (Engel, 2010). Other examples residents is still under debate; the most include higher organisms that adapted to plausible explanation is that they originally the subterranean environments via various descended from the surface, and entered the metabolic and morphological adaptations, subsurface either accidentally or were forced such as pigment loss, eye loss, wing loss, underground by catastrophic events. Once reduction in size, development of more sen- below, they adapted to the conditions. sitive sensory organs, limb extension, reduc- Depending on the available energy tion in metabolic rates and increased source over time and the survival ability of longevity (Culver and Pipan, 2009; Romero, the species, different types of adaptations in 2009; Fig. 16.11a–d). Interestingly, a broad response to the cave environment are likely taxonomic range of organisms obligately to have taken place for at least certain spe- adapted to the subsurface, in either terres- cies. A good example of this are microbial trial (troglobionts) or aquatic (sytgobionts) species that adapted metabolically to a strict habitats, share this suite of characters, chemolithoautotrophic life style, living off referred to as troglomorphy. These obligate inorganic compounds present in the rocks subsurface organisms generally also have 330 N.M. Lee et al.

How Cave Biology Works The Cave Food Pyramid

Predators Centipedes, cave spiders, salamanders

Omnivores and Herbivores

Microorganisms Millipedes, crustaceans, and Decomposers amphipods, planarians

Organic material, guano, fungus, microscopic bacteria

2009 HowStuffWorks

Fig. 16.10. The cave food pyramid (graph from www.howstuffworks.com). limited possibilities for dispersal, which can viruses in caves: (i) many prokaryotes in further constrain genetic populations to various types of extreme ecosystems are in local, and rarely regional, hydrostratigraphic general attacked by viruses (e.g. see exam- regions. ples in Rainey and Oren, 2007); (ii) large numbers of novel viruses have been detected Viruses in the subsurface and are thus postulated to play a crucial role (e.g. Kyle et al., 2008); Viruses are the most abundant type of ‘bio- and (iii) several infectious viruses have been logical entity’ on Earth, being found wher- reported from caves, in particular from ever there is life, and have probably existed animals like insects or mammals residing since the first cells evolved. Viruses are in caves. A classic example of this comes capable of infecting all types of organisms, from bats: the animals may themselves be from prokaryotes to plants and animals. It is attacked by viruses, such as the West Nile evident that viruses have had and still have virus, or alternatively serve as significant a strong impact on virtually all evolutionary reservoirs of viruses that infect humans and and ecological processes (Abedon, 2008; other animals, e.g. emerging zoonotic Forterre, 2010). Unfortunately, there have viruses, such as lyssaviruses, filoviruses been no detailed, holistic studies on the and paramyxoviruses (Quan et al., 2010). ecology of viruses in caves or on the role of Furthermore, most of the outbreaks of viruses or the mechanisms of their impact. hemorrhagic fever caused by the lethal There are a few, sporadic reports of high Marburg virus in humans and associated abundances of viruses in various extreme with visits to caves and mines in Africa environments or caves, with at least three (Kuzmin et al., 2010). The reason for this is related categories of case study pointing unknown because the source of the initial toward the potentially high significance of infection is unclear, but a reasonable lesson Caves and Karst Environments 331

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Fig. 16.11. Examples of cave animals: (a) the amphibious non-pigmented isopod Titanethes albus, from Planinska Jama, Slovenia, © Megan Porter; (b) dead cave frog overgrown by fungi, Photographer Peter Luptacik, © Elsevier; (c) the blind amphibious, non-pigmented, cave salamander Proteus anguinus, Slovenia, © Megan Porter; (d) jaw of the Late Pleistocene cave bear Ursus spelaeus, Krizna Jama, Slovenia, © Megan Porter. to be learned from this case is that the evo- The first microbiological studies in caves lution of a rather special gene pool (in this were performed in the late 1940s using pre- case viral) in isolated portions of caves may dominately microscopy and enrichment have profound impacts on other organisms techniques, and these approaches contin- once they enter these isolated areas. Thus, ued for almost three decades. The research research into the unique, isolated ecosys- revealed few spectacular insights. For exam- tems presented by caves may yield many ple, these studies demonstrated that microbes interesting and relevant insights into a were prevalent but not as diverse as in sur- number of other biological disciplines. face habitats, and that several geological processes, such as speleothem formation, Bacteria and Archaea cave deposits such as saltpetre and moon- milk (Fig. 16.12), iron oxidation (Fig. 16.13) The prokaryotes, which comprise the domains and sulfur oxidation, may be microbially Bacteria and Archaea, are the most abun- mediated (Northup and Lavoie, 2001; Barton dant group of organisms on our planet. It is and Northup, 2007; Engel and Northup, 2008; well understood that these microbial groups Engel, 2010). Unfortunately, quantitative have played a key role in the development and undisputable evidence for these hypoth- of our planet since the early beginnings. eses was missing from these earlier studies. 332 N.M. Lee et al.

