L. Espinasa and N.H. Vuong ± A new species of cave adapted Nicoletiid (Zygentoma: Insecta) from Sistema Huautla, Oaxaca, Mexico: the tenth deepest cave in the world. Journal of Cave and Karst Studies, v. 70, no. 2, p. 73±77. A NEW SPECIES OF CAVE ADAPTED NICOLETIID (ZYGENTOMA: INSECTA) FROM SISTEMA HUAUTLA, OAXACA, MEXICO: THE TENTH DEEPEST CAVE IN THE WORLD

LUIS ESPINASA AND NGUYET H. VUONG School of Science, Marist College, 3399 North Road, Poughkeepsie, NY 12601, [email protected] and [email protected]

Abstract: Anelpistina specusprofundi, n. sp., is described and separated from other species of the subfamily Cubacubaninae (Nicoletiidae: Zygentoma: Insecta). The specimens were collected in SoÂtano de San AgustõÂn and in Nita Ka (Huautla system) in Oaxaca, MeÂxico. This cave system is currently the tenth deepest in the world. It is likely that A.specusprofundi is the sister species of A.asymmetrica from nearby caves in Sierra Negra, Puebla. The new species of nicoletiid described here may be the key link that allows for a deep underground food chain with specialized, troglobitic, and comparatively large predators suchas thetarantula spider Schizopelma grieta and the 70 mm long scorpion Alacran tartarus that inhabit the bottom of Huautla system.

INTRODUCTION 760 m, but no human sized passage was found that joined it into the system. The last relevant exploration was in Among international cavers and speleologists, caves 1994, when an international team of 44 cavers and divers that surpass a depth of minus 1,000 m are considered as pushed its depth to 1,475 m. For a full description of the imposing as mountaineers deem mountains that surpass a caves of the Huautla Plateau, see the bulletins from these height of 8,000 m in the Himalayas. Among deep caves, periods of exploration in the National Speleological those in the Huautla Plateau, Oaxaca, MeÂxico, are among Society (NSS), the Association for Mexican Cave Studies the most profound in the world. Exploration of their vast (AMCS), and the book by Stone et al. (2002). With its underground passages has taken more than 40 years. To current 1,475 m depthand as of June, 2007, theNSS explore their depths, a revolution in cave techniques such compilation of the world's deepest caves, the Huautla as the use of multiple underground camps and the use of system is the second deepest cave in MeÂxico (just 9 m below computer controlled rebreathers was required. It also took Sistema Cheve/Cuicateco), and the tenth deepest cave in the its human toll. In 1980 a Polish team suffered two serious world (www.caverbob.com/wdeep.htm as of 2007). accidents below 600 m and an international effort was From a biological perspective, deep caves are a successful in rescuing them, but Josef Cuber was paralyzed particularly harsh environment where few organisms can witha fractured spinal column. Thenin 1994, Ian Rolland survive. The few species adapted to this extreme environ- died while diving a sump at minus 1,325 m (Stone et al., ment have to endure continuous darkness, but more 2002). significantly, a lack of food (HuÈppop, 2005). Since most Cavers are relentless in their pursuit to break depth energy sources ultimately come from the sun and records in caves. Year after year they discover new and photosynthesis, the further away from the surface, the deeper caves, thus the list of the deepest caves is always more diluted the energy supplies become. A large changing. Throughout the history of cave exploration, the proportion of cave adapted species (troglobites) actually Huautla caves have always been in the forefront. Starting live comparatively close to cave entrances where they in 1967, cavers exploring the SoÂtano de San AgustõÂn ran depend for their sustenance on bat droppings as guanobites out of rope at a depth of 449 meters, establishing it as the (Gnaspini, 2005) or on surface debris that is washed in or second deepest cave in NorthAmerica. Thenin 1969 cavers percolates from a nearby surface source (Trajano and reached a sump in San AgustõÂn at a depthof 612 m, Bichuette, 2006). making San AgustõÂn the deepest cave in the Americas. By In a pyramid of productivity, each higher level of 1977, exploration had reached a depth of 800 m. In 1980, producers, primary consumers, secondary consumers, Bill Stone made the celebrated sump dive that connected Li tertiary consumers, etc., has a multiplicative loss of energy Nita cave to SoÂtano de San AgustõÂn, giving the combined in the food chain that severely limits the overall biomass of system a depth of 1,220 m and making it the world's third top level carnivores that any ecosystem can support deepest cave. Ten years later, another sump dive joined (Campbell and Reece, 2002). In extreme environments Nita Nanta cave to the system, bringing its depth to where the net primary productivity of autotrophs is low, or 1,353 m and briefly making the Huautla system the world's in places where primary consumers have restricted access to second deepest cave. In 1989 Nita Ka was explored to nutrients, suchas in caves, food chainstend to be simple, Journal of Cave and Karst Studies, August 2008 N 73 A NEW SPECIES OF CAVE ADAPTED NICOLETIID (ZYGENTOMA:INSECTA) FROM SISTEMA HUAUTLA,OAXACA,MEXICO:THE TENTH DEEPEST CAVE IN THE WORLD productivity pyramids typically have few tiers, and specialized large predators are typically absent. The Huautla system is inhabited by ten troglobites (Stelle and Smith, 2005) but two of them are particularly remarkable; the scorpion Alacran tartarus Francke and the tarantula spider Spelopelma grieta Gertsch. In his descrip- tion of the new scorpion, Francke (1982) comments, ``The surprising fact is the depth at which these scorpions exist: about 750 to 820 m below the cave entrance! This is one order of magnitude deeper than the previous depth record for troglobitic scorpions, Typhlochactas ellioti Mitchell from SoÂtano de Yerbaniz (minus 75 m) ¼ Equally remarkable, and unexpected, is the rather large size these deep dwelling scorpions attain: 60±70 mm in total length. Typhlochactas spp. are less than 20 mm long.'' The troglobitic tarantula is also found deep in the caves (Stelle and Smith, 2005). That such specialized and relatively large predators can survive at suchdepthis startling. To explain their presence, there must also be a suitable source of large prey. The new species of nicoletiid described here may be the key that permits the rather complex food chain to exist at such depth in the Huautla system. It is hoped that this study will contribute to a better understanding of trophic levels and food webs of extremophiles in harsh environ- ments, suchas troglobites in deep caves.

MATERIAL AND METHODS

Samples examined are from the Texas Memorial Museum of Invertebrate Zoology collection. They were left in vials with ethanol. All illustrations were made with the aid of a camera lucida attached to a compound microscope. Figure 1. Anelpistina specusprofundi n. sp., Microchaetae partially shown. A, Adult female. B, Dorsal side of head. Antennal insertion sockets, although not illustrated, are RESULTS AND DISCUSSION positioned before the clypeus, under the edges of both sides of the head with five macrochaetae. C, Ventral face of antennae MATERIAL in adult males. D, Outside lateral face of antennae in adult SoÂtano de San AgustõÂn, Huautla Cave System, San males. E, Mandible. F, Labial palp and labium. G, Galea AgustõÂn (5 km SE Huautla de JimeÂnes), Oaxaca, MeÂxico. (with two different conules) and lacinia. Scale on upper right 96.773206 longitude, 18.079458 latitude. corner for all illustrations, except where indicated. Holotype male, body 17 mm long, antennae 42 mm, caudal appendages 30 mm, hind tarsus 3.1 mm. 01/28/88 Tex. Mem. Mus. Invertebrate Zool. Coll # 23,595. Allan Maximum conserved lengthof antennae 42 mm. Maxi- Cobb col. mum conserved lengthof caudal appendages 36 mm. Paratypes: 2 females; 10 and 19 mm long. May 1985. General color light yellow to white. Body very abundantly Tex. Mem. Mus. Invertebrate Zool. Coll # 23,597. A. G. covered withsmall microchaetae(Figs. 1B, 2E and 3A±C). Grubbs, L. Wilk, J. H. Smith, F. Holladay, and J. Blaksley Head withapproximately 5 + 5 macrochaetae on border of cols. 3= and 2R; 7, 9.2, 16 mm long and 15, 18 mm long. insertion of antenna (Fig. 1B). Pedicellus in adult males 1988. Tex. Mem. Mus. Invertebrate Zool. Coll # 23,596. slightly smaller than scapus and with unicellular glands on A. G. Grubbs col. ventral surface, clustered approximately in five groups and Other localities: Nita Ka cave, Huautla. 1 male; 12 mm witha row of microchaetaebordering themin form of a long. 1988. Tex. Mem. Mus. Invertebrate Zool. Coll # ``U'' (Fig. 1C). On outside lateral border an extra three 23,600. A. G. Grubbs col. clusters (Fig. 1D). Female basal articles of antenna simple and pedicellus about half the length of scapus. DESCRIPTION Mandible chaetotaxy as in Figure 1E, with four distinct Overall morphology long, slender, and with long macrochaetae. Mouthparts long and slender (Figs. 1F and appendages (Fig. 1A). Maximum body length19 mm. 2A). Labial palp as in Figure 1F. Apical article subtrian- 74 N Journal of Cave and Karst Studies, August 2008 L. ESPINASA AND N.H. VUONG

Figure 2. Anelpistina specusprofundi n. sp., Microchaetae partially shown. A, Maxilla. B, Hind leg. C, Endopodium and lateral claws. D. Thoracic terga. E, Urosterna I±III. Figure 3. Anelpistina specusprofundi n. sp., Microchaetae Scale on upper right corner for all illustrations, except where partially shown. A, Urosterna VIII±IX, parameres and penis indicated. in adult male. B, Urosterna VIII±IX, subgenital plate and ovipositor in adult female. C, Urotergum X. D, Male cercus. gular and distinctly longer than at its largest width at its E, Spines in cercus. Scale on upper right corner for all tip. The article is also distinctly longer than penultimate illustrations, except where indicated. article. Penultimate article witha barely conspicuous bulge containing two macrochaetae. Labium and first article of inal urosterna II±VII subdivided into coxites and sternites labial palp withmacrochaetae.Maxilla as in Figures 1G (Fig. 2E) and sterna VIII and IX of male entire (Fig. 3A), and 2A. Apex of galea withtwo conules, one longer than as in other members of subfamily. No apparent modifica- wide and the other wider than long (Fig. 1G). Caution tions in urosternum III and IV of adult male. Urosternum should be used when assessing this character, because at VIII of adult male emarginated and its projections are most angles of observation, bothconules appear longer rounded to slightly acute (Fig. 3A). Stylets IX slightly than wide (Fig. 2A). Ultimate article of maxillary palp larger than others, with two macrochaetae and an extra approximately 3/4 lengthof penultimate article and subapical pair. Other stylets with one macrochaeta plus the approximately six times longer than wide (Fig. 2A). subapical pair (Fig. 3A±B). Terminal spine withsmall Legs long and slender (Figs. 1A and 2B). Hind tibia teeth. Stylets IX without modifications in males and approximately eight times longer than wide and approxi- females. mately 2/3 lengthof tarsus. Claws witha hairyappearance Penis and parameres of adult males as in Figure 3A. (similar to other Anelpistina (Espinasa et al., 2007)) and Parameres attaining about 1/3 the length of stylets IX. very long (Fig. 2C). Mesonotum witha single distinct Point of insertion of parameres in urosternum IX shallow, macrochaetae on lateral borders and on posterolateral slightly below level of styli in this segment. Coxal processes borders several setae of varied sizes, although none and middle posterior portion of urosternum IX without distinctly more robust than the other (Fig. 2D). Abdom- any distinct setae (Fig. 3A). Adult female genital area as in Journal of Cave and Karst Studies, August 2008 N 75 A NEW SPECIES OF CAVE ADAPTED NICOLETIID (ZYGENTOMA:INSECTA) FROM SISTEMA HUAUTLA,OAXACA,MEXICO:THE TENTH DEEPEST CAVE IN THE WORLD

Figure 3B. Subgenital plate square-like in appearance, flat- (Espinasa and Rishmawi, 2005)), and A.yatbalami rounded distally (Fig. 3B). Ovipositor in adult female Espinasa et al. 2007 a unique combination of characters surpassing stylets IX by1/2 of stylets lengthand gonapo- that separates them from all other described Anelpistina physes with about 18 annuli. species: a) Head withabout five macrochaetae by insertion Urotergite X shallowly emarginate in both sexes, of antennae (Fig. 1B); b) Mandible withfour macrochaetae posterior angles withseveral macrochaetaeand a few (Fig. 1E), and c) Galea withconules of different size relatively strong setae. Lengthof inner macrochaetae (Fig. 1G). All other species of Anelpistina lack this unique almost 1/2 distance between them (Fig. 3C). Cercus of set of characters. adult male typically witha longer thanwide basal annulus Anelpistina yatbalami was collected from under rocks in followed by a very long annulus withspines (Fig. 3D). The the Maya ruins of Yaxchilan, Chiapas. Since it is a surface spines' composition is homogeneous, with no spine being species, it can easily be differentiated from A.specuspro- distinctly longer or inserted in distinctly larger tubercles fundi because it lacks the adaptive modifications of a (Fig. 3E). Female cercus simple. troglobite, suchas a long, slender body and appendages. Postembryonic development of males: specimens of 12, Furthermore, the point of insertion of parameres in 16, and 17 mm body length share similar morphology, with urosternum IX is deep in the epigean species and the body the exception of a Nita Ka specimen (12 mm), in which the is covered by few microchaetae, while in the new hypogean penis is proportionally bigger, reaching almost the apex of species insertion of parameres is shallow and the micro- the parameres. It is unknown if this difference reflects chaetae covering their body are much more abundant. individual or population differences or if all medium sized Although only immature females were available when A. males have this size ratio. Smaller male specimens (7 and yatbalami was described, it also appears that its gonapo- 9.2 mm) with smaller pedicellus, about half the length of physis may be subdivided into more annuli (about 20±25) scapus, but still withgland clusters, and cerci without in A.yatbalami than in the new species (about 18). spines. Anelpistina parkerae was described from El SoÂtano Postembryonic development of females: specimens of 15 Hondo del Pinalito, in Hidalgo. Also being a troglobite, it and 18 mm body lengthhaveovipositors surpassing stylets shares with A.specusprofundi the long and slender IX by about 1/2 of the stylets length and gonapophyses proportions of its body and appendages. Males can be withabout 18 annuli. In a female 10 mm body length, differentiated because in the former species the pedicellus is ovipositor just beginning to develop and barely reaching distally muchenlarged, creating a lobe, and its cerci have base of stylets IX. Gonapophyses without distinct annuli. heterogeneous spines inserted in tubercles of different length. In the new species the pedicellus, although DISTRIBUTION modified, is not enlarged to the extent of creating a lobe Specimens of this species have been collected from (their maximum width is less than double of the following SoÂtano de San AgustõÂn, Nita Ka, and Li Nita, which are articles of the antennae) and the cerci spines are part of the Sistema Huautla cave system. Only the homogeneous. Females from Pinalito appear to have a specimens from SoÂtano de San AgustõÂn and Nita Ka were gonapophysis subdivided into less annuli (about 10±11) available for examination during this study. It is likely that than in the new species. Finally, the body in specimens of A.specusprofundi is endemic to caves within the karstic the new species is also covered by more microchaetae. area of Huautla, Oaxaca, MeÂxico. Anelpistina specusprofundi is more difficult to differen- tiate from A.asymmetrica . A.asymmetrica has been ETYMOLOGY described from three caves: TP4 13, XalteÂgoxtl, in Puebla, From specus5cave and profundi5deep, in genitive and Cueva de Gabriel, in Oaxaca (Espinasa et al., 2007). singular. It makes reference to its habitat within one of the They both share a labial palp with a subtriangular apical deepest caves in the world. article, homogeneous spines in the cerci, and an ovipositor of similar lengthand subdivision (17±18 annuli). Females REMARKS of bothspecies are mostly undistinguishablebased on The new species described here is a member of the sexual secondary characters as the increase in length of the subfamily Cubacubaninae, which has a neotropic distribu- ovipositor withrespect to stylets IX follows a similar rate. tion. It has stylets on urosternite II, but lacks scales, Males are more easily differentiated based on sexual sensory pegs in the appendix dorsalis, and conspicuous secondary characters because similarly sized adult males lateral lobes bearing numerous glandular pores. As such, of the new species lack the curved cerci and the asymmetric its generic allocation is within Anelpistina Silvestri, 1905 as pedicellus of adult A.asymmetrica males. Furthermore, in defined by Espinasa et al. (2007). A.asymmetrica the penis is much enlarged and almost The genus Anelpistina has 23 species described. Within reaches the apex of the parameres, while in the new species these members of the genus, A.specusprofundi n. sp. shares (except in the Nita Ka specimen) the penis is of normal only with A.asymmetrica (5 Cubacubana asymmetrica dimensions when compared with other Anelpistina and (Espinasa, 2000)), A.parkerae (5 Cubacubana parkerae reaches only about 1/2 the length of the parameres. 76 N Journal of Cave and Karst Studies, August 2008 L. ESPINASA AND N.H. VUONG