Fig. 16.12. Moonmilk in Altamira Cave, Spain, most likely of biological origin (Cañaveras et al., 2006).

Furthermore, because standard microbio- biogeography and endemism, as well as logical approaches are only capable of iden- microbial adaptation mechanisms to extreme tifying culturable species and these showed conditions without light. One hypothesis that several identified cave microbes were being evaluated suggests that older caves similar to surface-derived groups, e.g. from may serve as a long-term reservoir of microbes soils, it was assumed that the microbes in the subsurface and thus offer unique pos- identified in the caves had merely been sibilities to explore the vast unknown biodi- transported into the cave. The conclusion versity of the subsurface. In this way, cave was, therefore, that no unique cave microbes research provides analogues for marine and existed. deep-sea hydrothermal vent systems and Fortunately, with the development of possible life on other planetary bodies molecular and other analytical techniques (Northup and Lavoie, 2001; Barton and since the 1980s, the exploration of non-cul- Northup, 2007; Engel and Northup, 2008; turable organisms has become possible. Engel, 2010). Despite the fact that only ca. 10% of all Many different types of biogeochemical caves discovered so far have been biologi- reactions driven by microorganisms have cally explored, many different types of bac- been observed from distinct ecological cave terial and archaeal groups have now been zones (e.g. ammonification, denitrification, detected in caves (Engel, 2010). Evidence nitrification, sulfate reduction, anaerobic has mounted that these cave microbes may sulfide oxidation, metal oxidation, metal indeed be unique and genetically divergent reduction, methane cycling, photosynthe- from surface groups, which has important sis; Northup and Lavoie, 2001; Barton and implications regarding the role of microbes Northup, 2007; Engel and Northup, 2008; in distinct geochemical and geobiological Engel, 2010). Depending on the rock type, processes. Consequently, several hypothe- the concentration and nature of electron ses have been proposed that address ques- donors and acceptors, availability of oxygen tions related to surface–subsurface linkages, and flux of organic material derived from Caves and Karst Environments 333

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Fig. 16.13. Precipitates of biological origin produced by various cave bacterial species: (a) biogenic iron precipitated by a dense microbial community on Leptothrix sheaths, Borra caves, India; (b) calcium carbonate precipitate. Calcite crystals precipitated in vitro by Bacillus pumilis isolated from Sahastradhara caves, Dehradun, India (Baskar et al., 2006). © Sushmitha Baskar and Ramanathan Baskar. 334 N.M. Lee et al.

the surface, chemolithoautotrophs and het- planktonic life stages to impressive aggre- erotrophs play different ecological roles. gates, such as biofilms, forming either mas- Extreme environmental conditions also influ- sive microbial mats on cave springs or pools ence the metabolism of oligotrophic, acido- (Fig. 16.14), or microbial draperies (‘snot- philic, thermophilic and/or sulfidophilic tites’) on cave wall surfaces (Fig. 16.9). species. Different types of growth patterns An important question that is being may be observed, including single-celled, investigated by current research is what the

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Fig. 16.14. Microbial mats in caves: (a) white filamentous microbial mats in the sulfidic stream of the Pozzo di Cristali in the Frasassi Caves, Italy, © Annette S. Engel; (b) black microbial mats in the sulfidic stream of the Lower Kane Cave, Wyoming, USA, © Natuschka M. Lee; (c) orange microbial mat with iron oxidizing bacteria growing on the flow water dripping out from a drill hole (the subsurface hard rock laboratory, Äspö, Sweden), © Natuschka M. Lee; (d) fluorescence in situ hybridization of different types of S-oxidizing filamentous bacteria (novel Epsilonproteobacteria, Thiothrix and other unknown species) in a microbial mat in Lower Kane Cave, Wyoming, USA, © Natuschka M. Lee. Caves and Karst Environments 335

source and transportation modes for micro- in caves. Microbes may promote these pro- organisms are that bring cells into, distrib- cesses in either an active (e.g. by enzymes) ute cells throughout, and carry cells out of, or passive way. Organisms (live or dead) or cave environments. Possible inoculation their products, such as extracellular sub- sources include soil, water (i.e. groundwater, stances (EPS), serve as nucleation sites for sea spray and rainwater), plants, animals, chemical reactions by sorbing various com- deep-seated fluids circulating in sedimen- pounds (e.g. metals to amphoteric func- tary basins, and possibly even the rock tional groups such as carboxyl, phosphoryl itself. Microbial transport is itself linked to and amino constituents, or on to negatively various circulation systems, such as vertical charged cell wall surfaces, sheaths or cap- and horizontal fluid flow, which affects sules). These biochemical functional groups retention in zones of slow movement and provide additional sites for chemical inter- circulation. Slow movement is strongly actions, reduce activation energy barriers, influenced by sorption onto biofilms that change the system pH, or remove solutes form on nearly every solid and semi-solid from solution by causing solid phases, like surface (e.g. rocks or the shells of macro- minerals, to precipitate. Precipitation proc- organisms; Northup and Lavoie, 2001; Barton esses result in the formation of different and Northup, 2007; Engel and Northup, types of products of various sizes, from 2008; Engel, 2010). microscopic structures to large speleothems, As in other ecosystems, biogeochemi- and compositions (e.g. calcium carbonate cal processes in caves are controlled by a (Fig. 16.13) such as moonmilk (Fig. 16.12) complex interaction between geochemistry, or silicates and clays, iron and manganese geophysics and system ecology (for a more oxides, sulfur compounds, or nitrates detailed review, see Northup and Lavoie, such as saltpetre). Microbially influenced 2001; Barton and Northup, 2007; Engel and dissolution and corrosion processes can be Northup, 2008; Engel, 2010). This makes it mediated by iron-, manganese- and sulfur- difficult to distinguish between abiotically oxidizing bacteria, occurring via mechani- and biogenically driven processes in the cal attachment and secretion of exoenzymes cave environment. None the less, microbes or from organic or mineral acids (e.g. sulfu- have been shown to influence many geo- ric acid) that generate considerable acidity chemical processes in caves at various stages (Northup and Lavoie, 2001; Barton and of speleogenesis, because microbes are con- Northup, 2007; Engel and Northup, 2008; sidered to be agents of concentration, Engel, 2010). whereby they localize the accumulation of The number of bacterial and archaeal inorganic minerals (e.g. CaCO3 deposits such 16S rRNA gene sequences retrieved by as moonmilk, or FeS2 formation from sulfate standard clone libraries and pyrosequenc- reduction); dispersion, whereby they initiate ing from various caves thus far constitute the solubilization, mobilization and disper- only a small fraction of all 16S rRNA gene sion of insoluble minerals (e.g. Fe(III)-oxide sequences retrieved from the environment reduction); fractionation, whereby they on a global basis (Engel, 2010). Despite this preferentially use one component in a mix- fact, several interesting insights have been ture, resulting in the fractionation of ele- gained. Approximately half of the bacterial ments and isotopes; and reduction, whereby phyla, and less than half of the archaeal they form new compounds due to the use of phyla, are identifiable to a certain extent. certain other compounds (e.g. acids from The rest of the 16S rRNA gene sequences so respiration (H2CO3), from S0 oxidation far retrieved from cave and karst environ-

(H2SO4), or H2S production from sulfate ments represent novel, so far unculturable reduction) (Ehrlich, 1996). species with unknown function. However, With this understanding, the focus of certain patterns can be discerned within a previous research has been to distinguish number of known phylogentic groups. For what effects and interactions microbes have instance, some epigenic caves appear to on precipitation and dissolution processes contain Deltaproteobacteria, Acidobacteria, 336 N.M. Lee et al.