The clearest character that allows the differentiation County, Texas, found that the most common troglobitic between bothspecies, independent of thepostembryonic fauna in caves withno natural entrances are nicoletiids, development of sexual secondary characters, is that suggesting that they can subsist in low energy habitats (P. although both have long and slender bodies and append- Sprouse, pers. comm.). It is likely that nicoletiids are one of ages, as expected from troglobitic species, A.asymmetrica the few taxa of troglobites that can efficiently convert appears to be proportionally longer and more slender than enough chemical energy from the minimal supplies found A.specusprofundi . For example, in A.asymmetrica the in deep caves to produce enoughsecondary productivity to ultimate article of the maxillary palp is approximately 14 sustain a food chain that includes a specialized and times longer than wide, while in A.specusprofundi the relatively large predator. The new species of nicoletiid ultimate article of maxillary palp is approximately six times described here is most likely the key link that allows for the longer than wide. rather complex food chain present at such depth in the It is likely that A.specusprofundi belongs within the Huautla system. SouthMexico and Antilles phyletic species group presented in the cladogram obtained from a phylogeny study of the ACKNOWLEDGMENTS Cubacubaninae using five molecular loci (Espinasa et al., 2007). Both A.yatbalami and A.asymmetrica belong to this We thank James Reddell for providing samples and phyletic group. Furthermore, it is even likely that A. Ellise C. Cappuccio, Monika Baker and Dr. Stewart Peck specusprofundi and A.asymmetrica are sister species for reviewing the manuscript. We also thank the School of because of their morphological similarity and because they Science at Marist College and Dr. Michael Tannenbaum, inhabit caves that are only about 30 km away in the its Dean, for supporting the publication of this manuscript. mountain range that divides the states of Puebla and Finally, we thank Andy Grubbs, the collector of the Oaxaca. specimens. He is one of the few persons, who after enduring all the hardship of deep cave exploration, still DISCUSSION has the patience and care to dutifully collect biological specimens. The rather long (< 90 mm including antennae and caudal appendages) new species of nicoletiid described here REFERENCES is a good candidate to explain the presence of a specialized and relatively large (< 70 mm) deep dwelling predator such Campbell, N.A., and Reece, J.B., 2002, Biology, San Francisco, Benjamin as the troglobitic scorpion found at depths of 750 to 820 m Cummings, p. 1206±1207. Espinasa, L., 2000, A new species of the genus Cubacubana (Insecta: below the cave entrance in the Huautla system. For such a Zygentoma: Nicoletiidae) from a Mexican cave, in Proceedings of the large predator to survive, there must also be a suitable Biological Society of Washington, v. 1, no. 113, p. 218±223. source of large prey. This prey should have a very efficient Espinasa, L., and Rishmawi, I.J., 2005, A new species of the genus secondary productivity (rate at which consumers convert Cubacubana (Insecta: Zygentoma: Nicoletiidae) from a cave in Hidalgo, Mexico, in Proceedings of the Biological Society of the chemical energy of the food they eat into their own new Washington, v. 4, no. 118, p. 803±808. biomass within an ecosystem). As mentioned before, Espinasa, L., Flick, C., and Giribet, G., 2007, Phylogeny of the American because most energy sources ultimately come from the silverfishCubacubaninae (Hexapoda: Zygentoma: Nicoletiidae): a combined approachusing morphology and five molecular loci: sun and photosynthesis, the further from the surface, the Cladistics, v. 1, no. 23, p. 22±40. more diluted the energy supplies become, and only an Francke, O.F., 1982, Studies of the scorpion subfamilies Superstitioninae organism withvery efficient secondary productivity could and Typhlochactinae, with description of a new genus (Scorpiones, Chactoidea): Association for Mexican Cave Studies, v. 8, p. 51±61/ provide the overall biomass to support top level carnivores. Texas Memorial Museum Bulletin v. 28, p. 51±61. Andy Grubbs, one of the collectors of both the Gnaspini, P., 2005, Guano communities, in Culver, D.C., and White, W. scorpions and the nicoletiids from Li Nita and San AgustõÂn B., eds., Encyclopedia of Caves, Burlington, Elsevier, p. 276±283. HuÈppop, K., 2005, Adaptation to low food, in Culver, D.C., and White, caves, has corroborated that both species are ``definitely W.B., eds., Encyclopedia of Caves, Burlington, Elsevier, p. 4±10. down in the same areas'' near where both caves have an Reddell, J.R., 1981, A review of the cavernicole fauna of Mexico, underground connection (A. Grubbs, pers. comm.). He Guatemala and Belize: Texas Memorial Museum Bulletin, v. 27, also mentioned that the nicoletiids were found in the wet 327 p. Silvestri, F., 1905, Materiali per lo studio dei Tisanuri. VI. Tre nuove areas withmud banks. Mud banks are a common habitat specie di Nicoletia appartenenti ad un nuovo sottogenero: Redia of nicoletiids (Reddell, 1981) where specimens eat the soil (Firenze), v. 2, p. 111±120. and extract from its minimal supplies of organic com- Stelle, C.W., and Smith, J.H., 2005, Sistema Huautla, Mexico, in Culver, D.C., and White, W.B., eds., Encyclopedia of Caves, Burlington, pounds enoughenergy to survive, grow and reproduce. Elsevier, p. 514±521. Furthermore, Nicoletiids as a group are common Stone, W.C., am Ende, B.A., and Paulsen, M., 2002, Beyond the Deep: inhabitants of the cave environment. They are broad The Deadly Descent into the World's Most Treacherous Cave, New York, Warner Books, 351 p. generalists in their feeding habits and a recent bioinventory Trajano, E., and Bichuette, M.E., 2006, Biologia SubterraÃnea. IntroducËaÄo, of entranceless voids on a highway project in Williamson SaÄo Paulo, Redespeleo, 92 p.

Journal of Cave and Karst Studies, August 2008 N 77 D.S. Jones, E.H. Lyon, and J.L. Macalady ± Geomicrobiology of biovermiculations from the Frasassi Cave System, Italy. Journal of Cave and Karst Studies, v. 70, no. 2, p. 78±93. GEOMICROBIOLOGY OF BIOVERMICULATIONS FROM THE FRASASSI CAVE SYSTEM, ITALY

DANIEL S. JONES*,EZRA H. LYON 2, AND JENNIFER L. MACALADY 3 Department of Geosciences, Pennsylvania State University, University Park, PA 16802, USA, phone: tel: (814) 865-9340, [email protected]

Abstract: Sulfidic cave wallshostabundant, rapidly-growing microbial communiti es that display a variety of morphologies previously described for vermiculations. Here we present molecular, microscopic, isotopic, and geochemical data describing the geomicrobiology of these biovermiculations from the Frasassi cave system, Italy. The biovermiculations are composed of densely packed prokaryotic and fungal cellsin a mineral-organic matrix containing 5 to 25% organic carbon. The carbon and nitrogen isotope compositions of the biovermiculations (d13C 5235 to 243%,andd15N 5 4to 227%, respectively) indicate that within sulfidic zones, the organic matter originates from chemolithotrophic bacterial primary productivity. Based on 16S rRNA gene cloning (n567), the biovermiculation community isextremely diverse,including 48 representative phylotypes (.98% identity) from at least 15 major bacterial lineages. Important lineagesinclude the Betaproteobacteria (19.5% of clones),Ga mmaproteobacteria (18%), Acidobacteria (10.5%), Nitrospirae (7.5%), and Planctomyces (7.5%). The most abundant phylotype, comprising over 10% of the 16S rRNA gene sequences, groupsin an unnamed clade within the Gammaproteobacteria. Based on phylogenetic analysis, we have identified potential sulfur- and nitrite-oxidizing bacteria, as well as both auto- and heterotrophic membersof the biovermiculation community. Additionally ,manyofthe clonesare representativesof deeply branching bacterial lineageswith n o cultivated representatives. The geochemistry and microbial composition of the biovermiculations suggest that they play a role in acid production and carbonate dissolution,thereby contributing to cave formation.

INTRODUCTION The oxidation of hydrogen sulfide provides a rich energy source for chemolithotrophic microorganisms. In Sulfidic cavesare limestone cavesthat form via the sulfidic caves, these microorganisms form the trophic base oxidation of hydrogen sulfide to sulfuric acid. Although of aphotic ecosystems that support macroinvertebrate life, this mechanism is not as widespread as epigenic, carbonic- and in some cases, even vertebrates (Sarbu et al., 1996; acid karstification (Palmer, 1991), sulfidic processes have Hose and Pisarowicz, 1999). Because these microbial created many spectacular caverns, unforgettable for their communities are isolated in the subsurface and ultimately massive, resplendent rooms and unusual sulfur mineral supported by energy from reduced sulfur compounds, deposits. Cave formation occurs near the water table where sulfidic caves are important analogues for early Earth sulfide-bearing phreatic water mixes with oxygenated environments, and potentially, for sulfur-rich extraterres- vadose waters and cave air. As hydrogen sulfide is oxidized trial environments(Bostonet al., 2001). Microbial to sulfuric acid (e.g., Equation (1)), the sulfuric acid then communities associated with sulfidic caves include viscous, reacts with limestone to form gypsum (Equation (2)). highly acidic ``snottites'' that drip from overhanging surfaces, white microbial mats in cave streams, less H S z 2O ? H SO 1† 2 2 2 4 conspicuous microbial communities in gypsum wall crusts H2SO4 z CaCO3 z H2O ? CaSO42H2O z CO2 2† and stream sediments, and biovermiculations Ð anasto- Where cave streams and lakes are undersaturated with mosing, microbe-packed films of organic-rich sediment on gypsum, calcium and sulfate are carried away in solution. cave ceilingsand walls(Hoseand Pisarowicz, 1999; Engel Above the water table, however, gypsum crusts form as et al., 2004a; Hose and Macalady, 2006). degassing hydrogen sulfide oxidizes in condensation Biovermiculationsresemble vermiculationscommon to droplets (Egemeier, 1981). Gypsum deposits and forma- carbonic-acid caves. Vermiculations are broadly described tionsin CarlsbadCavern and Lechuguilla Cave in the as discontinuous, worm-like deposits of mud and clay Guadalupe Mountains, New Mexico, are interpreted as found on cave wallsand ceilings(Hill and Forti, 1997). evidence for past sulfidic processes (Hill, 1998). For insightful discussions of cave formation by sulfuric acid * Corresponding author. dissolution, see Egemeier (1981) and recent geomicrobiol- 2 E-mail: [email protected]. ogy research by Engel et al. (2004b). 3 E-mail: [email protected]. 78 N Journal of Cave and Karst Studies, August 2008 D.S. JONES, E.H. LYON, AND J.L. MACALADY

However, Hose et al. (2000) distinguished between vermic- ulation-like microbial formationsin the sulfidic Cueva de la Villa Luz and other vermiculationsbecausebiovermicula- tionsform more rapidly and hosta rich and active microbial flora. We have adopted the term biovermiculation because the dynamic geochemical and biological processes in sulfidic cavescontrastsharply with the oligotrophic conditions typical for carbonic acid caves. Although it is possible that all vermiculationsare biologically mediated (Anelli and Graniti, 1967; Northup and Lavoie, 2001; Camassa and Febbroriello, 2003), this study focuses on vermiculations found in sulfidic environments. Substantial research has focused on describing micro- bial communities in sulfidic caves. Stream biofilms are the most widely studied (Thompson and Olson, 1988; Hubbard et al., 1990; Sarbu et al., 1994; Angert et al., 1998; Hose et al., 2000; Engel et al., 2001; Rohwerder et al., 2003; Engel et al., 2004a; Macalady et al., 2006). Snottitesfrom several different caves have also recently been described using molecular methods (Hose et al., 2000; Vlasceanu et al., 2000; Macalady et al., 2007). However, existing microbi- ological research on biovermiculations is limited to microscopy and culture-based surveys (Hose and Pisar- owicz, 1999; Hose et al., 2000) and preliminary molecular work reported by Hose and Northup (2004). This report Figure 1.Map of the Grotta Grande del Vento-Grotta del builds on observations in the Frasassi cave system made by Fiume (Frasassi) cave system showing sample locations Galdenzi (1990), and represents the first in-depth microbi- (circles).Topographic lines and elevations (m) refer to ological study of biovermiculations using molecular and surface topography above the cave.Grotta del Mezzogiorno- isotopic methods. Grotta di Frasassi complex (not shown) is located approx- imately 500 m northeast of Grotta Grande del Vento.Base

METHODS map courtesy of the Gruppo Speleologico CAI di Fabriano.

complex, developed at higher elevation in the same FIELD SITE,SAMPLE COLLECTION AND GEOCHEMISTRY The Grotta Grande del Vento-Grotta del Fiume geologic formation (Galdenzi and Maruoka, 2003). The (Frasassi) cave system comprises over 20 km of ramifying Grotta del Mezzogiorno-Grotta di Frasassi system is and irregular passage in Jurassic limestone of the Calcare located over 300 metersabove the presentday water table, Massiccio Formation (Galdenzi and Maruoka, 2003) of the and it is unclear whether this cave system originated by Frasassi Gorge. Sulfuric acid speleogenesis is actively sulfuric acid speleogenesis (S. Galdenzi, pers. comm.). occurring near the water table, which isaccessiblevia Because the GM-GF system is not currently experiencing technical caving routes. For group safety, we carry an EN- sulfidic processes, it is not clear what, if any, biological role has been played in their formation. Therefore, we classify MET MX2100 portable gasdetector with H 2S, O2,and vermiculations from this system simply as a vermiculations, SO2 sensors. We wear respirators on the rare occasions that rather than biovermiculations. H2S levels exceed OSHA safety standards (10 ppm). Sampling locations are shown in Figure 1. Cave streams Sampleswere collected between 2002 and 2006 in sterile in Ramo Sulfureo (RS) are fast flowing and turbulent while tubes using sterilized metal spatulas. Snottite and stream Grotta Sulfurea (GS) and Pozzo dei Cristalli (PC) streams biofilm samples in Table 1 were collected as described are characterized by slowly flowing water and stagnant previously (Macalady et al., 2006; Macalady et al., 2007). pools. Dissolved sulfide levels in the cave streams are Wherever possible, pH was measured with pH paper (range between 2±3 timeshigher at PC than the other two 5±10 and 4±7). In 2005 and 2006, cave air concentrationsof locations(Macalady et al., 2006), but hydrogen sulfide gas H2S, CO2,NH3, and N2O were measured at each sample concentrationsare highestin the cave air at RS (Macalady location using DraÈeger short-duration tubes and an Accuro et al., 2007). Gypsum crusts are extensive at PC and RS, hand pump (DraÈeger Safety Inc., Germany). Detection but at GS gypsum crusts are found only within two meters limitsfor H 2S and CO2 are between 0.2±60 ppm and 100± above the water table (unpublished observations). A 6000 ppm, respectively. NH3 and N2O were undetectable vermiculation sample was collected from the older Grotta at all sites. Lower detection limits were 0.25 for NH3 and del Mezzogiorno-Grotta di Frasassi (GM-GF) cave 0.5 for N2O. Journal of Cave and Karst Studies, August 2008 N 79 GEOMICROBIOLOGY OF BIOVERMICULATIONS FROM THE FRASASSI CAVE SYSTEM,ITALY

ELEMENTAL AND ISOTOPIC ANALYSES CLONE LIBRARY CONSTRUCTION AND DNA SEQUENCING Samples for elemental and isotopic analysis were stored at A clone library of bacterial 16S rRNA geneswas 4 uC immediately after collection and dried at 70 uCwithin constructed from biovermiculation sample GS03-5, col- 24 hours. Samples were acid-washed with HCl to remove lected in Grotta Sulfurea in 2003 at the same location as carbonate minerals.Acid washing wasperformed asfollows: samples for geochemical and isotopic analyses (Table 2). Sampleswere finely ground and reacted with ACS grade 2M DNA wasextracted at the OsservatorioGeologico di HCl for two hours under constant agitation. Afterwards, Coldigioco Geomicrobiology Lab within 24 hoursof samples were centrifuged and the supernatant decanted. collection using the MoBio Soil DNA extraction kit Samples were then washed several times with distilled, (MoBio Laboratories, Inc., USA) according to the deionized water to remove residual chloride ions, then manufacturer's instructions. Small subunit rRNA genes freeze-dried and homogenized. Elemental analysis for carbon, were amplified and cloned asin Macalady et al. (2007) nitrogen, phosphorous, and sulfur were performed at the using bacterial forward primer 27f (59-AGAGTTT- Agricultural Analytical ServicesLaboratory at Pennsylvania GATCCTGGCTCAG-39) and universal reverse primer State University, where total carbon and nitrogen were 1492r (59-GGTTACCTTGTTACGACTT-39). Cloneswere determined by combustion in a Fisions NA 1500 Elemental sequenced at the Penn State University Biotechnology Analyzer and phosphorous and sulfur were determined by Center using T3 and T7 plasmid-specific primers. Partial microwave digestion (Miller, 1998). Isotopic analyses were sequences were assembled with Phred base calling using performed via an Elemental Analyzer ± Isotope Ratio Mass CodonCode Aligner v.1.2.4 (CodonCode Corp., USA) and Spectrometer (EA-IRMS) at the Stable Isotope Biogeochem- manually checked. Assembled sequences were submitted to istry Laboratory at Penn State. Samples were combusted in a the online analyses CHIMERA_CHECK v.2.7 (Cole et al., Costech 4010 Elemental Analyzer, and subsequently intro- 2003) and Bellerophon 3 (Huber et al., 2004). Putative duced by continuousflow inlet into a Thermo-Finnegan Delta chimeras were excluded from subsequent analyses. Plus XP Isotope Ratio Mass Spectrometer (IRMS). Stable isotopic ratios are reported in delta (d) notation asfollows: PHYLOGENETIC AND DIVERSITY ANALYSES  The nearly full-length gene sequences were compared R against sequences in public databases using BLAST X ~ 103 sample { 1 3† Rstandard (Altschul et al., 1990) and the online workbench greengenes (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi; DeSantis et where X is d13Cord15N, and R denotesthe abundance ratio of 13 12 15 14 al., 2006). Sequenceswere aligned using the NAST aligner the heavy to light isotope, i.e., C/ Cor N/ N. Standards available at the greengenesweb site. NAST-aligned for carbon and nitrogen are Vienna PeeDee Belemnite sequences were added to a phylogenetic tree containing (VPDB) and atmospheric nitrogen (AIR), respectively. Based .150,000 bacterial sequences using the ARB_parsimony on internal standards and replicate samples, precision of all tool (Ludwig et al., 2004). Further phylogenetic analyses measurements is 60.2%. were generated using maximum parsimony. Maximum parsimony phylograms (heuristic search) and bootstrap FLUORESCENCE MICROSCOPY AND SCANNING ELECTRON consensus trees (heuristic search, 500 replicates) were MICROSCOPY (SEM) computed using PAUP* 4.0b10 (Swofford, 2000). Analyses Samples for microscopy were stored at 4 uC and either included closest BLAST matches of environmental sequenc- fixed in 4% paraformaldehyde (pfa) within 24 hoursof es and cultivated representatives. Phylogenetic relationships collection, or stored at 220uC in a 1:5 dilution of of the clonesare referenced to the Hugenholtz taxonomy RNAlater (Ambion, U.S.A.) and later fixed in 4% pfa. (Desantis et al., 2006; available at http://greengenes.lbl.gov). Fixation wasperformed asin Macalady et al. (2007). The Clonesin the 16S rRNA library were grouped into fluorescent dye 49,69-diamidino-2-phenylindole (DAPI) operational taxonomic units (OTUs) based on .98% was used to label cells for microscopy. Fixed samples and nucleotide identity. Rarefaction analyses were computed control cellswere applied to multiwell, Teflon-coated glass using EstimateS 7.5.0 (Colwell, 2005). slides, air-dried, and dehydrated with ethanol. Ten mLof Representative 16S rRNA gene sequences for each DAPI (1 mg/mL) wasapplied to each well, incubated for phylotype determined in this study have GenBank acces- 5 minutes, rinsed with distilled water, dried, and mounted sion numbers DQ499275 to DQ499330 and EF530677 to with Vectashield (Vectashield Laboratories, USA). Fluo- EF530681. rescence in situ hybridization (FISH) analyses were performed using methods and probes exactly as described RESULTS in Macalady et al. (2007). Slideswere viewed on a Nikon E800 epifluorescence microscope. Air-dried and gold- DISTRIBUTION AND MORPHOLOGY OF BIOVERMICULATIONS coated samples were also examined with a Hitachi S- Biovermiculations occur throughout the Frasassi cave 3000N SEM. Secondary electron imageswere taken under system and are found immediately above the water table to high vacuum mode at an accelerating voltage of 10 KeV. over 40 metersabove it where no sulfideisdetectable by 80 N Journal of Cave and Karst Studies, August 2008 D.S. JONES, E.H. LYON, AND J.L. MACALADY