Nitrospira and Betaproteobacteria, whereas Eukaryotes some hypogenic caves, such as sulfidic caves, appear to be dominated either by Many different types of eukaryotes have Epsilonproteobacteria, or Gammaproteo- been detected in caves worldwide, from single- bacteria and Betaproteobacteria in microbial celled species, such as different types of mats, or by Acidimicrobium, Thermoplasmales, Protozoa, to a large variety of multicellular Actinobacteria, bacterial candidate lineages, species ranging from fungi, invertebrates and some Archaea in ‘snottites’ on cave wall (flatworms, annelids, millipedes, centipedes, surfaces (Engel, 2010; Fig. 16.15). diplurans, insects, collombolans, spiders, Along with this, several different bacte- mites, crustaceans, scorpions) and verte- rial genera, such as Firmicutes, Bacillus, brates (amphibians, reptiles, fish, mammals Clostridium and various enteric bacteria/ such as bats). For extensive details, see indicator bacteria have been discov- Culver and Pipan (2009). Actively growing ered in cave systems that are believed to plants, algae, or microscopic phototrophs result from some kind of contamination via, are only found in cave entrances, in streams for example, local wastewater treatment or other locations where sufficient amounts plants, storm events, or visitors. Fortunately, of light are available, whether natural or arti- many of these microbial contaminants have ficial due to cave lighting, where as little as a low persistence in the cave environment, 1 μmol photon m−2 may support growth indicating that caves may have the potential (Grobbelaar, 2000). to recover from short-lived, occasional con- Depending on the location within a tamination events. Nevertheless, there are cave zone, adaptation level and residence also a number of caves, especially caves time in the caves, organisms may be classi- with valuable rock-art paintings, that have fied as trogloxenes – terrestrial organisms had enormous difficulty recovering from that use the cave merely for occasional shel- invasive species, such as heterotrophic bac- ter, such as bats – or troglophiles – terrestrial teria, phototrophs and fungi, spread by organisms that may complete their life cycle improper cave management, insects, or ani- in the cave but are still able to survive out- mals (Section 16.4). side the caves. Many troglophiles therefore Further research is needed to resolve maintain at least some of their original the questions remaining about the presence senses. Stygoxenes are aquatic trogloxenes of endemic and invasive microbial species and stygophiles are aquatic troglophiles. in caves and their possible roles. Despite all Trogloxenes and troglophiles are usually developments in terms of molecular and found in the entrance or the twilight zone microbial analytical tools, only a fraction of (Howarth, 1980; Culver and Pipan, 2009). the real taxonomic and functional diversity Troglobites and stygobites, obligate ter- of Bacteria and Archaea in caves has been restrial and aquatic subsurface-dwellers, thoroughly and appropriately described. respectively, have developed astonishing This is due to several methodological prob- adaptation mechanisms to life in caves lems, ranging from inadequate sampling (Culver and Pipan, 2009). Troglobites are gen- technology, insufficient distribution data, erally exclusively found in the deeper parts of and the lack of appropriate methodology to the caves where it is permanently dark and address a research question. Testing specific the humidity often high (up to 95–100%). hypotheses requires modifications to tradi- Aquatic stygobites are generally geographi- tional approaches using more holistic, full- cally more wide-ranging than troglobites, and cycle methods, in order to more truly stygobites are more commonly found in caves identify, quantify and determine the func- with tropical and subtropical climates than tion and activity of culturable, as well as troglobites (Lamoreux, 2004). Both groups unculturable, species and thus provide cor- have developed impressive morphological relations and evidence of the role of adaptations, as well as physiological mecha- microbes in different types of speleogenetic nisms, to survive e.g. darkness, high humidity processes. and limited food supplies. Typical food Caves and Karst Environments 337

(a)

33 34 35 32 1 31 11 36 10 29 30 12 28 2 47 52 54 27 55 3 48 51 53 13 26 37 49 4 21 50 5 15 24 58 1425 23 22 38 16 45 6 39 43 44 57 7 8 56 17 40 42 41 9 18

19 46 20 59

60

cave type (simplified): Main categories – symbol code: non-sulfidic sulfidic cave Mineralogy – colour code: ice orthoquartzite lava miscellaneous limestone

number cave name, country seq./diversitya number cave name, country seq./diversitya 1 Wind Cave, SD, USA 83/14 31 Swallow hole, Yverdon-les-Bains, 134/>10 Switzerlandb 2 Hellespont Cave, WY, USA 11/1 32 Scladina case (cave layer), 63/10 3 Lower Kane Cave, WY, USAb, (→ Fig. >1500/>17 33 Herrenberg Cave, Germanyc 9/3 16.3d; 16.14b, d) 4 Glenwood Springs Cave, CO, USA 334/14 34 Alpine Ice Caves, Werfen, Austriab, c 2/1 5 Fairy Cave, CO, USA 37/6 35 Domice Cave/, Slovakia 52/3 6 Kartchner Cave, AZ, USAb, c 164/10 36 Movile Cave, Romaniab 372/14 7 Millipede Cave, AZ, USA >12/1 37 Pajsarjeva jama Cave, Sloveniab 50/11 8 Carlsbad Lechuguilla Cave and Spider 46/9 38 Frasassi Cave system, Italy (→ Fig. 1039/>18 Cave, NM, USAb 16.9d; 16.14a) 9 Hinds Cave, TX, USA 50/4 39 Cave of Acquasanta Therme, Italyb 173/11 10 Mammoth Cave, KY, USAb 254/10 40 Saint Callixtus Catacombs, Rome, Italyb 8/1 11 Tytoona Cave, PA, USA 13/7 41 Grotta Azzurra of Palinuro Cape, Italyb 39/10 12 Parker Cave, KY, USA 17/4 42 Grotta dei Cervi, Italyb, c (→ Fig. 16.2a) 2/1 13 Cesspool Cave, VA, USAb 129/>6 43 Cave Koutouki, Attika, Greecec 1/1 14 Limestone Cave, KY, USA 4/2 44 Cave Kastria, Peloponnese, 15 Big Sulfur Cave, KY, USA >30/1 45 Shallow-water Cave, Paxos, Greece 148/9

16 Blowing Spring Cave, AL, USA 9/4 46 Lime Cave, Baratang, Indiab, c 1/1 17 South Andros Black Hole Cave, 3/1 47 Meghalaya Caves, Indiac 3/2 Bahamasb, c 18 El Zacaton sinkhole, Mexicob 1589/27 48 Thai Cave, Thailandb, c 9/1 19 Roraima Sur Cave, Venezuelab 42/9 49 Pha Tup Cave Forest Park, 2/1 20 Gruta da Caridade, Rio Grande do 2/1 50 Reed Flute Cave, Guilin, Guangxi, 2/1 Norte, Chinab, c 21 Cave Papellona, Barcelona, Spainb, c 1/1 51 Niu-Cave, China 142/14 22 Ardales Cave, Spain (→ Fig. 16.16) 19/7 52 Heshang Cave, China 111/13 23 Santimamiñe Cave, Spain 10/10 53 Cave Apatite Deposits,South Korea 65/7 24 Covalanas, Cantabria and La Haza 325/3 54 Pseudo-limestone Cave, South Korea 2/1 Caves, Spain 25 Lava Tubes, Azores, Terceira, 515/>10 55 Gold Mine Caves, Kongju, South 4/2 Koreab, c

Fig. 16.15. (a) Worldmap showing all caves which have been explored with molecular biological methods. (b) 16s rRNA phylogenetic tree showing nearly full sequences retrieved from various caves all over the world. Abbreviations: BPB/GPB = Betaproteobacteria/Gammaproteobacteria; DPB = Deltaproteobacteria; APB = Alphaproteobacteria; EPS = Epsilonproteobacteria; WS3 and OP 10 = novel candidate divisions. 338 N.M. Lee et al.

26 Altamira Cave, Spainb, c (→ Fig. 16.12) >581/12 56 Ryugashi Cave, Shizuoka, 1/1 27 Monedas and Chufin Caves, Spain 125/10 57 Cave Sabichi, Ishigakijima, Japanb 1/1 28 Llonin Cave and La Garma Cave, Spain 85/10 58 Jomon and Joumon Limestone Caves, 9/2 Gifu, Japanb, c 29 , Spain (→ Fig. 16.17) 41/10 59 Lava Caves and Tubes, HI, USAb 302/>18 30 Cave, 681/>4 60 Nullarbor Caves, Australia 52/8

a: The ratio demonstrates the total amount of 16S rRNA gene sequences from the domains Bacteria and Archaea versus the amount of identifiable phyla. This information is based on ~ 8,000 partial 16S rRNA gene sequences retrieved from the Gene bank (http://www.ncbi.nlm.nih.gov/) in February 2011. A detailed list of the sequences and the references can be downloaded from the website http://www.microbial-systems-ecology.de/Lee_et_al_2011_cave.html b: A representative selection of these sequences (> 1,400 nucleotides) were used to calculate an overall 16S rRNA phylogenetic tree (see figure 16.15b and http://www.microbial-systems-ecology.de/Lee_et_al_2011_cave.html) showing all so far retrieved 16S rRNA gene sequences from different parts of the world.

c: This study also describes 16S rRNA gene sequences from isolates.