Table 1.BLAST summary of 16S rRNA clone library. Representative clonea # clonesTop BLAST match identity Accession% Gammaproteobacteria 12 Piscirickettsiaceae 1 CV92 1 Gas hydrate associated sediment clone Hyd89-87 AJ535246 93 Nevskiaceae 1 CV109 1 polychlorinated biphenyl-polluted soil clone WD280 AJ292674 95 Acidithiobacillaceae 9 CV44 2 Guanting Reservoir (China) sediment clone 69-25 DQ833501 98 CV45 7 Acid mine drainage sediment clone H6 DQ328618 94 Unclassified 1 CV77 1 Uranium contaminated aquifer clone 1013-28-CG33 AY532574 99 Betaproteobacteria 13 CV10 3 Frasassi stream biofilm WM93 DQ415787 98 CV11 3 Green Bay ferromanganousmicronodule clone MND1 AF293006 98 CV21 3 Uranium contamination clone AKAU3480 DQ125519 93 CV30 1 siliceous sedimentary rock clone MIZ05 AB179496 95 CV41 2 African subsurface water clone EV221H2111601SAH33 DQ223206 95 CV71 1 Manganese-oxidizing soil clone JH-GY05 DQ351927 97 Alphaproteobacteria 4 CV17 1 farm soil clone AKYH1192 AY921758 98 CV25 1 farm soil clone AKYH1192 AY921758 97 CV43 1 freshwater sponge Spongilla lacustris clone SS31 AY598790 97 CV81 1 denitrifying quinoline-removal bioreactor clone SS-57 AY945873 94 Deltaproteobacteria 3 CV1 1 Mangrove soil clone MSB-5C5 DQ811828 93 CV34 1 Arabian sea picoplankton clone A714011 AY907798 89 CV90 1 Soil clone WHEATSIP-CL55 DQ822247 95 Cytophaga-Flexibacter- 3 Bacteroides CV24 1 Guanting Reservoir (China) sediment clone 69-27 DQ833502 98 CV70 2 flooded poplar tree microcosm soil clone 21BSF16 AJ863255 92 Nitrospira 5 CV22 2 Chinese deep sea sediment nodule clone E75 AJ966604 96 CV64 1 Green Bay ferromanganousmicronodule clone MNC2 AF293010 98 CV82 2 U contaminated aquifer clone 1013-28-CG51 AY532586 95 Verrucomicrobia 2 CV35 1 coastal marine sediment clone Y99 AB116472 95 CV80 1 Freshwater clone PRD01a004B AF289152 93 Planctomycetes5 CV14 1 Broad-leaf forest soil clone AS47 AY963411 89 CV47 1 Lake Kauhako Hawaii 30 m clone K2-30-19 AY344412 90 CV73 1 marine Pirellula clone 5H12 AF029076 94 CV97 1 Soil bacteria CWT SM02_H07 DQ129094 94 CV104 1 Anaerobic ammonia oxidizing community clone LT100PlB4 DQ444387 93 Acidobacteria 7 CV12 1 Bor Khlueng hot spring (Thailand) clone PK-85 AY555795 93 CV18 2 drinking water biofilm clone Biofilm_1093d_c1 DQ058683 97 CV68 1 Lake Washington sediment clone pLW-64 DQ066994 97 CV76 1 Hamelin Pool stromatolite clone HPDOMI1G01 AY851769 91 CV79 1 PCB polluted soil clone Lhad8 DQ648907 98 CV94 1 Amazon soil clone 1267-1 AY326533 97 Actinobacteria 2 CV54 2 San Antonio urban aerosol clone AKIW984 DQ129354 95

Journal of Cave and Karst Studies, August 2008 N 81 GEOMICROBIOLOGY OF BIOVERMICULATIONS FROM THE FRASASSI CAVE SYSTEM,ITALY

Table 1.Continued. Representative clonea # clonesTop BLAST match identity Accession% Chloroflexi 1 CV37 1 Lost City Hydrothermal Field clone LC1537B-77 DQ272585 94 OP11 2 CV31 1 deep groundwater clone KNA6-NB23 AB179676 92 CV46 1 Massachusetts river clone PRD01a009B AF289157 96 RCP2-18 1 CV51 1 Deep sea cold seep sediment clone BJS81-120 AB239028 90 SPAM 1 CV52 1 Lake Washington sediment clone pLW-68 DQ067010 97 TM7 2 CV63 1 batch reactor clone SBR2013 AF269000 94 CV78 1 Sulfur-oxidizing community clone IC-42 AB255066 89 WS3 2 CV39 1 Chinese deep sea sediment nodule clone MBAE32 AJ567590 91 CV106 1 antarctic sediment clone MERTZ_2CM_290 AF424308 91 Unclassified 2 CV40 1 3.5 Ma oceanic crust clone CTD005-79B-02 AY704386 91 CV60 1 siliceous sedimentary rock clone MIZ45 AB179536 94 a Taxonomic groupings based on Hugenholtz taxonomy (Desantis et al., 2006). Representative clones correspond to OTUs. portable gassamplingequipment (0.2 ppm detection limit). samples to enable comparison with other microbial Hydrogen sulfide concentrations at sample collection sites communities within the same cave system. In addition to ranged from undetectable to 4.3 ppm (Table 2), and in the values presented in Table 2, we measured the pH of general, gas concentrations in Frasassi are seasonally numerous biovermiculations in sulfidic zones over the past variable (Galdenzi et al., 2007). Biovermiculationshave five yearsand all measurementswere between 5.5 and 6.5 only been observed on limestone surfaces where no gypsum (data not shown). Replicate gas measurements made in a crust is present. Several attempts to identify biovermicula- given location on the same day were within 20%. tions or similar structures beneath gypsum crusts on walls Previously measured carbon and nitrogen isotopic immediately adjacent to biovermiculationshave been ratiosof invertebratesand microbial communitieswithin unsuccessful. We also noted the strict absence of biover- sulfidic zones indicate a chemolithotrophic energy base for miculations on the surface of chert nodules, even when the cave ecosystem (Galdenzi and Sarbu, 2000). Biovermi- adjacent limestone walls were thickly colonized (Fig. 2c). culation d13C and d15N values measured in this study Frasassi biovermiculations exhibit several of the mor- (Fig. 3) fall within or near the range of sulfidic zone phologies described by Parenzan (1961): bubble-like spots, samples analyzed by Galdenzi and Sarbu (2000), Sarbu et elongated spots, leopard spots, and tiger skin. In some al. (2000), and Vlasceanu et al. (2000). In contrast, the places, we observed biovermiculations so dense that they GM-GF (GM05-1) sample falls within the isotopic range formed continuous wall-covering sheets. Sample PC06-106 of materialscollected from the surface and near cave (Fig. 2e), sampled at the high water mark one meter above entrances(Fig. 3). the stream level in Pozzo dei Cristalli, was very light brown to grey. Other biovermiculationswere dark brown or FLUORESCENCE MICROSCOPY AND SEM occasionally black. In contrast, vermiculations sampled We used the fluorescent DNA stain DAPI to examine from GM-GF (GM05-1, Fig. 2f ) displayed dendritic several biovermiculation samples. Characteristic photomi- morphology and were surrounded by an obvious light- crographs of DAPI-stained samples are shown in Figure 4. colored halo. The GM-GF vermiculationswere light grey A range of cell morphologiesand sizescan be observed, in color, and covered a small area of the limestone walls although small coccoid-shaped cells (,1 mm) are the most relative to sample sites in sulfidic zones. common. Short rod-shaped (1 to 3 mm) cellsare also abundant and sometimesform filaments(Fig. 4b and c). GEOCHEMICAL ANALYSES Larger, undifferentiated filamentswith multiple nuclei are Geochemical data for biovermiculation samples are common and likely represent fungal hyphae. Attempts to listed in Table 2. Vermiculations from GF-GM (sample analyze biovermiculation samples using fluorescence in situ GM05-1) are listed separately because this cave has not hybridization (FISH) were largely unsuccessful because of experienced sulfidic processes in over 200,000 years, if ever. high autofluorescence in the biovermiculation matrix. We also provide data from stream biofilm and snottite However, cellsbinding to bacteria-specific probeswere 82 N Journal of Cave and Karst Studies, August 2008 Table 2.Summary of elemental and isotopic analysis of biovermiculation s amples. Sample Sample Sample Sample Sample Sample Sample Sample Parameter GS04-58 GS06-31 PC02-24 PC06-106 RS03-18 GM05-1 GS06-15 RS06-120 Sample Collection Info. Epigenic Beggiatoa Snottite Biovermiculations Vermiculation Stream Mat Sample Type Locationa GS GS PC PC RS GM GS RS Date Collected 7/22/04 8/16/06 12/8/02 8/21/06 10/28/03 8/15/05 8/16/06 8/23/06 Height Above Water table 2 m 2 m 20 m 1 m 10 m .200 m 0 m 2 m pH nm 6 6.0±6.5 6 nm nm 7.1 0±1 Elemental Analysis Percent Carbon 6.67 5.72 19.57 24.96 19.63 27.82 12.98 5.83 Percent Nitrogen 0.76 0.62 2.47 3.12 2.59 4.92 2.11 0.31 Percent Phosphorous 0.2 0.16 nm nm nm nm 0.15 0.04 Percent Sulfur 1.27 0.49 nm nm nm nm 8.25 21.08 C:N 8.8:1 9.2:1 9.2:1 9.3:1 8.8:1 6.6:1 6:1 19:1 C:N:Pb 33:4:1 35:4:1 nm nm nm nm 85:14:1 139:7:1 ora fCv n as Studies, Karst and Cave of Journal Isotopes d13C 237.3 236.4 235.1 236.9 242.7 220.5 237.1 nm d15N 1.2 2.5 2.7 226.5 4.2 16.1 29.9 nm Air Chemistryc J D.S.

[H2S] ppm ,0.2 ,0.2 ,0.2 2±4.3 0.4 nm nm 17±25 ONES [CO2] ppm 1000 1000 1300 1600 .3000 nm nm 3300 ..L E.H. , a Sampling locationsare given in Figure 1. GS 5 Grotta Sulfurea, PC 5 Pozzo dei Cristalli, RS 5 Sulfureo, GM 5 Grotta Madonna. b Rounded to nearest whole number. c Based on gas measurements at sample site in 2005±2006.

nm 5 Not measured. YON , AND uut2008 August ..M J.L. ACALADY N 83 GEOMICROBIOLOGY OF BIOVERMICULATIONS FROM THE FRASASSI CAVE SYSTEM,ITALY detected in a few regionswith low autofluorescence, of the library, which suggests it is an important member of indicating at least some active microbial populations the biovermiculation community. Phylogenetic analysis (results not shown). Scanning electron microscopy (SEM) (Fig. 6) shows that CV45 groups in an unnamed clade (Fig. 4a) revealed the presence of clay minerals and within the Gammaproteobacteria. Figure 6 also includes corroded calcite grains. We also observed abundant phylotype CV44 (2 clones) and the iron-oxidizing acido- prokaryotesand noted the presence of larger eukaryotic philic isolate m-1, which was originally misclassified as cells. In some regions, amorphous and filamentous Acidithiobacillus ferrooxidans (Johnson et al., 2001). The microbial biofilm material coated mineral grains(Fig. 4a). closest BLAST match and phylogenetic neighbor to CV45 isclone H6, sequenced from acid mine drainage (Hao et al., 16S rRNA CLONE LIBRARY 2007), an environment in which the primary electron We created a clone library of bacterial 16S rRNA genes donorsare reduced iron and sulfur. Numerousother from biovermiculation sample GS03-5, collected in Grotta sequences from acid mine drainage are also found in the Sulfurea in 2003. Thissample had pH 5.6, and wascollected clade as shown in Figure 6. These phylogenetic relation- from the same location as the samples for geochemical and ships suggest that CV45 may be a sulfur or iron oxidizer. isotopic analysis (Table 2). The 67 non-chimeric 16S rRNA Analysis of another phylotype, CV10, suggests that it is a sequences were analyzed by adding NAST-aligned sequenc- sulfur-oxidizer (Fig. 7). CV10 is closely related to Thioba- esto a phylogenetic tree containing .150,000 bacterial cillus species within the Betaproteobacteria. Named Thio- sequences using the ARB_parsimony tool (Ludwig et al., bacillus species in Figure 8 are sulfur-oxidizing autotrophs 2004). The resulting 48 phylotypes (defined as monophyletic (Drobner et al., 1992; Beller et al., 2006), including cladeswith .98% sequence identity) were consistent with Thiobacillus thioparus, which isimportant in the sulfidic taxonomy inferred based on BLAST similarities. Taxonom- Movile Cave, Romania (Vlasceanu et al., 1997). Figure 7 ic affiliations and closest BLAST matches (Altschul et al., also includes the heterotrophic, sulfur-oxidizing genus 1990) of the clonesare summarized in Table 1. Clones Spirillum, (Podkopaeva et al., 2006), several other sulfur- grouped within 15 major bacterial lineagesincluding the oxidizing isolates, and Frasassi stream biofilm clones Gamma(beta)-, Alpha-, and Deltaproteobacteria, Acido- sequenced by Macalady et al. (2006). In addition to the bacteria, Planctomyces, Cytophaga-Flexibacter-Bacteroi- phylogenetic evidence for sulfur-oxidation in biovermicu- des, Verrucomicrobia, Nitrospirae, Actinobacteria, and lations presented here, Hose et al. (2000) observed growth Chloroflexi (Fig. 5a). More than 10% of the clonesbelong in both thiosulfate and sulfide enrichment cultures to deeply-branching, phylum-level cladeswith no cultivated inoculated with biovermiculationsfrom sulfidicCueva de representatives, including TM7, OP11, RCP2-18, WS3, and Villa Luz, Mexico. For other sequences in the clone library SPAM. Further phylogenetic analyses were conducted for (Table 1), distant relationships to cultivated species do not phylotypeswhich fell within cladeswith a consistent allow further speculation about their metabolisms. Work in metabolic strategy, or which had close relationships with progress by our group will attempt to enrich microbes from cultivated species. These analyses are shown in Figure 6 sulfidic cave biovermiculations to further explore impor- through 9 and are discussed in the text below. tant metabolisms. We also performed a maximum parsimony analysis on DISCUSSION the genus Nitrospira, which includesfive biovermiculation clonesrepresentedby phylotypesCV22, CV64, and CV82 BIOVERMICULATION COMMUNITIES (Fig. 8). The genus Nitrospira includesnamed species N. Rarefaction analysis of the CV clone library (Fig. 5b) moscoviensis and N. marina, both characterized by indicatesthat biovermiculationsare among the most autotrophic nitrite reduction (Watson et al., 1986; Ehrich diverse microbial communities analyzed thus far from the et al., 1995; Altmann et al., 2003). Many of the other Frasassi cave complex, and the shape of the rarefaction environmental clonesin the Nitrospira were sequenced curve indicatesthat additional diversity in the biovermicu- from nitrifying environments. The presence of this group lations remains to be described in future efforts. The suggests that oxidation of reduced nitrogen compounds majority of 16S rRNA gene sequences retrieved from the may be an important type of microbial energy generation biovermiculation sample are distant (,90% identity) from in biovermiculation communities. cultivated isolates, and many are distant from environ- Organotrophy isanother potential meansfor energy mental sequences as well (Table 1). This makes it difficult production in biovermiculations. Phylogenetic analysis of to hypothesize about potential metabolisms for most of the CV109 (Fig. 9) shows that it groups in the Nevskiaceae biovermiculation clones. However, inferences about the family, related to the organotrophic genera Nevskia and physiology of certain clones can be made on the basis of Hydrocarboniphaga (GloÈckner et al., 1998; Palleroni et al., geochemistry and phylogenetic relationships. 2004) aswell asother hydrocarbon-degrading isolates. We performed a maximum parsimony analysis target- In addition to the phylotypes discussed above, the clone ing CV45, the most abundant phylotype in the clone library contained diverse members of the phyla Acidobac- library. CV45 represents seven clones, comprising over 10% teria and Actinobacteria. Membersof thesetwo lineageswere 84 N Journal of Cave and Karst Studies, August 2008 D.S. JONES, E.H. LYON, AND J.L. MACALADY

Figure 2.Biovermiculations from the Frasassi cave system.(A) Biovermic ulations covering cave walls in Grotta Sulfurea.(B) Biovermiculations covering an isolated patch of limestone surrounded by thick gypsum wall crust, in Pozzo dei Cristalli.Long arrow indicates biovermiculations and fat arrows indicate white gypsum crust. (C) Biovermiculations from Grotta Sulfurea.Note the absence of biovermiculations on chert nodules (white arrows).(D) Biover miculations from Ramo Sulfureo.(E) Sample PC06-106, from Pozzo dei Cristalli.This sample was forming below the high water mark within 50 cm above the cave stream surface.(F) Sample GM05-1.Vermiculations from the Grotta del Mezzogiorno-Grotta di Frasassi complex are over 300 meters above the current water table.

Journal of Cave and Karst Studies, August 2008 N 85 GEOMICROBIOLOGY OF BIOVERMICULATIONS FROM THE FRASASSI CAVE SYSTEM,ITALY

these ratios along with measurements of autotrophic versus heterotrophic production to conclude that organicsfrom Cesspool Cave have low nutritional quality. Compared to Cesspool Cave, stream biofilms from Frasassi have been considered a nutritious energy source for heterotrophic organisms (Galdenzi and Sarbu, 2000; Engel et al., 2001). Although biovermiculation C/N ratiosare not aslow as those of stream biofilms (Table 2), 9:1 is a low C/N ratio compared with many detrital organics(e.g., Taylor and Roff, 1984; Janssen, 1996). Additionally, C/N/P ratios indicate phosphorous is abundant in biovermiculations. These results suggest that biovermiculations could repre- sent an important food resource for terrestrial inverte- brates on the cave walls. Further research is currently underway by our group to explore trophic relationships between biovermiculationsand cave wall invertebrates. Nitrogen isotopic ratios indicate that there are two different nitrogen sources for the biovermiculation com- munities. Biological nitrogen fixation has little fraction- Figure 3.Carbon and nitrogen stable isotopic ratios of ation associated with it (+0.7%, Sharp, 2007). Four of the organic matter from sulfidic cave zones and outside samples have d15N valuesgrouped near zero (Fig. 3), environments.Biovermiculations analyzed in this study are suggesting that N2 fixation isthe dominant mechanismfor marked by black squares, and values associated with sample nitrogen supply to the communities. In sharp contrast, names are given in Table 2.Bars indicate the range of values sample PC06-106 has a d15N value of 226.5%. Thisisin reported by Galdenzi and Sarbu (2000) for other sample the range of nitrogen isotopic ratios from extremely acidic types (n = 18 for surface samples, n = 32 for sulfidic cave wall biofilmsreported by Galdenzi and Sarbu (2000) samples).Note the clear separation between values from and Vlasceanu et al. (2000). Stern et al. (2003) hypothesized sulfidic regions compared to surface and cave entrances. that depleted d15N values on sulfidic cave walls could result from volatilization of NH3 gas from streams, with subsequent assimilation by subaerial microbial communi- reported in biovermiculationsfrom sulfidic Cueva de Villa ties. If so, biovermiculations near cave streams may Luz (Hose and Northup, 2004). In our clone library, assimilate isotopically depleted ammonium, while those Acidobacteria and Actinobacteria represent 10.5% and 3% forming farther away or near more diluted watersprimarily of clones, respectively (Table 1; Fig. 5a). In addition to the fix N2 from the cave air. Our data support this hypothesis. prokaryotic community in biovermiculations, we also Near-zero d15N valuesfor Grotta Sulfurea biovermicula- observed larger nucleated cellswith morphologiestypical tions sampled just above the water table are consistent with for protists and fungal hyphae. We also noted several types of low cave air H2S concentrationscompared to sample macroinvertebratesliving on the cave wallsin closeproximity PC06-106 (Fig. 3, Table 2). Work in progress will test the to biovermiculations, including spiders (Nesticus sp.), isopods relationship between stream degassing and the d15N of wall (Androniscus sp.), springtails (Order Collembola), annelid biofilmsincluding biovermiculations. worms(ClassOligochaeta), and nematodes(Phylum Nem- atoda) (Sharmishtha Dattagupta, unpublished results). INFLUENCES ON CAVE FORMATION Egemeier (1981) theorized that sulfidic cave formation ELEMENTAL AND ISOTOPIC COMPOSITION occursprimarily by replacement-solution, in which hydro- Based on elemental analysis, Frasassi biovermiculations gen sulfide is oxidized to sulfuric acid in condensation have an organic carbon content between 5.7 to 25% percent dropletson cave wallsand ceilings.Sulfuric acid reacts dry weight (Table 2), in the upper range of valuesreported with limestone to produce gypsum, which builds up as for soil ``A'' horizons and as high as values for some peat thick crusts that eventually detach and accumulate on the soils (Singer and Munns 2002). Carbon to nitrogen (C/N) cave floor or dissolve in cave streams. Subsequently Engel ratios are similar across all samples, roughly 9:1, and et al. (2003) suggested that the buildup of gypsum crusts carbon to nitrogen to phosphorous (C/N/P) ratios are may protect limestone walls from sulfide vapors, and that roughly 34:4:1. Engel et al. (2001) measured C/N ratios of subaerial corrosion is fastest at exposed limestone surfaces. organics from Cesspool Cave, VA, and report ratios of 13.5 This hypothesis suggests an important role for biovermi- and 16.06 for stream and wall biofilms, respectively. Low culation microorganisms in subaerial sulfuric acid speleo- C/N ratiosare indicative of a high quality food source for genesis. In sulfidic zones of the cave system, nearly all detrital feeders(Taylor and Roff, 1984); Engel et al. used exposed limestone surfaces are covered with biovermicula- 86 N Journal of Cave and Karst Studies, August 2008 D.S. JONES, E.H. LYON, AND J.L. MACALADY

Figure 5.Bacterial diversity of biovermiculation sample GS03-5, based on 16S rRNA gene sequencing.(A) Percentages of biovermiculation clones in major bacterial lineages.(B) Rarefaction curves depicting observed microbial richness of Frasassi microbial communities.Operational taxonomic units (OTUs) are defined by .98% sequence identity.Biovermiculation library CV (n =67, 48 OTUs), this study; stream biofilms WM (n=86, 56 OTUs) and zEL (n=78, 36 OTUs), Macalady et al.(2006); snottite sample RS2 (n=107, 9 OTUs), Macalady et al.(2007).Error bars represent 95% confidence intervals based on variance of OTUs drawn (y-axis) by clone library size (x-axis), from 100 randomizations/sample.

tions, even isolated patches completely surrounded by gypsum crust (Fig. 2b). Sulfur-oxidizing microorganisms in stream biofilms have been shown to accelerate cave formation (Engel et al., 2004b; Joneset al., 2006), suggesting that acid-producing microorganisms in biover- miculationscould play a similar role. Biovermiculation clones retrieved in this study include close relatives of

r Figure 4.(A) SEM image of biovermiculation matrix. Small arrows indicate putative amorphous extracellular photomicrographs showing the range of DAPI-stained cell organic material, and large arrows indicate mineral grains. morphologies observed in Frasassi biovermiculations.Arrows Scale bar is 20 mm (B and C) Representative fluorescence indicate cells linked into filaments.Scale bars are 5 mm. Journal of Cave and Karst Studies, August 2008 N 87 GEOMICROBIOLOGY OF BIOVERMICULATIONS FROM THE FRASASSI CAVE SYSTEM,ITALY

Figure 6.Maximum parsimony phylogram of 16S rRNA gene sequences in an unna med lineage within the Gammaproteo- bacteria.GenBank accession numbers are listed with the sequence names.M aximum parsimony bootstrap values .70% are shown.Number of clones represented by each phylotype is given in parenthe ses. sulfur- and nitrogen-oxidizing species that would be who proposed that they form during the drying of wet, expected to produce strong acids (Table 1). In addition, sediment-covered cave walls as the sediments shrink and many organoheterotrophic microorganisms, including fun- flocculate. Different typesoccur depending on the initial gi, produce organic acidsasa by-product of their oxidative water to sediment ratio, although other factors may or fermentative metabolism. Because biovermiculations influence pattern morphology (Hill and Forti, 1997). In occur directly on limestone walls, any organic or inorganic the Bini et al. model, many materialscan form vermicu- acidsproduced by microorganismswouldinteract directly lations. Sources include residual silicate mineral grains with the carbonate wall surface and contribute to remaining after corrosion of the parent limestone and speleogenesis. Significantly, subaerial limestone dissolution allochthonousmaterial transportedto the cave interior by in Frasassi has been shown to be occurring at roughly the air or water. Based on our observations, we hypothesize that same rate as subaqueous limestone dissolution, at least biological processes are very important in the formation of near the water table where walls are exposed to sulfide biovermiculationsin sulfidic zonesof the cave system. We degassing (Galdenzi et al., 1997). have identified two typesof potentially important biological effects, (1) microorganisms may directly enrich the biover- BIOVERMICULATION FORMATION miculation matrix by supplying organic material, and (2) The generally accepted model for vermiculation forma- micro- and macroorganisms may play an active role in tion in epigenic caveswasdescribed by Bini et al. (1978), trapping and binding sediment particles. 88 N Journal of Cave and Karst Studies, August 2008 D.S. JONES, E.H. LYON, AND J.L. MACALADY

Figure 7.Maximum parsimony phylogram of 16S rRNA gene sequences related t o the genus Thiobacillus in the Betaproteobacteria.GenBank accession numbers are listed with the seque nce names.Maximum parsimony bootstrap values .70% are shown.Number of clones represented by each phylotype is given in pa rentheses.

The biovermiculationswe sampled have a high organic different surfaces (Hedges, 1993), the biovermiculations we matter content (between 5±25% organic carbon). Further- observed occur on limestone surfaces but not on chert more, biovermiculation carbon and nitrogen in sulfidic (Fig. 2c and d). Thisimplieseither a requirement for zones is isotopically distinct from sources outside of the buffering offered by the carbonate rock or a difference in cave system (Fig. 3), indicating that the organic matter is the mineral surface properties that affect adhesion of the produced in situ. Hose and Northup (2004) suggested that biovermiculations. biovermiculationsmight form via microbial trapping of If biological activity were important for biovermicula- sediments in a manner analogous to stromatolite forma- tion formation, we would expect to see more abundant tion. Fluorescence microscopy and SEM imaging of biovermiculationsin regionsof higher biological produc- biovermiculationsin thisstudyrevealed filamentous tivity. If this hypothesis is correct, the richest development microorganisms and amorphous extracellular organic of biovermiculations should be closest to the sulfidic water matter entwining and binding mineral grainsin the table. Such a relationship is broadly consistent with biovermiculation matrix (Fig. 4a). Nematodesin the observations we have made throughout the Frasassi cave biovermiculations may also play an important role because system. However, two factors may impose limitations on many species of nematodes are known to agglutinate this pattern. In the areas with the highest sulfide gas sediments (Riemann and Helmke, 2002). Whereas vermic- concentrations, biovermiculation formation is in competi- ulationshave been previouslyobservedon a variety of tion with gypsum crust formation. Therefore, growth of Journal of Cave and Karst Studies, August 2008 N 89 GEOMICROBIOLOGY OF BIOVERMICULATIONS FROM THE FRASASSI CAVE SYSTEM,ITALY

Figure 8.Maximum parsimony phylogram of 16S rRNA gene sequences within th e Nitrospirae.GenBank accession numbers are listed with the sequence names.Maximum parsimony bootstrap values .70% are shown.Number of clones represented by each phylotype is given in parentheses. biovermiculations might be outstripped by rapid gypsum culationsbegan to grow into the clear area after only a few accumulation in some locations (e.g., Fig. 2b). Addition- weeks. ally, seasonal and decadal high water events may wash Previous authors have described microorganisms in away biovermiculationsforming within several metersof vermiculations from non-sulfidic caves and speculated the average stream level. Nonetheless, we often observed about biological influenceson their formation (Anelli and biovermiculationsbeginning to form below high water Graniti, 1967; Camassa and Febbroriello, 2003). The work marks(for example, samplePC06-106, Fig. 2e). This presented here offers evidence that microbial activity is observation is consistent with high biological productivity important for creating biovermiculations in sulfidic caves. and rapid formation of biovermiculationsin the presence However, the origin of the geometric patternsthat make of high sulfide gas concentrations, and suggests that there biovermiculations so visually striking remains to be are conditionsunder which biovermiculationsform more established. Our study does not exclude the possibility rapidly than gypsum crusts. Hose and Northup (2004) that the physical morphology of biovermiculations is cleared biovermiculationsfrom a patch of cave wall in influenced by drying processes described by Bini et al. sulfidic Cueva de Villa Luz and noted that new biovermi- (1978). Our work does suggest that enhanced biological 90 N Journal of Cave and Karst Studies, August 2008 D.S. JONES, E.H. LYON, AND J.L. MACALADY

Figure 9.Maximum parsimony phylogram of 16S rRNA gene sequences related t o the genera Nevskia and Hydrocarboniphaga within the Gammaproteobacteria.GenBank accession numbers are listed wi th the sequence names. Maximum parsimony bootstrap values .70% are shown. activity in sulfidic caves enables biovermiculation forma- and carbonate dissolution, thereby contributing to cave tion on a time scale of years to decades, and that a formation. The 16S rRNA gene sequences analyzed to date significant fraction of their dry mass is organic matter indicate that biovermiculation communitiescan be ex- produced in situ. Thus, in sulfidic cave environments, tremely diverse, and that they contain representatives of biovermiculation morphology could also be influenced by deeply branching, phylum-level lineageswith no cultivated microbial growth patterns. representatives. Further work isrequired to elucidate the microbial role CONCLUSIONS AND FUTURE DIRECTIONS in biovermiculation formation and limestone dissolution in sulfidic caves. Biovermiculations collected in different Carbon and nitrogen isotope values indicate that within areas of the cave system and in other sulfidic caves should sulfidic zones, the abundant organic matter in biovermi- be compared to determine whether they have a character- culationsoriginatesfrom in situ chemolithotrophic bacte- istic assemblage of microbial species. Mineralogical anal- rial productivity rather than from sources outside the cave yses are needed to better describe the composition and system. The high density of microbial cells and the likely provenance of inorganic constituents of biovermiculations presence of sulfur and nitrite oxidizing bacteria suggests such as clay minerals. Future research should also include that biovermiculationsmay play a role in acid production comparisons with vermiculations sampled in non-sulfidic Journal of Cave and Karst Studies, August 2008 N 91 GEOMICROBIOLOGY OF BIOVERMICULATIONS FROM THE FRASASSI CAVE SYSTEM,ITALY environmentsto determine whether similar biogeochemical Ehrich, S., Behrens, D., Lebedeva, E., Ludwig, W., and Bock, E., 1995, A new obligately chemolithoautotrophic, nitrite-oxidizing bacteria, processes, including sulfur cycling, are responsible for their Nitrospira moscoviensis sp. nov. and its phylogenetic relationship: formation in both sulfidic and non-sulfidic karst systems. Archivesof Microbiology, v. 164, p. 16±23. Egemeier, S.J., 1981, Cavern development by thermal water: National Speleological Society Bulletin, v. 43, p. 31±51. ACKNOWLEDGEMENTS Engel, A.S., Porter, M.L., Kinkle, B.K., and Kane, T.C., 2001, Ecological assessment and geological significance of microbial communities from Cesspool Cave, Virginia: Geomicrobiology Journal, v. 18, p. 259±274. We thank S. Dattagupta for field assistance, especially Engel, A.S., Stern, L.A., and Bennett, A., 2003, Condensation on cave with invertebrate collection and identification. C. Junium walls: Implications for cave enlargement and sulfuric acid speleogen- and J. Fulton performed the isotopic analyses. We thank esis: Goldschmidt Conference, Kurishiki, Japan, Geochemica et Cosmochimica Acta, v. 67, A455 p. A. Montanari for logistical support and the use of facilities Engel, A.S., Porter, M.L., Stern, L.A., Quinlan, S., and Bennett, P.C., at the Osservatorio Geologico di Coldigioco (Italy) and S. 2004a, Bacterial diversity and ecosystem function of filamentous Galdenzi and L. Hose for insightful discussions. S. microbial mats from aphotic (cave) sulfidic springs dominated by chemolithoautotrophic ``Epsilonproteobacteria'': FEMS Microbiolo- Mariani, S. Cerioni, M. Mainiero, and S. Recanatini gy Ecology, v. 51, p. 31±53. provided expert field assistance. We also thank K. Meyer Engel, A.S., Stern, L.A., and Bennett, P.C., 2004b, Microbial contribu- for help with SEM analyses and T. Stoffer and I. tions to cave formation: New insights into sulfuric acid speleogenesis: Geology, v. 32, p. 369±372. Schaperdoth for laboratory assistance. This study was Galdenzi, S., 1990, Un modello genetico per la Grotta Grande del Vento, funded by the Biogeosciences Program of the National in Il Carsismo della Gola di Frasassi, Mem. Ist. It. Spel., Vol. II, Science Foundation (EAR 0311854) and the NASA Astro- No. 4, p. 53±64. Galdenzi, S., Cocchioni, M., Marichetti, L., Amici, V., and Scuri, S., 2007, biology Institute (PSARC, NASA award NNA04CC06A). 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Journal of Cave and Karst Studies, August 2008 N 93 S. Galdenzi, M. Cocchioni, L. Morichetti, V. Amici, and S. Scuri ± Sulfidic ground-water chemistry in the Frasassi Caves, Italy. Journal of Cave and Karst Studies, v. 70, no. 2, p. 94±107. SULFIDIC GROUND-WATER CHEMISTRY IN THE FRASASSI CAVES, ITALY

SANDRO GALDENZI1*,MARIO COCCHIONI2,LUCIANA MORICHETTI2,VALERIA AMICI2, AND STEFANIA SCURI2

Abstract: A year-long study of the sulfidic aquifer in the Frasassi caves (central Italy) employed chemical analysis of the water and measurements of its level, as well as

assessments of the concentration of H2S, CO2,andO2 in the cave air. Bicarbonate water seepage derives from diffuse infiltration of meteoric water into the karst surface, and contributesto sulfidic ground-water dilution, with a percentage that va riesbetween 30% and 60% during the year. Even less diluted sulfidic ground water was found in a localized area of the cave between Lago Verde and nearby springs. This water rises from a deeper phreatic zone, and itschemistry changesonly slightly with the seasonswith a contr ibution of seepage

water that doesnot exceed 20%. In order to understand how the H 2S oxidation, which is considered the main cave forming process, is influenced by the seasonal changesin the cave hydrology, the sulfide/total sulfur ratio was related to ground-water dilution and air composition. The data suggest that in the upper phreatic zone, limestone corrosion due to

H2S oxidation is prominent in the wet season because of the high recharge of O2-rich seepage water, while in the dry season, the H2S content increases, but the extent of oxidation is lower. In the cave atmosphere, the low H2S content in ground water during the wet season inhibits the release of this gas, but the H2S concentration increases in the dry season, favoring its oxidation in the air and the replacement of limestone with gypsum on the cave walls.

INTRODUCTION water samples in the laboratory. In order to understand the influences of surface recharge on the sulfidic ground-water The Frasassi Caves are one of the best examples of caves characteristics, we identified three sampling points for with an active flow of H2S-rich water. Sulfuric acid derived seepage water and four for sulfidic ground water, two in from the oxidation of H2S isconsidered the main cave- the cave and two at the emergences. The choice of these forming agent, and the oxidation process is enhanced by the sampling points was based on preliminary field analysis of supply of oxygen transported in seepage water or directly in water characteristics in different zones of the cave. The the cave atmosphere (Galdenzi, 1990). The active speleoge- surface water in the Sentino River was sampled in order to netic processes can be directly observed in the lower, sulfidic ascertain whether the river directly influences the ground- partsof the cave. Experimental measurement using lime- water characteristics. During the period November 2001± stone tabletsplaced in the cave for five yearsdetermined the December 2002, water wassampledmonthly and analyzed present rate of sulfuric acid speleogenesis (Galdenzi et al., in order to identify the most significant seasonal changes. 1997). The weight loss reached values of up to In addition, some further samples coming from other cave 22 21 20 mg cm yr , both in the tabletsexposedto H 2S vapors lakeswere studied. above the water table and those placed directly in the sulfidic The water temperature and conductivity were measured ground water. Sulfide oxidation involvesbacterial activity; in situ using probes (Delta OHM, Italy). In the laboratory, and organic matter produced by bacteria isthe main source chemical and physicochemical parameters of the water of food for the fauna inhabiting the sulfidic sections of the samples were measured using the methods shown in cave (Sarbu et al., 2000). Table 1. Detailed study of water chemistry and water-level To facilitate the interpretation of seasonal changes in changes, carried out over the course of a year in different water chemistry, temperature, conductivity and water level sampling points inside the cave, identified seasonal changes of sulfidic ground water were measured continuously over in the ground-water characteristics and formed the basis the same period. A remote logger for temperature and for assessing their influence on the cave environment and conductivity (DAS, Italy) waslocated in a small artificial the processes guiding cave development. The purpose of tunnel near the springs. This probe was exposed to direct thispaper isto document the ground-water characteristics contact with the river water during the main floodsof the that influence the cave environment and speleogenesis.