(b)

Actinobacteria

BPB / GPB OP10

Spirochaetes

Firmicutes

Cyanobacteria

EPB

OUTGROUPS APB Nitrospira

Acidobacteria

WS3

Chlorobi / Bacteroidetes

Planctomycetes DPB

DPB

Verrucomicrobia

Acidobacteria

Chloroflexi

Fig. 16.15. Continued. sources may include microorganisms, other Because of their low metabolic rates, some- animals, faeces (e.g. bat guano), carcasses times sedentary lifestyle and infrequent from trogloxenes, or other matter, such as reproduction, trogoblites and stygobites usu- twigs and plant residues delivered into the ally live longer than many non-cave species. cave by other animals or aquatic streams. Several animal categories, such as carabid Caves and Karst Environments 339

beetles, gastropods, collembolans and spi- ders are found in many caves worldwide, while some animal species have so far only been detected in certain caves, e.g. scorpi- ons are mostly found in Mexican caves. The most common troglobite so far described is the carabid beetle of the subfamily Trechinae, which measures approximately 5–7 mm (Barr and Holsinger, 1985). The largest troglobite found so far is the cave salaman- der (Proteus anguinus), commonly referred to as the ‘Olm’, that can measure up to 30 cm in length (Krajick, 2007; Fig. 16.11c). To date, nearly 8000 species of troglo- bites have been described (Krajick, 2007). However, since approximately only 10% of the caves thought to exist worldwide have been discovered, it should be anticipated Fig. 16.16. Photo of fungi (Beauveria felina) on that many more species and interesting eco- rodent excrement, Ardales Cave, Spain. © Cesareo Saiz-Jimenez. logical interactions remain to be discovered. A striking example for this is the unknown diversity of micro-eukaryotes in caves. catastrophes, through different types of envi- Currently, 18S rRNA gene sequence data ronmental pollution. Even an ecosystem suggest that most of the micro-eukaryotes so impacted by a minor disturbance, such as a far are not identifiable (Engel, 2010). Their natural flood (Fig. 16.7), can become suscep- role is unknown, but the limited knowledge tible to additional, more severe disturbance, retrieved suggests that at least some of the like disease or being out competed for nutri- micro-eukaryotes may have a severe impact ents and resources by invading species. on speleothems and deterioration of valua- In general, microbial colonization of a ble cave walls and paintings. Different types cave is a natural process that has occurred a of other ecological functions can be ascribed, long time before a cave’s discovery. However, depending on the species and activity level. as soon as a cave, with its already established Some eukaryotic structures, such as fungal ecosystem that is finely balanced in terms of hyphae, may serve passively as nuclei for ecological interactions and environmental crystallization or as sites for attachment of conditions (such as nutrient input) is opened crystals and thus, indirectly, contribute to a and connected with the exterior environment, speleological process (Figs 16.11b and 16.16). the ecosystem becomes subjected to an unac- customed input of abundant organic matter, or may be impacted by invading communi- ties coming from the surface. This can lead to 16.4 Natural and Anthropogenically significant food web changes because the Disturbed Caves and the Future of Cave newcomers may exert an enormous pressure Preservation on the original inhabitants. In the worst-case scenario, the newcomers may even displace Unfortunately, our chance of realizing the the original populations and communities. true biodiversity of caves is endangered by There are several dramatic examples of this many factors, including the environmental (Saiz-Jimenez, 2010). problems caused by mining, drilling, pump- The strongest disturbance recorded so ing of aquifers, contamination, or invasions far has been commercial cave mass tourism, by other organisms, including humans via which commenced during the second half tourism and even research activities. Many of the 19th century. At that time, minimal cave environments appear to be endangered consideration was given to conservation. by several parameters, ranging from climatic As a consequence, practices adopted for 340 N.M. Lee et al.