* Corresponding author. METHODS 1 Viale Verdi 10, 60035 Jesi - Italy, [email protected] 2 Department of Hygiene and Health-Environmental Sciences± Universityof The field work was based on field measurement of Camerino ± Italy, [email protected], [email protected], valeria. environmental parametersand on the chemical analysisof [email protected], [email protected] 94 N Journal of Cave and Karst Studies, August 2008 S. GALDENZI,M.COCCHIONI,L.MORICHETTI,V.AMICI, AND S. SCURI

Table 1.Chemical and physicochemical parameters of water samples and mea surement methods. Parameter Method Temperature Digital thermometer, Hanna Instruments model HI805 Conductivity Conductivity meter, Crison model 524 pH Digital pH-meter, Hanna Instruments model HI 824 Hardness Titrimetric method, Standard Methodsa 2340 C Calcium Titrimetric method, Standard Methodsa 3500 B Magnesium Calculation method, Standard Methodsa 3500 B Sodium, Potassium Flame photometric method with Perkin Elmer model 3300 Chloride, Sulfate Ion chromatography with Dionex 120 DX Carbon dioxide Titrimetric method and calculation method, Standard Methodsa 4500 C/D Bicarbonate Titrimetric method, Standard Methodsa 2320 B Sulfide Iodometric method, Standard Methoda 4500 F Dissolved Oxygen Iodometric method, Standard Methodsa 4500 C Oxygen Saturation % Calculation method BOD Oxygen consumed in a five day SM 5210 B a Standard Methods for the Examination of Water and Wastewater 20th Edition. river itself. Unfortunately, sensor failure compromised the steps in the deepening of the river valley and related base conductivity data for a long period. level. The lower levels, present mainly in the Fiume-Vento Two water-level loggers(Meccatronica, Italy; measure- system, originated during the Middle-Upper Pleistocene, as ment range: 0±2.5 m) were located inside the cave to did the alluvial deposits just outside the Frasassi Gorge monitor changesin ground-water level in different zonesof (Bocchini and Coltorti, 1990). These lower cave levels host the cave. In the following years, measurements of water- speleothems dating back 200,000 years BP (Taddeucci et table levels were repeated at different sites to ascertain the al., 1992) and developed under geomorphological and timing of water-level changesbetween the different zones hydrogeological conditions similar to current ones. The of the cave and the river. upper levelsdeveloped mainly in the Buco Cattivo and

The concentrationsof H 2S, CO2,O2 and SO2 in the cave air were measured with a hand pump (Gastec Corporation, Japan) and detector tubes(measurement range: H2S, 0.1±4 and 0.25±120 ppm; CO2, 300± 5,000 ppm; O2, 3±24%; SO2, 0.05±10 ppm). Measurements of H2S, CO2, and O2 were repeated throughout the sampling period at the same time as water chemistry analyses. Sulfur dioxide (SO2) wasnot detected in the cave atmosphere, and measurements were repeated occasional- ly. Two monthly measurements were missed because of difficulties in accessing the sampling sites. Some measure- mentswere alsodone to verify changesof the air composition in the upper non-sulfidic cave sections.

GEOLOGIC SETTING

The Frasassi Caves, one of the most famous show caves in central Italy, are located on the eastern slope of the Apennine mountain chain, 40 km from the Adriatic Sea. They are developed in the small area around the Frasassi Gorge, a 500 m deep and 2 km long canyon that the Sentino River cutsin a small anticline ridge. The caves consist of over 25 km of cave passages located at altitudes between 200 (corresponding to the riverbed) and 500 m (Fig. 1). The cave complex consists of superimposed and interconnected levels, the genesis of which is related to Figure 1.Cave map, with water sampling locations. Journal of Cave and Karst Studies, August 2008 N 95 SULFIDIC GROUND-WATER CHEMISTRY IN THE FRASASSI CAVES,ITALY

Figure 2.Geologic cross-section of the Frasassi Anticline.The marl laye r in the eastern anticline limb causes ground-water flow toward the gorge.CM) Calcare Massiccio Formation; JL) low permeabil ity Jurassic limestones; M) Calcare Maiolica Formation; MF) Marne a Fucoidi Formation; S) Scaglia Bianca and Scaglia Rossa Formations.

Mezzogiorno-Frasassi caves. In these caves, the features Overlying the Calcare Massiccio Fm. is a 60 m thick originated by sulfidic water near the water table are less formation of Jurassic limestone (Bugarone Fm.), contain- common, probably because they evolved under a partly ing a marly and a cherty level. Thislow-permeability different hydrogeological setting. formation can influence ground-water drainage locally. In The area ismountainous,with altitudesranging the eastern limb of the anticline, however, a fault permits between 200 m at the bottom of the valleysto about hydraulic continuity between the Calcare Massiccio and 1000 m on the surrounding peaks. The climate is Apenni- the overlying, permeable Cretaceouslimestones(Fig. 2). nic subcontinental, and the annual average temperature is The Cretaceousformationsare thin-layered, permeable ,13 uC, while the annual temperature range reaches20 uC. limestones (Maiolica Fm., upper Jurassic ± lower Creta- The average rainfall is ,1000 mm yr21 with maximum ceous, and Scaglia Fm., upper Cretaceous ± Eocene) with precipitation generally in autumn and spring. Evaporation an interbedded, low-permeability 50 m thick marl forma- typically exceedsprecipitation in summer. Stormsoften tion (Marne a Fucoidi, middle Cretaceous). The Marne a occur at the end of summer, and sometimes these months Fucoidi Fm. isthe local aquiclude and definesa recharge can be the rainiest of the year. In the winter, snow often area of about 5 km2 feeding the main aquifer hosted in the covers the mountain surface for short periods. Calcare Massiccio Fm. and, partly, in the Maiolica Fm. The Sentino River reaches the Frasassi gorge 40 km from (Fig. 2). itssource. The recharge area extends ,250 km2. Limestone The ground-water drainage isinfluenced by the geologic outcropsin ,40% of the basin, claystone and sandstone in setting. In the recharge area, diffuse infiltration prevails, ,30% of the basin, and the remaining surface is covered by and the drainage network islimited, with only temporary alluvial and detrital deposits (Dramis et al., 1976). flowsin a few channelsafter major rainstorms.A few low discharge springs originate from small aquifers perched on THE KARST AQUIFER the Jurassic or Cretaceous marly levels. Most seepage water, however, reachesthe main aquifer, whoserecharge The steep cliffs of the Frasassi Gorge clearly show the area corresponds to the core and the eastern limb of the geologic structure of the region (Fig. 2). The Calcare anticline (Fig. 2). Ground water in thisaquifer istypically

Massiccio Fm., a thick, massively bedded, lower Jurassic enriched in H2S and salts due to the rise of mineralized limestone, constitutes the core of the anticline and outcrops water in the core of the anticline. The spring area is located across the whole gorge. It is a very pure limestone, in the eastern side of the gorge where the down-cutting of consisting mainly of wackestone and packstone facies the river valley caused the erosion of the marly cover in the without any significant clay or silica minerals. It is very limb of the anticline (Fig. 3). permeable, due to high syngenetic porosity and a well- The cavesdevelop in the zone between the core and the developed network of fractures, and it hosts the main eastern side of the anticline in the Calcare Massiccio Fm. aquifer in the area. and partly in the Maiolica Fm. Here the ground-water 96 N Journal of Cave and Karst Studies, August 2008 S. GALDENZI,M.COCCHIONI,L.MORICHETTI,V.AMICI, AND S. SCURI

Figure 3.View of the Frasassi Gorge.CM) Calcare Massiccio Formation; M) C alcare Maiolica Formation; MF) Marne a Fucoidi Formation; S) Scaglia Bianca and Scaglia Rossa Formations; Pl) continental Pleistocene deposits. drainage isconcentrated near the large fault dividing the vadose zone and in small aquifers perched on interbedded two formations(Fig. 2). The marly cover in the limb of the marls. This water has a low salinity (about 200± anticline facilitates drainage toward the springs. 400 mg L21), low sulfate, and high dissolved oxygen The hydraulic gradient for the ground water islow due (about 0.32 mmol L21). to the high permeability of the host rock and water flow is The sulfidic water is found in the main aquifer. It is cold generally very slow. Flowing sulfidic water is only found in (about 13 uC), and hasa higher salinity than the bicarbo- the eastern part of the cave. The water table is close to the nate water, up to 2 g L21. It isenriched in sodium and river level and can be reached in many locationsin the chloride, and contains sulfate and hydrogen sulfide. These lower section of the cave. dissolved components are acquired before the ground water rises upward, and they are generally believed to be a GROUND WATER consequence of flow through an underlying Triassic anhydrite formation. Isotopic data on d18O, d2H, and GENERAL CHARACTERISTICS OF GROUND WATER tritium discussed by Tazioli et al. (1990) suggested a Ground water in the Frasassi area consists of two main meteoric origin for the sulfidic ground water. These types: bicarbonate and sulfidic, which differ in their authorsestimateda recharge area located at altitudesof chemical composition and origin (Tazioli et al., 1990; 600±1000 m, with a brief residence time in the aquifer. Sighinolfi, 1990; Cocchioni et al., 2003). The bicarbonate The very low water flow in a large part of the cave often water derives from the diffuse infiltration of surface leads to ground-water stratification. A surface layer of meteoric water through the limestone, and is found in the water stays near the surface of the water table due to its Journal of Cave and Karst Studies, August 2008 N 97 SULFIDIC GROUND-WATER CHEMISTRY IN THE FRASASSI CAVES,ITALY

Figure 4.Main Spring.The white color is due to the microbial mats that cove r the floor below the running water. lower salinity. The thickness of this stratified surface layer surface. The sulfidic ground water was sampled both at the can reach 5 m (Cocchioni et al., 2003). springs and at sampling points inside the cave. The conductivity and temperature of the sulfidic water The springs discharge along the river bank very close to are correlated with precipitation (Sarbu et al., 2000). These the river channel. The spring water either flows from the observations indicate that water recharge derived from limestone directly to the river or rises through alluvial surface precipitation dilutes the sulfidic ground water deposits. Preliminary measurements revealed two different (Tazioli et al., 1990; Sighinolfi, 1990). A role of the river spring water types. Most of the springs have the same in the ground-water dilution, however, hasalsobeen conductivity and temperature (13.0±13.5 uC), but a small hypothesized (Tazioli et al., 1990; Ciancetti and Pennac- one rising directly from the limestone (Fissure Spring) had chioni, 1993; Caprari et al., 2001). a temperature about 0.5 uC higher and conductivity about 30% higher than the other springs. These differences persist SAMPLING SITES throughout the seasons. For this reason, two springs were The preliminary field analysis of water characteristics sampled: Main Spring (Fig. 4), which is the largest surface was used to identify seven different sampling points, three spring along the river, and Fissure Spring (Fig. 5). for seepage water and four for sulfidic ground water Sampling of the Fissure Spring was not possible on two (Fig. 1). In addition, the Sentino River water wasanalyzed occasions due to high river levels. upstream from the sulfidic springs. Seepage water was In the cave, sulfidic water was sampled in Lago Verde collected inside the touristic part of the cave at three and Ramo Sulfureo. Lago Verde is a deep stagnant sulfidic different sampling points (Abisso Ancona, Lago Smeraldo, pool, fed from the bottom (Fig. 6), where the water has Sala Pagliai) located at increasing distances from the chemical characteristics similar to those of Fissure Spring. 98 N Journal of Cave and Karst Studies, August 2008 S. GALDENZI,M.COCCHIONI,L.MORICHETTI,V.AMICI, AND S. SCURI

Figure 5.Fissure Spring.Thissmall spring rises in the same Figure 6.Lago Verde.Thewater was sampled in the surface limestone outcrop as Main Spring, a few meters away. of this stagnant deep lake.

Ramo Sulfureo is the most studied cave room (Galdenzi et 0.2 L min21. Weak seasonal changes occurred in the al., 1997; 1999; Sarbu et al., 2000), with an active flow of chemical parametersexamined (Fig. 7). sulfidic water. Here the water is similar to that of Main Water temperature isabout 13 uC, like the air tempera- Spring, and the direct influence of meteoric water on its ture in the cave. The pH valuesin the AbissoAncona vary temperature and conductivity hasalready been document- between 7.5 and 8.4 and are consistently about 0.4 higher ed (Sarbu et al., 2000). than those of the other sampling points. In April and In addition to these sampling sites, samples were August, the pH in all the locations shows a small decrease collected in other cave lakes. Lago dell'Orsa and Laghi di associated with an increase in dissolved CO2 content. It Lucia proved to be quite interesting and will be described should be pointed out that during these periods there are below. A detailed description of the sampling locations is more tourists visiting the cave, although the relationship provided in Cocchioni et al. (2003), which also provides the between the number of tourists and the CO2 concentration complete set of the data obtained. Data on chemical in the cave iscomplex, and isalsoinfluenced by the direct characteristics of water samples were integrated with the arrival of air with low CO2 from the surface through the continuousmonitoring of the temperature and conductiv- artificial tunnel (Galdenzi and Menichetti, 2002). The total ity of the sulfidic ground water in an artificial tunnel near dissolved ions and the conductivity are very similar in the the springs, and with water-table level monitoring in Lago Abisso Ancona (average values: 251.3 mg L21 and Verde and Ramo Sulfureo. 359 mScm21) and Lago Smeraldo (average values: 247.3 mg L21 and 354 mScm21), while they are a little SEEPAGE WATER lower in the more interior sampling point of Sala Pagliai Seepage water at all the sampling points had uniform (average values: 219.8 mg/L and 314 mScm21). There are characteristics with only minor differences. The flow rate minor differencesin the ion concentrationsbetween Lago of Abisso Ancona dripping water was more variable, while Smeraldo and Abisso Ancona. The Abisso Ancona water Sala Pagliai dripping water had a constant rate of about has a higher concentration of sodium, potassium, chloride Journal of Cave and Karst Studies, August 2008 N 99 SULFIDIC GROUND-WATER CHEMISTRY IN THE FRASASSI CAVES,ITALY

Figure 8.Correlation between rainfall, ground-water level, ground-water temperature, and dilution ratio between rising sulfidic water and descending seepage water.In calculating dilution ratio, the late autumn Lago Verde water was considered not diluted.

Figure 7.Seasonal changes in conductivity (A) and chloride that of Main Spring. All these relationships remain stable concentration (B) in the cave waters. during the year. Cation and anion compositions confirm the differences and sulfate, while the calcium and bicarbonate content is between the two typesof water. Chloride, sodiumand lower. These ionic variations offset each other and the potassium have the same trend described for conductivity waters of the two locations have the same salinity. Sala (Fig. 7). Average values for chloride in Fissure Spring ± Pagliai water hasa lower content of each ion in accordance Lago Verde are almost identical (19.05±19.12 mmol L21), with the lower salinity. The concentration of O2 in the while in Main Spring they are a little higher (12.04 water is consistently high in all the samples and during the mmol L21) than in Ramo Sulfureo (9.75 mmol L21). year the saturation index is between 93±99% (0.31± Sodium and potassium concentrations show the same 0.33 mmol L21). variations. The lower ion concentrations in Ramo Sulfureo The water in the Sentino River was sampled upstream can be related to a more significant dilution with seepage from the sulfidic springs. This calcium bicarbonate water water. hasa salinity of 230±300 mg L 21 without important The sulfate and sulfide concentrations follow the same seasonal changes. The relative constancy of the chemical trend asother ions,but the range of variability iswider, composition and the similarity with the characteristics of depending also on the oxidation-reduction processes the dripping water (Fig. 7) make it difficult to use chemical involving sulfur in the ground water. This subject will be parameters to discuss the river's influence on the sulfidic discussed in detail in the following text. ground-water composition. In contrast, the total hardness has a different trend from other parameters. The highest values are reached in SULFIDIC WATER Main Spring, with low variations(42±47 uF), while the Chemical data on sulfidic water confirmed the prelim- lowest values are in Ramo Sulfureo (30±39 uF). The water inary observations on the existence of two main types of is always undersaturated with respect to gypsum (Satura- sulfidic ground water. Water samples from Lago Verde and tion Index # 1.2 during the whole year in all the sampling Fissure Spring had very similar characteristics during the sites) while the saturation index for calcite is close to 0. The whole year, asdid thosefrom Ramo Sulfureo and Main ground-water temperature in the sulfidic ground water Spring (Fig. 7). The water temperature in Main Spring is stays a little higher compared with the seepage water and almost constant throughout the year, varying between 13.1 the cave atmosphere. In Ramo Sulfureo, the temperature and 13.7 uC. The water in Fissure Spring has the same decrease as a consequence of rapid water recharge after seasonal trend, but is always 0.5±1 uC higher. Sulfidic rainfall hasbeen well documented (Sarbu et al., 2000). The ground water is slightly warmer than both the seepage data acquired at the springs show a general decrease in water and the cave atmosphere. ground-water temperature during the rainy winter season Average conductivity valuesare the samefor Lago (Fig. 8). The sharp temperature decrease in the graph is Verde and Fissure Spring (2360 mScm21) and similar for due to the direct invasion of cold river water during major the two other waters(1482±1617 mScm21). Sulfidic water floodsin the artificial tunnel fitted with the temperature in Ramo Sulfureo has slightly lower salinity compared to logger. 100 N Journal of Cave and Karst Studies, August 2008 S. GALDENZI,M.COCCHIONI,L.MORICHETTI,V.AMICI, AND S. SCURI

Figure 9.Replacement gypsum on the cave walls.A continuous rim of slushy g ypsum covers the cave walls exposed to acidic vapors.The dark color is likely due to a layer of microbially produced orga nic matter (image width ,10 cm).

GROUND-WATER STRATIFICATION weak, but significant, differences from both the seepage water sampled in the cave and the river water. A small In many isolated pools of the cave, the water increase in Na+ and Cl2 content could be attributed to composition is affected by local factors. Because the contamination from small amounts of washing water used ground-water flow isgenerally very slow,ground-water in the show cave or directly from the sulfidic water. stratification occurs in many places within the cave. The However, the low Ca2+ and bicarbonate content correlates chemistry of the surface layer of water resembles that of well with the characteristics of the seepage water in this seepage water or an intermediate composition between that area, which hasunusuallylow contentsof theseions of sulfidic and seepage water. It stays near the surface of (Cocchioni et al., 2003). the water table due to itslower salinity, and itsthickness differsin each lake. Thissurface ground water isgenerally LAGHI DI LUCIA rich in dissolved O2, although in the Laghi di Lucia H2S The Laghi di Lucia are two small sulfidic lakes was detected. Brief descriptions of two lakes sampled in (,20 m2) that can be reached by descending a 20 m deep this study (Lago dell'Orsa and Laghi di Lucia) follow shaft. They are fed from the bottom and slow water flow below. can generally be observed. Widespread corrosion of the

limestone above the lakes is due to H2S vaporsthat oxidize LAGO DELL'ORSA and produce abundant slushy gypsum on the cave walls The Lago dell'Orsa consists of a group of pools and ceilings (Fig. 9). During some periods, however, the interconnected at depth. These pools are inside the show- H2S in the air becomes very low or disappears. cave zone, and each pool can be reached by descending In December 2000, the chemical characteristics of the through a separate shaft about 20 m deep. We sampled the surface water in the lake were intermediate between the northern pool, far from the tourist walkway. The water sulfidic and bicarbonate water, and the H2S waslow in the depth isabout 8 m and a surface layer of bicarbonate water water (0.1 mmol L21) and not detected in the air. This ,5 m deep overlies the sulfidic water. Samples of sulfidic intermediate composition, however, cannot be attributed and bicarbonate water were collected during two different merely to the dilution of the sulfidic water with a periods(April and September 2001). Sulfidic water showed significant amount of seepage water. In fact, while the a composition similar to Main Spring ± Ramo Sulfureo concentration of the principal ions(K +,Na+,Mg2+,Cl2) water. The composition of the surface layer demonstrated wasreduced to ,20% of those measured in Main Spring on Journal of Cave and Karst Studies, August 2008 N 101 SULFIDIC GROUND-WATER CHEMISTRY IN THE FRASASSI CAVES,ITALY

The ground-water composition in Main Spring is representative of the water in the shallow phreatic zone throughout the cave, as also documented by the similarity of water chemistry in most springs and in Ramo Sulfureo, Lago dell'Orsa, and other cave lakes or streams. On the contrary, water in Lago Verde and Fissure Spring is less diluted sulfidic water, with minor seasonal changes in water chemistry and less influenced by the variable recharge of bicarbonate water. Thismore concentrated water represents the composition in a lower part of the phreatic zone where the dilution is of lesser importance. This supposed origin is confirmed by its similarity to the water coming from a 30 m deep well drilled for thermal baths, analyzed monthly by one of the authors. The fairly stable chemical composition of seepage water in the different sampling zones throughout the year made it possible for us to verify by analytical calculation that the concentration of the primary ionsin Main Spring ± Ramo Sulfureo ground water can derive from the natural mixture of bicarbonate water and high salinity sulfidic water. Since we observed that the river water has a chemical compo- sition similar to that of seepage water (Fig. 7), the calculated dilution ratiosare not influenced by the causes of the dilution itself. To calculate dilution ratios, we compared chloride concentration in each sample with the values measured in the Lago Verde at the end of the summer after a long dry Figure 10.Schematic section of Laghi di Lucia.The high period. Thiswater wasconsidered to be pure deep phreatic sulfate content in the diluted sulfidic ground water is water, although there might be a slight dilution by surface consistent with seepage water that is sulfate-enriched after water. A comparison of ion concentrations in the studied flowing through the replacement gypsum crust covering the period shows that the Lago Verde water is diluted with cave walls above the water table. seepage water to a maximum percentage of 20% during the wet season. In Main Spring, the water dilution due to seepage-water recharge varies between 30% in the autumn the same date, the sulfate concentration in the water and 60% during the spring (Fig. 8). maintained valuesashigh asat Main Spring. The anomalouscomposition of the water might be due to a WATER-TABLE LEVEL AND GROUND-WATER DILUTION dilution of the sulfidic ground water with seepage water that The elevation of the water table in the cave varies hasdissolved replacement gypsumasit descends(Fig. 10). during the year (Fig. 8). Itsmain range waslower than 50 cm, but during major flooding eventsin the Sentino SULFIDIC GROUND-WATER DILUTION River, the level rose quickly up to 1.5 m for short periods. Measurements in Lago Verde and in Ramo Sulfureo DILUTION RATIOS showed the same trend, both in the range and in the timing The data show that water characteristics vary signifi- of the water-level changes, and the two curves representing cantly in the different zonesof the cave, and that the water-level changes can be superimposed (data not shown). surface composition of ground water is often influenced by The same results were obtained in 2003±2004, comparing local factors, including stratification. Furthermore, the water-table levelsin Lago Verde and Lago dell'Orsa(data water in Ramo Sulfureo isslightly more diluted than in not shown). These uniform variations of water level in the Main Spring (Fig. 7). The water moving from the Ramo different cave zonesafter meteoric eventsreveal the high Sulfureo to Main Springs mixes with less diluted sulfidic permeability of the karst aquifer. water. These data suggest that the variable recharge of Some interesting conclusions can be drawn by compar- seepage water from the karst surface is the main cause of ing seasonal changes in the ground-water temperature, the dilution of sulfidic ground water in the shallow phreatic water-table level, and the dilution curve of sulfidic ground zone. The pattern of ground-water dilution we observed is water. Periodswith a high water-table level correspond not consistent with river recharge because the dilution ratio with lower temperaturesand higher dilution. All these isnot correlated with the distanceto the river. parameters are also related to the distribution of rain 102 N Journal of Cave and Karst Studies, August 2008 S. GALDENZI,M.COCCHIONI,L.MORICHETTI,V.AMICI, AND S. SCURI eventsduring the year. Thesedata clearly show the influence of seasonal rainfall evolution on the ground- water temperature, level, and chemistry. The dilution curvesof Lago Verde and Main Spring have a similar seasonal trend, but the maximum dilution occurred in different periods(Fig. 8). In Lago Verde, the maximum dilution occurred at the same time as the maximum water-table level. In Main Spring, the maximum dilution wasreached in April at the end of the rainy period and at the beginning of the depletion period. In the following months, the water temperature rose slowly, returning to the autumn valuesin unisonwith the decrease of dilution. The differencesbetween the trend of dilution curvesin Main Spring and Fissure Spring and their different relationswith water temperature and water-table level (Fig. 8) show that Main Spring represents the overall discharge of the shallow phreatic zone, where ground- water dilution variesaccording to the general hydrologic cycle of the spring. On the contrary, the water in Lago Verde (and in Fissure Spring), rising from deeper zones of the aquifer, isdirectly diluted by seepagewater during the principal rainfalls, but only for short periods and with a lower intensity (Fig. 8).

FLOODING EVENTS A detailed discussion of the relationship between ground water and river levelsisnot the main focusof the present research. Studies on this subject are currently in progress, including absolute measurements of water levels in the cave. However, the data obtained on the water levels Figure 11.Data on flood events.The sharp lowering of in the cave and water temperature at the springs already water temperature observed on December 27 and January 31 give some useful indications about the modality of ground- was due to the direct contact of river water with the water dilution. thermometer near the sulfidic springs.The river level was During the monitoring period (2000±2001) three main registered a few km downstream of the springs.In the flood eventsoccurred (Fig. 8). During theseevents,the Frasassi Gorge, near the springs, the river level increased river level increased up to ,2 m near the spring, while in ,2m. the cave the maximum rise of the water table was ,1.50 m. Detailed analysis of these flooding events shows that they Although the river discharge may influence the cave had similar effects on water level and temperature. water-table level, the data acquired on flooding eventsdo Figure 11 represents the events of December 27, 2000 not suggest a direct invasion of river water or a direct role and January 31, 2001. In the cave, at the beginning the of the river in the dilution of the ground water in the water level increased without changes in water tempera- shallow phreatic zone. Furthermore, the chemical data ture. In fact, the sharp lowering of the temperature in the discussed above suggest that this superficial dilution is spring was due to the cold river water's direct invasion mainly due to the variable recharge of seepage water from during the flood. The maximum level of ground water was the karst surface. These data, however, do not exclude the reached after the flood in the river, and it relatesto the possibility that the river participates in the recharge of the lowering of water temperature. The water level remained ground water, as already proposed by some authors high for some days after the end of the river flood. The rise (Tazioli et al., 1990; Ciancetti and Pennacchioni, 1993; of the ground-water level isonly in part influenced by the Caprari et al., 2001). Thisrecharge might occur because increase of the river level, which causes a drainage upstream in the gorge, the river bed is above the sulfidic impediment at the springs. The increased recharge of cold ground-water level and, consequently, a permanent weak seepage water in the few days following the rainfalls water loss from the river might occur. Measurements of appearsto be the main causeof the decreasingtemperature river discharge, however, highlight a constant slight and increasing level in the ground water. increase of the river discharge from upstream to the Journal of Cave and Karst Studies, August 2008 N 103 SULFIDIC GROUND-WATER CHEMISTRY IN THE FRASASSI CAVES,ITALY

Figure 13.Schematic section of Lago Verde.This lake, near the cave entrance, is fed by rising sulfidic ground water. The irregular recharge of seepage water from the thin, highly karstfied overlying limestone causes a weak dilution at the lake surface only during the wet season or after major storms.

Figure 12.Seasonal changes in sulfide and sulfate concen- from ,0.1 up to 0.5 mmol L21 (Fig. 12). The sulfide/total tration in the ground water. sulfur ratio varies mainly as a result of changes in the sulfide concentration, which ranges between 5% and 30% downstream end of the Gorge (Ciancetti and Pennacchioni, seasonally. These changes are correlated with the relative 1993) or only minor changes(Caprari et al., 2001). dilution of ground water caused by the amount of seepage- water recharge (Fig. 12). In the wet period, the rapid SULFATE AND SULFIDE IN THE GROUND WATER recharge of water from the surface decreases the total

sulfur dissolved in the ground water. Furthermore, the O2- The sulfidic ground water contains both oxidized and rich seepage water enhances H2S oxidation, also reducing reduced sulfur with significant variations during the year. the sulfide/total sulfur ratio. The total amount of sulfur species in the water shows a In the dry period, the reduced recharge of water from seasonal trend similar to chloride, although the ratio the surface slackens H2S oxidation, and both sulfide between total sulfur and chloride (in mol) is not constant concentration and sulfide/total sulfur ratios increase. (,0.13 6 0.03), with a wider range in Main Spring than in Furthermore, the activity of sulfate-reducing bacteria can the other sampled localities (Fig. 12). The amount of the contribute to increased H2S concentrationsin the water dissolved sulfur species can be modified by the release of with more importance during the periodsof low oxygen H2S to the cave atmosphere and by the dissolution of availability. Sulfate reducing bacteria were identified in the replacement gypsum on the cave walls due to a rise of the ground water (Macalady et al., 2006) where they live water table or to seepage water. The isolated increase of the closely associated with sulfide oxidizing bacteria in the ratio of total sulfur/chloride observed at Lago Verde in microbial biofilmsthat cover wallsand floor below the June 2001 is a consequence of increased sulfate in the water table (Fig. 14). water. Thisevent occurred two daysafter a strongrain (Fig. 8), which should have caused the arrival of small AIR COMPOSITION IN THE SULFIDIC SECTIONS amounts of seepage water enriched in sulfate from This first series of gas concentration measurements in dissolution of the thin gypsum crystals covering the cave the air of sulfidic zones (including H2S, O2,CO2 and, walls(Fig. 13). occasionally, SO2) were made in the Ramo Sulfureo at the The amount of oxidized and reduced sulfur species same location as water sampling for chemical analyses. The shows more significant variations because their concentra- Ramo Sulfureo was chosen because the wide surface of the tion is not simply controlled by the amount of seepage- flowing sulfidic water facilitates gas exchange and enhances water dilution (Fig. 12). Biotic and abiotic oxido-reduction active replacement of limestone with gypsum. The first reactions can modify the ratio between sulfate and sulfide measurement (November 2000) was made at the top of a in the water. The sulfide concentration undergoes the small pit above a sulfidic lake with the most intense gas widest fluctuations on a percentage basis and can increase rise. All the following measurements were made at a nearby 104 N Journal of Cave and Karst Studies, August 2008 S. GALDENZI,M.COCCHIONI,L.MORICHETTI,V.AMICI, AND S. SCURI

Figure 14.Microbial mats in the sulfidic ground water (image width ,30 cm). sulfidic lake in the same room as water sampling. This from 1 to 8 ppm. The CO2 content varied during the year change of gas sampling site was based on the desire to from 1500 to 5600 ppm. SO2 wasnot detected. reduce the influence of the rise path on the gas The air composition in the sulfidic room is strongly concentration. Measurements for December 2000 and influenced by the ease of exchange with the upper non-

November 2001 are lacking because of difficulties in sulfidic sections. Therefore, the CO2 content in the air accessing the room, but the available data cover the entire decreases rapidly towards the upper cave levels, a fact that hydrologic seasonal cycle (Fig. 15). wasverified in two different periods(April 9, 2001 (data

The oxygen concentration in the air wasalmostthe not shown) and October 7, 2001 (Fig. 16)). The H2S in the same as that at the surface (about 20%). The H2S air can only be detected close to the sulfidic sections where concentration in the sampled room had seasonal changes, it isquickly oxidized (Fig. 16); carbon dioxide, on the

Figure 16.Schematic profile through the sulfidic section of

Figure 15.Seasonal changes in the H 2S and CO2 concen- the cave (after Galdenzi, 2001).CO 2 and H2S can diffuse trations in the cave air.The H 2S concentration in the water from the sulfidic lakes to the cave atmosphere.The and in the air has the same seasonal trend, and the lowest concentration of H2S rapidly decreases, because it oxidizes values correspond with high ground-water dilution.In the and causes the growth of gypsum replacement crusts on the same period, the maximum CO2 concentration occurs. cave walls. Journal of Cave and Karst Studies, August 2008 N 105 SULFIDIC GROUND-WATER CHEMISTRY IN THE FRASASSI CAVES,ITALY contrary, can maintain higher valuesin the surrounding taneous at all the monitored sites. The Sentino River forms roomsaswell. Gasconcentrationsexpected for a room the base level and can influence the cave water-table level, close to air exchange were simulated using an air bell but the main cause of ground-water dilution in the shallow floating on the water table. Here, after a few months, the phreatic zone probably lieswith the amount of seepage- concentration of H2S exceeded 120 ppm, while O2 de- water recharge. creased to 7% (Galdenzi, 2001). Despite high permeability, significant differences were Changesin cave air composition are correlated with found in water chemistry among sample sites. Water rising seasonal changes in sulfidic-water discharge and chemistry. from a deep zone of the aquifer directly feedssomepoolsof

The concentration of H2S in the air isrelated to the dissolved the cave, including Lago Verde and Fissure Spring. Low content of the H2S in the water (Fig. 15). For thisreason, water flow causes seepage water to remain on the surface there isalsoa good correlation with ground-water dilution. due to its lower density across a wide zone of the cave, During the spring, when the ground water is highly diluted by Seepage-water dilution variesbetween 30% and 60% the seepage water, H2S reachesitslowestvaluesin the cave air. during the year in Ramo Sulfureo and in Main Spring. In The CO2 concentration attainsmaximum valuesduring contrast, water chemistry in Lago Verde and Fissure the spring when water dilution is greater (Fig. 15). In this Spring ismore constantand isdirectly influenced for short case, there is not a clear relationship with the bicarbonate periods by the direct arrival of seepage water, causing a concentration in the water, which fluctuatesbetween 3.9 and dilution of less than 20%. 4.6 mmol L21 with higher valuesin the autumn. This Seasonal changes in water chemistry interact with seasonal increase of the CO2 content probably isnot due speleogenesis in the cave. The recharge of oxygen-rich to atmospheric CO2 carried in by seepage water during the seepage water promotes H2S oxidation during the wet wet and warm season. In the upper levels, where there is only season when the sulfide/total sulfur ratio decreases from seepage water, in fact, CO2 concentration iswell known 30% to 5%. During thisperiod, corrosionin the upper (Galdenzi and Menichetti, 2002) and remainslower than in phreatic zone ismostintense,promoting the production of the Ramo Sulfureo throughout the year. We believe that the CO2, which is then released into the air. In these wet air concentration of CO2 in the sulfidic sections is related to periods, the low H2S concentration in the water also the amount of gasrisingfrom the depthsand releasedfrom reducesH 2S in the cave atmosphere, slowing the growth of the ground water. An additional source of CO2 in the the replacement gypsum crusts on the cave walls above the shallow phreatic zone may derive from the active speleoge- water table. In the dry season, the high H2S concentration in the water probably testifies to a lower intensity of netic processes of the area. In the wet season, while the CO2 content in the air is increasing, the sulfide in the ground oxidation processes and limestone corrosion in the ground water decreases because of sulfide oxidation (Fig. 15): water, while the higher concentration of thisgasin the air likely facilitates limestone corrosion and gypsum produc- H S z 2O ? H SO 1† 2 2 2 4 tion due to H2S oxidation above the water table.

z 2{ ACKNOWLEDGMENTS H2SO4 z 2H2O ? 2H3O z SO4 2†

H2S oxidation (Equation (1)), which reducesthe sulfide/ This research was funded by a grant from Consorzio + total sulfur ratio, also produces H3O ionsthat react with Frasassi (the public corporation operating the Frasassi { limestone (Equations (2) and (3)), increasing the HCO3 show cave) to Camerino University and Istituto Italiano di concentration in the water and consequently, the release of Speleologia ± Frasassi section for researches on the CO2 into the cave atmosphere (Equations (4) and (5)): Frasassi ground water, and by a grant from the Marche H Oz z CaCO ? Ca2z z HCO{ z H O 3† Region for the project ``Chemistry and hydrodynamic 3 3 3 2 analysis of sulfidic ground water in the Frasassi caves'' of FSM (Federation of Speleological Groupsof Marche { z HCO3 z H3O ? H2CO3 z H2O 4† region), Istituto Italiano di Speleologia ± Frasassi section, and Gruppo Grotte Recanati. Field work wassupported H2CO3 ? H2O z CO2 †g 5† by the speleologists of the same Recanati cave group, whom we wish to thank. We also thank an anonymous The increase in cave air CO isconsistentwith a high rate of 2 reviewer, whose useful comments helped to clarify some H S oxidation in the ground water during the rainy season 2 conceptsand the text. compared to the dry season.