visits to caves often resulted in irreversible ous biocides (e.g. the Lascaux Cave, France; damage caused by thousands of visitors, for for more detailed information see review by example, in the form of lint, litter, and even Saiz-Jimenez, 2010; Fig. 16.17). increased carbon dioxide (from exhaled Another striking example of the impact breath) and altered temperature levels of disturbance on cave ecosystems is the (due to human body heat) in passages with unexpected outcome of the occurrence of low circulation. In addition, destructive the fungal species Geomyces destructans, construction works have removed tonnes of in some caves in the USA. Although still rocks and other materials from the topsoil under investigation, it is highly probable the and cave entrances to allow access for tour- G. destructans is a natural fungal species to ists to many caves worldwide. Lastly, the humid, cool subsurface environments. In introduction of artificial lighting in some North America, its link to the ‘white nose tourist caves, sometimes left illuminated all syndrome’, which has been associated with day, is sufficient to turn the darkness into a the sudden deaths of more than a million terrarium in some caves and support the bats in the USA, is mysterious (Blehert growth of various phototrophs and plants et al., 2009; Fig. 16.18). Strikingly, the first that would not otherwise be able to survive discovery was made in 2006 in a heavily (Fig. 16.17). One example of this is the green visited, commercial cave in Schoharie alga, Bracteacoccus minor, that occurred on County, New York, USA. After this, the the wall paintings in the Lascaux Cave, white-nose syndrome fungal infection France. For the most part, the ancient rock- spread astonishingly fast to over 100 other art paintings in the Lascaux Cave and others caves throughout the North American in Europe are not only threatened by algae, continent – including caves and mines that but also by fungal species introduced and are not generally accessible to humans. The spread by human activities. Due to this, a event, and more significantly the death of so number of caves had to be closed for several many different valuable bat species, has had years to treat invading organisms with vari- several serious consequences: it has led to

Fig. 16.17. Example of an invasive species on the ground in a cave, caused by artificial lighting. This promotes the growth of calcifying cyanobacteria (Scytonema julianum) and algae in tourist caves, as shown in Tito Bustillo Cave, Spain (Saiz-Jimenez, 1999). © Cesareo Saiz-Jimenez. Caves and Karst Environments 341

(a) website on the white nose syndrome). Clearly, research into the biology of caves is not only a matter of exploring unique and extreme ecosystems, but is also fundamen- tal to our understanding of the delicate eco- logical balances on Earth. Unfortunately, no tourist cave impacted by severe disturbances has ever been completely restored to its former ecological state (Elliot, 2006). This is particularly the case where the wrong decisions were made in selecting a biocide to employ in Lascaux Cave, since it caused severe irreversible (b) population shifts in the natural cave flora. For instance, in the treatment of the Lascaux Cave, benzalkonium chloride use resulted in the selection of Gram-negative bacterial species that were adapted to this biocide (e.g. Ralstonia, Pseudomonas and other pathogenic bacteria). The lesson to be learned from this case is that biocides that are generally considered acceptable for com- bating microorganisms in surface environ- ments are not necessarily appropriate for use in sensitive ecosystems such as caves. Interestingly, recently discovered rock-art Fig. 16.18. Cave bats infected by the ‘white nose caves such as Chauvet, France, and La syndrome’, caused by the fungus Geomyces Garma, Spain, that have been protected destructans. (a) Close-up of little brown bat’s nose against mass tourism, have so far shown no with fungus, New York, USA. Photo courtesy Ryan sign of deterioration. Based on this, it is von Linden, New York Department of Environmental evident that it is important to protect cave Conservation (www.fws.gov/whitenosesyndrome/ images/3842close-upofnosewithfungus.jpg). (b) ecosystems from the outset following dis- Fungus on wing membrane of little brown bat, covery and perhaps the best protection is to New York, USA. Photograph courtesy Ryan von limit the amount of tourism (Saiz-Jimenez, Linden, New York Department of Environmental 2010). Conservation (www.fws.gov/whitenosesyndrome/ Principally, every visitor to a cave, from images/3845Fungusonwingmembrane.jpg). the professional speleologist to tourists, has the potential to exert a negative impact on the the near extinction of several species and cave ecosystem, especially if the rules for has had a tremendous negative ecological basic safe caving, as outlined by McClurg impact on agriculture owing to the fact that (1996), are not followed: ‘Don’t take anything, bats consume enormous numbers of insects. don’t leave anything; don’t break or remove The Forest Service has esti- cave formations; don’t handle or collect mated that about 1.1 million kg of insects cave life; or, in other words: take nothing but will go uneaten in the most heavily impacted pictures; leave nothing but foot prints and regions, and it is likely that insect infestation kill nothing but time’. It goes without saying, will become a great financial burden on the however, that for some regions of the world, agricultural sector. Without a natural regu- caves serve as the only viable commodity and lator of insect population, the use of insecti- tourism is the only source of revenue. For cides is likely to increase, which will in example, in the Mammoth Cave region, USA, turn increase existing environmental prob- over US$400 million were brought to the area lems (for more information see the USDA by tourists in 2009, up from an estimate of 342 N.M. Lee et al.