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Galdenzi, S., and Menichetti, M., eds., Il carsismo della Gola di Conference Karst 99, Grands Causses, Vercors, France: Cagep, Frasassi [The karst of Frasassi Gorge]: Memorie Istituto Italiano Universite de Provence, Etudesde Ge Âographie physique, travaux Speleologia, s. II, v. 4, p. 155±180. 1999, suppl. N. 28, p. 101±106. Caprari, M., Galdenzi, S., Nanni, T., Ramazzotti, S., and Vivalda, P., Galdenzi, S., and Menichetti, M., 2002, Il monitoraggio ambientale nelle 2001, La sorgente di Gorgovivo: analisi idrogeologica finalizzata Grotte di Frasassi: struttura della rete di acquisizione e nuove all'individuazione delle zone di tutela, rispetto e protezione [The indicazioni sul microclima [Monitoring the Frasassi Caves: structure Gorgovivo Spring: hydrogeologic analysis to define zones for of the remote net and new data on the microclimate]: Le Grotte preservation, respect, and protection]: Memorie SocietaÁ Geologica d'Italia, s. V, v. 3, p. 75±86. Italiana, v. 56, p. 157±169. Macalady, J.L., Lyon, E.H., Koffman, B., Albertson, L.K., Meyer, K., Ciancetti, G.F., and Pennacchioni, E., 1993, Idrologia superficiale ed Galdenzi, S., and Mariani, S., 2006, Dominant microbial popula- alimentazione della falda dell'area carsica di Frasassi [Surface tions in limestone-corroding stream biofilms, Frasassi cave system hydrology and groundwater recharge in the Frasassi karst area]: Italy: Applied and Environmental Microbiology, v. 72, no. 8, Geologia applicata ed idrogeologia, v. 28, p. 285±293. p. 5596±5609. Cocchioni, M., Galdenzi, S., Morichetti, L., Nacciarriti, L., and Amici, V., Sarbu, S.M., Galdenzi, S., Menichetti, M., and Gentile, G., 2000, Geology 2003, Studio idrochimico delle acque nel complesso ipogeo di Frasassi and biology of the Frasassi Caves in Central Italy, an ecological multi- [Hydrochemical analysis of cave waters in the Frasassi caves]: Le disciplinary study of a hypogenic underground ecosystem, in Wilkens, Grotte d'Italia, s. V, v. 4, p. 49±61. H., Culver, D.C., and Humphreys, W.F., eds., Ecosystems of the Dramis, F., Gentili, B., and Pieruccini, U., 1976, La degradazione dei World, Subterranean Ecosystems, New York, Elsevier, p. 359± versanti nel bacino del Sentino (Appennino Umbro - Marchigiano) 378. [Slope degradation in the Sentino River basin (Umbria-Marche Sighinolfi, G.P., 1990, Chimismo ed origine delle acque del sistema ipogeo Apennines)]: Studi Geologici Camerti, v. 2, p. 45±72. Galdenzi, S., 1990, Un modello genetico per la Grotta Grande del Vento ``Grotte di Frasassi'' (Ancona) - implicazioni speleogenetiche ed [A speleogenetic model for the Grotta Grande del Vento], in Galdenzi, ambientali [Chemistry and origin of the waters of the ``Grotte di S., and Menichetti, M., eds., Il carsismo della Gola di Frasassi [The Frasassi'' cave system (Ancona, Italy) Ð speleogenetic and environ- karst of Frasassi Gorge]: Memorie Istituto Italiano di Speologia, s. II, mental implications], in Galdenzi, S., and Menichetti, M., eds., Il v. 4, p. 123±142. carsismo della Gola di Frasassi [The karst of Frasassi Gorge]: Galdenzi, S., 2001, L'azione morfogenetica delle acque sulfuree nelle Memorie Istituto Italiano di Speleologia, s. II, v. 4, p. 109±122. Grotte di Frasassi, Acquasanta Terme (Appennino marchigiano - Taddeucci, A., Tuccimei, P., and Voltaggio, M., 1992, Studio geocrono- Italia) e di Movile (Dobrogea ± Romania) [Morphogenetic action of logico del complesso carsico ``Grotta del Fiume ± Grotta Grande del the sulphidic waters in the caves of Frasassi and Acquasanta Terme Vento'' (Gola di Frasassi, AN) e indicazioni paleoambientali (Marche ApenninesÐ Italy) and Movile (Dobrogea ± Romania)]: Le [Geochronologic study of the ``Grotta del Fiume ± Grotta Grande Grotte d'Italia, s. V, v. 2, p. 49±61. del Vento'' cave complex (Frasassi Gorge, central Italy) and paleo- Galdenzi, S., Menichetti, M., and Forti, P., 1997, La corrosione di environmental implications]: Il Quaternario, v. 5, p. 213±222. placchette calcaree ad opera di acque sulfuree: dati sperimentali in Tazioli, G.S., Cocchioni, M., Coltorti, M., Dramis, F., and Mariani, M., ambiente ipogeo [Limestone tablets corrosion due to sulphidic water: Circolazione idrica e chimismo delle acque sotterranee dell'area experimental measurements in cave environment], in Proceedings, carsica di Frasassi nelle Marche [The flowpath and chemistry of International Congress of Speleology, 12th, Le Chaux-de-Fonds, groundwater of the Frasassi karst area, in the Marche region, Italy], in Switzerland: v. 1, p. 187±190. Galdenzi, S., and Menichetti, M., eds., Il carsismo della Gola di Galdenzi, S., Menichetti, M., Sarbu, S., and Rossi, A., 1999, Frasassi Frasassi [The karst of Frasassi Gorge]: Memorie Istituto Italiano di caves: a biogenic hypogean karst system? in ProceedingsEuropean Speleologia, s. II, v. 4, p. 93±108.

Journal of Cave and Karst Studies, August 2008 N 107 L.N. Walker, J.E. Mylroie, A.D. Walker, and J.R. Mylroie – The Caves of Abaco Island, Bahamas: keys to geologic timelines. Journal of Cave and Karst Studies, v. 70, no. 2, p. 108–119. THE CAVES OF ABACO ISLAND, BAHAMAS: KEYS TO GEOLOGIC TIMELINES

LINDSAY N. WALKER1,JOHN E. MYLROIE2,ADAM D. WALKER1, AND JOAN R. MYLROIE2

Abstract: Abaco Island, located on Little Bahama Bank, is the most northeastern island in the Bahamian archipelago. Abaco exhibits typical carbonate island karst features such as karren, blue holes, pit caves, banana holes and flank margin caves. Landforms that resemble tropical cone karst and pseudokarst tafoni caves are also present. The three cave types of Abaco—flank margin caves, pit caves, and tafoni caves—are abundant, but each forms by very different mechanisms. Flank margin caves are hypogenic in origin, forming due to mixing dissolution at the margin of the freshwater lens. Since the lens margin is concordant with sea level, flank margin caves record the position of sea level during their formation. Flank margin caves exhibit phreatic dissolutional features such as bell holes, dissolutional cusps and spongework. Pit caves form as vadose fast-flow routes to the freshwater lens and are common on the Pleistocene eolianite ridges of the Bahamas. Pit caves are characterized by their near-vertical or stair-step profiles. Because pit caves form in the vadose zone, their position is not tied to sea level. Tafoni caves are pseudokarst features that form when the soft interior of an eolianite ridge is exposed to subaerial erosion. Since tafoni caves form by mechanical processes, they do not exhibit phreatic dissolutional features. Tafoni caves may be mistaken as flank margin caves by the untrained observer, which may cause problems when using caves as sea-level indicators. Each of Abaco’s unique cave types may preserve depositional and erosional features that are useful to the researcher in creating general geologic timelines. While these timelines may not give exact dates, they are useful in the field for understanding depositional boundaries and determining sequences of geologic events.

INTRODUCTION are major petroleum reservoirs. The Bahama Platform (Fig. 1A) is composed of a series of thick, shallow-water, The Commonwealth of the Bahamas (Fig. 1A), located carbonate banks along the subsiding margin of North southeast of Florida and northeast of Cuba, consists of 29 America (Mullins and Lynts, 1977). The current landscape islands, numerous keys, shallow banks and rocks (Albury, of the Bahamas is largely constructional and is greatly 1975). The northwest-southeast trending archipelago extends influenced by glacioeustatic sea-level fluctuations (Carew 1400 km from the stable Florida peninsula to the tectonically and Mylroie, 1997). Carbonate deposition occurs on the flat active Caribbean Plate boundary near Hispaniola (Carew bank tops during glacioeustatic sea-level highstands when and Mylroie, 1995). The Turks and Caicos Islands make up shallow lagoons dominate. During sea-level lowstands, sea the southeastern extent of the same archipelago, but are a level drops below the bank margins. Carbonate sedimenta- separate political entity. The Bahamian portion of the tion ceases and subaerial karst processes dominate on the archipelago is 300,000 km2 in area, 11,400 km2 of which is exposed bank tops. Lowstands are recorded in the subaerial land (Meyerhoff and Hatten, 1974). sedimentary record by the development of terra rossa Abaco Island (Fig. 1), located on Little Bahama Bank, paleosols. These fossil soil horizons are the result of the is the most northeastern island in the archipelago. It is concentration of insoluble materials, such as atmospheric bordered on the east by the deep waters of the Atlantic dust, due to pedogenic processes. Ocean, on the south by the deep waters of N.W. Providence Channel and N.E. Providence Channel, and BACKGROUND on the west by the shallow waters of the Little Bahama Bank (Fig. 1A). The landmass of Abaco consists of two Island karst is a result of the unique environments and main islands, Great Abaco Island and Little Abaco Island, associated processes that affect carbonates in small island and numerous outlying cays (Fig. 1B). settings (Mylroie et al., 2004; Jenson et al., 2006). Island The Bahamas have long been the focus of geologic work karst is different from typical karst landscapes that develop on modern carbonates (Illing, 1954; Multer, 1977; Tucker in continental settings, and from karst on islands, which and Wright, 1990; Carew and Mylroie, 1997 and references therein). The Bahama Platform has particular interest to 1 308-115 Elk Run Blvd., Canmore, AB T1W 1G8, Canada s03.lmccullough@ wittenberg.edu, [email protected] geologists as it provides a modern analog for the dynamics 2 Mississippi State University, Department of Geosciences, P.O. Box 5448, Mississippi of ancient carbonate depositional platforms, many of which State, MS 39762 [email protected], [email protected] 108 N Journal of Cave and Karst Studies, August 2008 L.N. WALKER, J.E. MYLROIE, A.D. WALKER, AND J.R. MYLROIE

Figure 2. Schematic diagram of island karst processes on a simple carbonate island showing the fresh water lens (modified from Mylroie and Carew, 1995).

forms in the interiors of large islands such as Puerto Rico, Cuba, and Jamaica (Vacher and Mylroie, 2002). Karst on islands is more similar to continental karst than carbonate island karst. The principles of island karst are summarized by the Carbonate Island Karst Model, or CIKM, described by Mylroie, et al. (2004) and Jenson et al. (2006). Most of the freshwater that exists on carbonate islands like the Bahamas is stored in the freshwater lens, which is an accumulation of meteoric water that floats on the underlying marine water due to the density contrast (Fig. 2). Surface streams are rare or completely absent. As a result, many typical karst processes, such as stream cave formation, do not take place. The karst features of carbonate islands can interact in complex ways forming a highly modified landscape (Fig. 2). Karst features known to occur on Abaco include karren, blue holes, pit caves, banana holes, flank margin caves, and landforms that resemble tropical cone karst. Tafoni, pseudo- karst voids formed by subaerial erosion, are also present. Only the three cave types—flank margin caves, pit caves, and tafoni caves—are discussed in this paper. Because these cave types form by very different mechanisms, each provides a unique understanding of island karst and geomorphic processes, as well as clues to the geologic history of Abaco. The purpose of this study is to describe the presence of these caves on Abaco and to demonstrate the importance of caves in constructing general geologic timelines.

FLANK MARGIN CAVES Flank margin caves form from mixing dissolution at the Figure 1. A: Map of the Commonwealth of the Bahamas margin of the freshwater lens under the flank of the (modified from Carew and Mylroie, 1995). B: Map of Abaco enclosing landmass (Mylroie and Carew, 1990). Because Island, Bahamas, showing cave locations. Town locations are the calcium carbonate saturation curve is convex upward, labeled in normal font. Cave locations are shown in italics. the mixing of two waters of varying concentrations creates When two or more caves are close together they are shown as a solution that is less saturated with respect to calcite one dot: Cedar Harbour Caves = Cedar Harbour Cave I–V; (Dreybrodt, 2000). The interaction of waters of different Little Harbour Caves = Azimuth Cave, Dripping Stones chemistries at the boundaries of the freshwater lens, Cave, Hunter’s Cave, Manchineal Cave, and Sitting Duck therefore, creates an environment of preferential carbonate Cave; Little Bay Caves = Little Bay Cave I–III. dissolution (Mylroie and Carew, 1990). Mixing dissolution occurs both at the top of the lens, where vadose freshwater mixes with phreatic freshwater, and at the bottom of the lens, where phreatic freshwater Journal of Cave and Karst Studies, August 2008 N 109 THE CAVES OF ABACO ISLAND,BAHAMAS: KEYS TO GEOLOGIC TIMELINES mixes with marine water (Mylroie et al., 2004). The top and 2004), San Salvador Island (Vogel et al., 1990), and South bottom of the lens are also density interfaces, which allow Andros Island (Carew et al., 1998). This paper reports the for the collection of organic material. The oxidation of first study of flank margin caves on Abaco Island (Walker, these organics produces CO2 and thus increases dissolu- 2006). tional capability (Mylroie et al., 2004). Evidence suggests The majority of flank margin caves that are currently that the presence of sulfate-reducing bacteria in the mixing exposed in the Bahamas have dissolutional ceilings between zone may have a significant role in the formation of mixing 1 m to 7 m above modern sea level, which is consistent zone porosity (Bottrell et al., 1993). Thus, both organic and with formation in a freshwater lens elevated by the +6m inorganic mixing are involved. The mixing zones at both Oxygen Isotope Substage (OIS) 5e highstand that occurred the top and bottom of the lens meet at the lens margin approximately 125 ka (Carew and Mylroie, 1995). This (Fig. 2), forming a site that is most favorable for mixing evidence, combined with a lack of speleothem age dates zone dissolution. Because the margin of the freshwater lens greater than 100,000 years, suggests that all flank margin is concordant with sea level (Fig. 2), flank margin caves caves currently exposed in the Bahamas were formed mark the position of sea level during their formation during the OIS 5e highstand, and agrees with reported (Carew and Mylroie, 1995). isostatic subsidence of the archipelago of 1–2 m per Flank margin caves are hypogenic (sensu Palmer, 1991). 100,000 years (Carew and Mylroie, 1995). They form as complex mixing chambers, not conduits for turbulent-flow underground drainage. As such, they PIT CAVES commonly form with no surface openings, although Pit caves are vadose shafts that result from the entrances may later be created when erosional retreat of gathering of meteoric water into discrete inputs in the the island flank intersects the cave. Their shape is globular epikarst (Mylroie and Carew, 1995). Pit caves are with an extended horizontal dimension and limited vertical characterized by their near vertical or stair-step profiles, development reflecting their origin along the thin lens vertical grooves on the walls, and the absence of curvilinear margin. As dissolution continues over time, individual dissolution surfaces that are characteristic of phreatic voids may intersect to create larger caves. The joining of conditions (Mylroie and Carew, 1995). Pit caves commonly the voids creates a characteristic cave pattern of a maze of have a well-developed system of feeder tubes within the larger chambers connected by smaller passages. These epikarst that deliver water to the pit (Harris, et al., 1995). small passages often radiate outward from the main The active lifetime of a pit cave is relatively brief as its chambers but end abruptly in bedrock walls. The walls development is interrupted by the formation of newer pits and ceilings exhibit large dissolutional cusps, bellholes, and upstream that pirate its recharge. The end members of this spongework as evidence of their phreatic formation. process are areas of high pit density called pit complexes, Though the dissolutional cusps that are common in which represent the accumulated pit cave development and flank margin caves have a similar appearance to the subsequent abandonment over time (Mylroie and Carew, scallops found in many stream caves, they are different in 1995). Pit caves in these complexes can occur at densities of several ways. Scallops form under turbulent flow condi- over 100 per km2 (Harris et al., 1995; Seale et al., 2004). tions, are asymmetrical (providing a flow direction Pit caves in the Bahamas occur as both simple vertical indicator), and their size is inversely proportional to shafts and complex features resembling solution chimneys velocity (Curl, 1966; Curl, 1974). In mixing zone caves, (Seale et al., 2004). The more complex pits alternate where flow is strictly laminar, scallops do not form. between angled reaches developed along eolianite foresets However, dissolution does act differentially on the bedrock and vertical reaches that cut through the depositional surfaces within the cave to produce dissolutional pockets structure of the bedrock to form a stair-step profile. Pit or cusps. caves rarely exceed 10 m in depth, but may connect with Flank margin caves were first recognized in the other pits to form horizontal extents of up to 50 m (Seale et Bahamas (Mylroie and Carew, 1990) and have since been al., 2004). On Abaco and other Bahamian Islands, pit caves found on Bermuda (Mylroie et al., 1995), Isla de Mona are well developed in Pleistocene eolianite ridges, suggest- (Frank et al., 1998), the Mariana Islands (Mylroie et al., ing a relatively fast rate of formation. 2001), the Yucatan Peninsula of Mexico (Kelly et al., 2004), and Cuba (Soto, et al., 2004; Downey and Walck, TAFONI CAVES 2005). The tectonically stable Bahamian archipelago Cavernous weathering is the result of differential continues to be the ideal location to study flank margin erosion on a rock surface that allows some areas of the caves and mixing-zone porosity. To date, flank margin rock to be preferentially removed while surrounding areas caves have been described in the Bahamas on Cat Island remain intact (Turkington and Phillips, 2004). The (Mylroie et al., 2006), Crooked Island (Lascu, 2005), resulting pseudokarst voids or caverns are traditionally Eleuthera Island (Lascu, 2005), Great Inagua Island (Roth, known as honeycombs, aveoli, and tafoni (McBride and 2004), Long Island (Mylroie et al., 1991), New Providence Picard, 2000). Honeycombs and aveoli are centimeter to Island (Mylroie et al., 1991), North Andros Island (Roth, decimeter in size while tafoni (singular: tafone) are meter- 110 N Journal of Cave and Karst Studies, August 2008 L.N. WALKER, J.E. MYLROIE, A.D. WALKER, AND J.R. MYLROIE scale voids (McBride and Picard, 2000). Tafoni have been Table 1. Areas and perimeters of mapped flank margin caves described from localities worldwide and a diverse range of on Abaco Island, Bahamas. climate regimes. They are especially common in arid and Cave Area (m2) Perimeter (m) coastal environments (Sunamura, 1996). Tafoni are also known to form in a variety of rock types including Bucket 21 38 sandstone, conglomerates, limestone, granite, and volcanic Bellycrawl 23 29 tuff (Campbell, 1999; McBride and Picard, 2000; Huinink Little Bay III 40 35 et al., 2004 and references therein; Turkington and Phillips, Cedar Harbour III 43 40 2004 and references therein). Cedar Harbour V 62 59 The formation of tafoni is poorly understood and Cedar Harbour II 67 45 efforts to identify a single causative mechanism have been Little Bay I 68 58 largely unsuccessful. Tafoni are most commonly attributed Dripping Stones 71 51 to salt weathering, although a variety of chemical Cedar Harbour I 133 67 weathering processes have also been described in tafoni Cedar Harbour IV 153 80 formation (Sunamura, 1996; Campbell, 1999; Huinink et Little Bay II 156 60 al., 2004; Turkington and Phillips, 2004 and references Azimuth 164 73 therein). Physical weathering may also be involved in Sitting Duck 185 83 tafoni formation, as many rock surfaces that are suscep- Manchineal 186 71 tible to cavernous weathering commonly exhibit a hard- Hunter’s 214 316 ened outer crust over a weaker interior (Turkington and Long Beach 428 386 Phillips, 2004 and references therein). Removal of the outer 8-Mile 919 692 crust allows for preferential erosion of the soft interior. It is Hole-in-the-Wall 3422 1941 likely that tafoni formation is controlled by complex conditions within the natural environment and that the flank margin caves. When possible, the elevations of the dominant causative mechanism(s) will vary depending upon the conditions present in that environment. Tafoni caves were also measured relative to sea level. When caves in the Bahamas have recently been characterized by Owen were located too far inland to use sea level as a reference (2007), who provides a comprehensive review of the point, their approximate elevations were determined by relevant literature. plotting on topographic maps. Finally, the caves were classified as flank margin caves, pit caves, or tafoni based on their morphological characteristics. METHODS