US$52 million in 1993 (Stynes, 1999). The tion and its mutual interactions with natural beauty of caves can also serve as a the upper spheres. mechanism for science education to the pub- • Astrobiology whereby caves may serve lic. Local to national strategies can be, and as interesting model systems (ana- should be, enacted to manage and protect logues) for hypotheses on the origin and vulnerable or at-risk cave ecosystems while development of life on Earth, as well as also balancing the use of caves for tourism in outer space. and education. • Geotechnological applications, such as drilling, caving and mining. • Environmental sciences addressing 16.5 Summary and Future Visions topics such as climatology, global of Cave Life Sciences warming, pollution and the screening of potentially novel species with inter- esting degradation traits (with the During the last decades, caves have emerged potential to allow water purification as fascinating model systems for a number and/or degradation of contaminants). of scientific disciplines. As only a minor • Cultural history such as archaeology and fraction of all caves on our planet have been development of conservation techniques. found and explored, much still remains to • Medical sciences and different types of be discovered. To accomplish this, a number biotechnological applications whereby of further developments in caving technol- caves could be explored for, e.g. useful ogy, sampling technology and analytical enzymes, pathogenic organisms, screen- tools are needed and these must be applied ing of antibiotic-producing organisms, in a systematic way to allow clear correla- to name but a few. tions to be made. Fortunately, this is being accomplished via increased research in Clearly, cave science is an exciting and ever- various scientific disciplines, as well as in expanding field with enormous future the emergence of various professional net- potential. working organizations (e.g. websites for CRF, the Speleogenesis Network and the UIS). While each cave may pose its own Acknowledgements specific research questions, some general examples for future fields of research We thank all enthusiastic members in our include: research teams for valuable contributions • Cave life sciences, in general, because over the years. Financial support was pro- our knowledge about cave biodiversity, vided to NL, DM and WL from the Tech- biogeography, endemism, function and nische Universität München, the Helmholtz activity status, geo-ecological interac- Foundation for the ‘virtual institute for iso- tions, population dynamics, nutrient tope biogeochemistry–biologically mediated cycling, adaptation mechanisms and processes at geochemical gradients and inter- life cycles of various cave adapted spe- faces in soil–water systems’ and the DFG cies is still very limited. project FOR 571; from the United States • Biology of different types of extremo- National Science Foundation (DEB-0640835) philic organisms in oligotrophic, dark and the Louisiana Board of Regents life environments. (NSF(2010)-PFUND-174) for AES, for RA • Different applications in geobiology, and LK from the project APVV 0251-07, such as interactions between life and Slovakia, for CSJ from the project TCP minerals and subsurface sciences, as CSD2007-00058, Spain, for SB from the caves may provide a unique opportu- Scientists Pool Scheme CSIR, New Delhi, nity for in situ exploration of overall India, and for RB University Grants subsurface habitat biodiversity, func- Commission (UGC), New Delhi, India. Caves and Karst Environments 343

Websites

Canyons Worldwide: www.canyonsworldwide.com/crystals/mainframe3.html CRF Cave Research Foundation: www.cave-research.org (accessed 3 May 2011). Speleogenesis Network: www.network.speleogenesis.info/index.php (accessed 3 May 2011). UIS International Union of Speleology: www.uis-speleo.org (accessed 2 May 2011). USDA report on the white nose syndrome: www.invasivespeciesinfo.gov/microbes/wns.shtml (accessed 3 May 2011). US Show Caves Directory: www.goodearthgraphics.com/showcave/menu.html (accessed 3 May 2011).

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