Preliminary fieldwork, focused on locating caves and RESULTS AND DISCUSSION important geologic outcrops, was conducted March 11 to 20, 2005. The remainder of the fieldwork, including FLANK MARGIN CAVES mapping of all known caves, was completed from May Eighteen flank margin caves were explored and mapped 2 15 to June 15, 2005. The Friends of the Environment on Abaco Island (Table 1), ranging in area from 21 m 2 organization in Marsh Harbor, Abaco, assisted with local (Bucket Cave; Fig. 3) to 3422 m (Hole-in-the-Wall Cave; logistical support. A permit to conduct the research was Fig. 4). Flank margin caves on Abaco fit the model of secured through the Bahamian government. Mylroie and Carew (1990) in nearly every case and exhibit Caves were surveyed using a compass, inclinometer, characteristic hypogenic features such as bell holes, tape, and sketchbook, following the guidelines of the dissolutional cusps, and spongework. They have limited National Speleological Society (NSS) outlined in Dasher vertical, and extensive horizontal dimensions (Figs. 3 and (1994). Survey data were entered into COMPASS software 4). Each cave that exhibited these characteristics, except for reduction and line plot generation. Maps were drafted one, was located between 1 and 7 meters above modern sea in Corel Xara 3 1.0 using the line plots, field sketches, and level. the Association for Mexican Cave Studies (AMCS) Bellycrawl Cave near Cooper’s Town, however, was Standard Cave Map Symbols (Sprouse, 1991). Since length located 10 m above modern sea level. This elevation was is not an appropriate classification for flank margin caves determined by surveying from the position of the cave or tafoni caves, the area and perimeter of each cave were entrance to sea level using a tape and a Suunto computed using AutoCAD software. The area of internal inclinometer. Bellycrawl Cave consisted solely of one low bedrock pillars, columns, and bedrock bodies caught in crawlway that quickly became too small for human passage loops was subtracted from the overall cave area. exploration. The presence of other small phreatic voids The perimeter of these features, however, was added to the along the same 10 m horizon implies that a freshwater lens overall cave perimeter, as these bedrock components were may have reached an elevation of 10 m in this location. part of the water/rock interaction surface that formed the The absence of phreatic voids at this elevation on the rest Journal of Cave and Karst Studies, August 2008 N 111 THE CAVES OF ABACO ISLAND,BAHAMAS: KEYS TO GEOLOGIC TIMELINES

Figure 3. Map of Bucket Cave, Cooper’s Town, Great Figure 5. Map of 8-Mile Cave, Great Abaco Island. Abaco Island, Bahamas. fronting the voids. However, it was not possible to of the island implies that this was most likely a local determine within the scope of this project if the paleosol phenomenon. The small size of the voids could mean that may have had a role in the development of the 10 m the lens did not occupy that position for an extended horizon. Perching of the freshwater lens is certainly a period of time and may even have been episodic or the phenomenon that needs further investigation throughout result of a small perched water table. the Bahamas. Gentry and Davis (2004) describe perching of wetland The keyhole passage in 8-Mile Cave is an unusual waters on San Salvador Island in association with low passage shape for a flank margin cave. It is located at the permeability paleosols. A +10 m dissolutional ceiling from northeast extent of the cave and consists of a long passage Hatchet Bay Cave on Eleuthera Island in the Bahamas was trending approximately north-south, and makes a nearly also explained by storm loading of the lens and perching of 90u turn continuing to the west (Figs. 5 and 6). In epigenic the water table by a paleosol (Lascu, 2005). Terra rossa (i.e., stream) caves, phreatic processes form tubular paleosols, due to their relative impermeability and common passages with ovoid cross-sections while vadose flow occurrence in the Bahamas, no doubt have at least local creates deep, narrow canyons with rectangular cross- control over freshwater lens position, just as a lower- sections (Palmer, 1991). Keyhole-shaped passages in permeability layer would affect water-table position in a continental setting. A paleosol was visible along the shore

Figure 4. Map of Hole-in-the-Wall Cave, Great Abaco Figure 6. The Keyhole passage in 8-Mile Cave, Great Island, Bahamas. Abaco Island. 112 N Journal of Cave and Karst Studies, August 2008 L.N. WALKER, J.E. MYLROIE, A.D. WALKER, AND J.R. MYLROIE stream caves are usually the result of modification of an originally phreatic passage by vadose flow (i.e., formation of a canyon in the floor of an ovoid tube). Because 8-Mile Cave was not formed by stream processes, but rather by mixing dissolution, this typical explanation does not apply. The keyhole passage has phreatic origins as indicated by dissolutional cusps on the walls, ceiling, and spongework (Fig. 6). The trench in the floor, which causes the keyhole- shaped cross-section, seems to have come later, as it does not exhibit the same phreatic features as the rest of the passage. The trench may be explained by water entering the cave during storm events through a large crack present through- out much of the passage ceiling. If this water were to pond on the floor of the passage, it may form a trench by dissolution of the floor. The deepest area of ponding occurs where the passage makes the 90u bend to the west (Fig. 5). A small pool of water was present in this location during the initial survey of the cave. If ponding of surface waters are Figure 7. Map of Blowhole Cave, Great Abaco Island, allowing for continued dissolution of the cave floor, 8-Mile Bahamas. Cave shows an interesting interaction with current hydro- logic processes on Abaco, which is not typical of flank Hole-in-the-Wall. From the entrance the passage extends margin caves. Beyond the pool at the bend the passage down a 35u slope, following the dip of the eolianite foreset continues as a solely phreatic feature before intersecting a beds. At the bottom of the slope a small horizontal large vadose pit that has developed from the surface (Fig. 5). crawlway continues to the southeast and ends in a steep Some flank margin caves on Abaco also were useful in drop into a sea cave where the waves can be seen breaking. helping to determine the geologic history of the area in It is not known if downward growth of the vadose pit which they are located and even the relative age of the dune penetrated the roof of the sea cave or if roof collapse in the deposits in which they are enclosed. This is especially true sea cave intersected the vadose pit. However, most likely in Little Harbour and Cedar Harbour where coastal flank both mechanisms were at work. The interaction of the sea margin caves contain stalactiflats (Elliott, 2007), breccia cave and the vadose pit creates a blowhole. Each time a facies, beach deposits, and paleosols. The importance of wave breaks into the sea cave below, Blowhole Cave goes these features is described later as part of the discussion of dark. The sound of the wave is funneled through the cave using these caves as geologic timelines. In addition, the along with a strong burst of air, spray, and sand. presence of flank margin caves in Little Harbour located on opposite sides of the same eolianite ridge, such as TAFONI CAVES Dripping Stones Cave and Hunter’s Cave, provides During the initial reconnaissance for this study, a group evidence that the margin of the freshwater lens was active of 14 caves, later named the PITA Caves (Table 2), was on both sides of the ridge at the same time. This suggests discovered high in an eolianite ridge along the beach west that the ridge itself may have become a small island during of Hole-in-the-Wall on the south end of the island. The the OIS 5e highstand as surrounding topographic lows PITA Caves were of interest at the onset of this study were inundated by the rising sea. because they originally appeared to be located along a continuous horizon about 20 m above modern sea level PIT CAVES (Fig. 8A). Their arrangement in a continuous horizon Pit caves and solution pits are extremely common on suggested that they were flank margin in origin. As Abaco. Some locations have particularly high solution pit discussed previously, current models of flank margin cave densities, although most of these are too small for human formation in the freshwater-saltwater mixing zone require exploration. Pit caves, or solution pits large enough for that caves will be located between 1 and 7 m above modern human exploration, are numerous in several locations, sea level in agreement with the +6 m OIS 5e highstand which includes the Hole-in-the-Wall area on the south side (Mylroie and Carew, 1990). Given the tectonic stability of of the island. Only one pit cave, Blowhole Cave (Fig. 7), the Bahamas, a sea-level highstand of at least 20 m would was mapped as part of this study because pit caves have be required to form flank margin caves at the elevation of been thoroughly covered by other workers (Mylroie and the PITA Caves. A +20 m highstand has been proposed for Carew, 1995; Harris et al., 1995; Seale et al., 2004). OIS 11, but the data have been controversial, especially in Blowhole Cave is located near Hole-in-the-Wall on the the Bahamas (Lascu, 2005; Mylroie, 2008). The major southern coast of Abaco Island (Fig. 1). The cave entrance problem with this argument is that no confirmed subtidal is found on the same headland as the sea arch known as deposits dating from highstands prior to OIS 5e, including Journal of Cave and Karst Studies, August 2008 N 113 THE CAVES OF ABACO ISLAND,BAHAMAS: KEYS TO GEOLOGIC TIMELINES

Table 2. PITA cave elevations, Abaco Island, Bahamas. OIS 11, have been found in the Bahamas. If the PITA Elevations were measured from entrance ceiling to sea level. Caves were shown to be flank margin caves, they would PITA Cave Elevation (m) have provided the first conclusive evidence of a sea-level highstand in the Bahamas higher than the +6 m OIS 5e that A 22.5 was still exposed above modern sea level despite subsi- B 21.5 dence. C 18.7 Upon further investigation it became clear that the D 19.9 PITA Caves are not found in a continuous horizon. The E 14.5 large amount of vegetation in the area had initially made it F 17.9 impossible to see many of the caves. Once the vegetation G 18.0 around the caves had been removed and the elevations of H 20.9 the caves were measured it became clear that cave I 17.7 elevations were more random (Fig. 8B and Table 2). Also, J 10.8 individual investigation of each cave shows that they lack K 18.8 characteristic flank margin cave phreatic dissolutional L 14.1 features such as cusps and bell holes (Fig. 8C). The rough M 20.3 surfaces of the cave walls and ceilings are more typical of N 17.4 mechanical erosion (Fig. 8C). The combination of these observations demonstrates that the PITA Caves are not

Figure 8. A: Apparent continuous horizon of the high PITA Caves as seen from the beach before vegetation was removed. White dots show cave entrances visible from the beach. B: Pita Caves F–J as seen from the beach. Notice the various elevations of the entrances. C: Interior of PITA Cave B showing evidence of mechanical erosion. 114 N Journal of Cave and Karst Studies, August 2008 L.N. WALKER, J.E. MYLROIE, A.D. WALKER, AND J.R. MYLROIE

mystery.’’ As noted by Owen (2007) and Mylroie, et al. (2007), tafoni in Bahamian carbonates are only found in eolian calcarenite ridges where cave collapse, wave erosion, or anthropogenic activity such as road cuts and quarries has exposed the soft interior of the ridge to subaerial weathering. The PITA tafoni were probably formed during the +6 m OIS 5e highstand, when the ridge in which they are found was attacked by wave energy (Fig. 9). This erosion undercut the hillside, which then collapsed to form a cliff, resulting in removal of the calcrete crust of the dune. The soft interior of the dune was consequently exposed to attack by the coastal elements, allowing voids to be created at variable elevations (Figs. 9 and 10A). Tafoni have also been identified on San Salvador Island, Bahamas on North Point (Fig. 10B) and in Watling’s Quarry (Mylroie, et al., 2007; Owen, 2007). North Point is Figure 9. Proposed formation of high PITA Caves on a modern sea cliff in Holocene eolianites with conditions Abaco Island, Bahamas. similar to those that would have been present on Abaco during the formation of the high PITA Caves. Watling’s flank margin caves and thus do not represent a +20 m sea- Quarry is an inland exposure of Pleistocene eolianites. In level highstand. both cases, the eolianite was carved into a cliff either by The PITA caves are most likely a form of tafoni: holes natural (North Point), or anthropogenic (Watling’s Quarry) or depressions up to a few meters in dimension that form processes, which exposed the soft interior to erosion. Owen on cliffs and steep rock faces by cavernous weathering; a (2007) demonstrates that wind erosion is the primary cause subaerial process (Ritter et al., 2002). As noted earlier, of tafoni formation in Quaternary eolianites. It is important tafoni, as described in the literature, are extremely varied to note that because North Point is a Holocene deposit it and no single agreed upon definition exists (Owen, 2007). could not have supported a past freshwater lens. Thus, the The tafoni definition supplied by the Glossary of Geology voids found in the North Point eolianite cannot be flank (Neuendorf et al., 2005, p. 655) is only one of many, and margin caves. This further supports the argument that the the depth value given, 10 cm, is almost certainly an error similar features on Abaco were formed in the same way as because all literature sources reviewed by Owen (2007) those on North Point and are not highly weathered flank show a depth value much greater than 10 cm. Ritter et al., margin caves. (2002, p. 88) also discuss the terminology and literature The presence of tafoni in the Bahamas, first recognized concerning the definition and origin of tafoni, and in this study, is important because such pseudokarst voids conclude ‘‘The exact origin of tafoni, however, remains a can easily be mistaken for flank margin caves by the

Figure 10. A. Exposed soft interior of a Pleistocene eolianite after removal of calcrete crust. B. Modern tafone-like feature in the Holocene eolianites of North Point, San Salvador Island, Bahamas. Journal of Cave and Karst Studies, August 2008 N 115 THE CAVES OF ABACO ISLAND,BAHAMAS: KEYS TO GEOLOGIC TIMELINES

Figure 11. A. Paleosol in the wall of Cedar Harbour Cave II. Vegemorphs appear to be modern but are, in fact, calcified. Arrow indicates a vegemorph. Tape for scale. B. Patch of paleosol on the floor of Cedar Harbour Cave II. Rock hammer for scale. untrained observer. Because flank margin cave elevations facies containing eolianite breccia blocks are also com- provide an estimate of sea-level height during their monly observed along the cave walls (Fig. 12B). The sum formation, identifying pseudokarst voids as flank margin of these observations allows for a geologic timeline to be caves can lead to incorrect estimates of past sea-level interpreted. positions. The elevation of the Cedar Harbour Caves above modern sea level suggests that they were developed CAVES AS KEYS TO GEOLOGIC TIMELINES ,125,000 years ago during the +6 m OIS 5e highstand in In many locations on Abaco, the presence of caves can an eolianite ridge that was already present. The 5e be used to create a timeline of past events; and thus, help to highstand offered a 12,000-year time window, 131 ka to unravel the complex geologic history of an area. While 119 ka (Chen et al., 1991). The eolianite formed either on these timelines may not allow the researcher to discern the the transgression of the OIS 5e highstand or during a exact ages of deposits, they are helpful in understanding previous highstand event. As sea level reached its depositional boundaries that are visible in the field. For maximum height, the stable position allowed for the example, the rock containing the cave must be older than development of flank margin caves within the eolianite the cave, but the cave deposits must be younger than the ridge. The developing caves were breached by wave action cave. If cave deposits can be tied to a specific geologic as the 5e highstand continued. Beach sands were deposited activity, such as breaching of the cave by wave activity, in the caves, entombing breccia blocks from the eroding then the relative age of geologic events may be tied to eolianite cliffs (Fig. 12B). As sea level fell at the end of the geologic deposits, and the deposit becomes the marker for 5e highstand, the beach environment moved seaward and the event. away from the caves. Speleothems grew as the caves were Near Cedar Harbour (Fig. 1B) on the northern coast of abandoned by marine waters. Vegetation colonized the Little Abaco Island, both pit caves and flank margin caves area, including the beach deposits within the Cedar preserve important geologic information. Here, the coast- Harbour Caves, as the moist cave environment provided line is dominated by a consolidated eolianite with few a favorable place for vegetative roots. A sandy soil vegemorphs (calcified remains of plant matter) overlain by eventually developed on the beach deposits. Stalactitic a terra rossa paleosol and containing flank margin caves material and flowstone covered some of this new sediment (the Cedar Harbour Caves I through V, Table 2). Within floor. several of the Cedar Harbour caves, remnants of a paleosol As sea level rose with the present day highstand, the with extensive vegemorph development are present along beach environment once again began to affect the caves the cave walls and in patches on the floors (Fig. 11). This and much of the vegetation was removed. Despite sea level paleosol is only found within the caves and does not extend being 6 m lower today than during OIS 5e, storm wave into the cliffs along the beach. Speleothems developed on a energy still reaches into the caves as is evident by modern previous cave sediment floor now hang suspended above beach deposits and organic matter in the caves. This wave the original bedrock floor as stalactiflats (Fig. 12A). Beach action removed much of the soil that had developed during 116 N Journal of Cave and Karst Studies, August 2008 L.N. WALKER, J.E. MYLROIE, A.D. WALKER, AND J.R. MYLROIE

Figure 12. A: Well-developed stalactiflat in the western entrance of Cedar Harbour Cave III. Tape for scale. B: Breccia facies in Cedar Harbour Cave II. Rock hammer for scale. the post OIS 5e lowstand. Today, only remnants are Harbour was not unique to one part of the island. This present as a paleosol along the walls of the caves and in confirms our position that flank margin caves can preserve small patches on the floors (Fig. 11). The excavation of this geologic information that can be used to discern the soil under speleothems allowed for the formation of depositional and erosional history of carbonate islands, as stalactiflats as the speleothems were left suspended above well as providing vital evidence of past sea-level positions. the new floor level (Fig. 12A). The rocky shore of Cedar Harbour contains numerous Similar features (breccia facies, beach deposits, stalacti- vertical structures that stand in relief to the surrounding flats, paleosols) found in the coastal flank margin caves of surface of the eolianite bedrock (Fig. 13). Such vertical Little Harbour (Azimuth Cave, Manchineal Cave and structures are common on other Bahamian islands and have Sitting Duck Cave) on the east coast of Great Abaco Island been identified by previous workers as relict vadose solution (Fig. 1B) suggest that the timeline that occurred at Cedar pits (Carew and Mylroie, 1994). These pits formed during

Figure 13. A–B: Vertical features on the coast near Cedar Harbour that represent relict solution pits. Machete for scale in A. Journal of Cave and Karst Studies, August 2008 N 117 THE CAVES OF ABACO ISLAND,BAHAMAS: KEYS TO GEOLOGIC TIMELINES the original karstification of the eolianite bedrock surface, developed in Pleistocene eolianite ridges, suggesting a which took place during the lowstand following deposition relatively rapid rate of formation. Relict solution pits can of the eolianite. During this lowstand, the insides of the pits provide clues to the age of the eolianite (Pleistocene versus became coated with insoluble soil material, mostly aerosol Holocene) in which they have formed. Tafoni are derived, from the surrounding karst surface. This soil pseudokarst voids that form by a variety of mechanisms. eventually hardened into a paleosol. During subsequent Bahamian tafoni form when the hard calcrete crust of an highstand events, the majority of the paleosol was removed eolianite is removed, usually by wave action, exposing the from the surface of the bedrock due to marine processes. soft interior to attack by erosional processes. While tafoni However, the paleosol in the pits was somewhat protected often form in coastal areas, their elevations cannot be tied from the waves. The removal of the paleosol from the to past sea levels. Mistaking tafoni for flank margin caves surrounding surface made that surface more susceptible to will result in incorrect estimates of past sea levels. erosion by both dissolutional and mechanical processes. The The effects of continuing erosional and depositional pits interiors, however, were protected by the hard paleosol processes that occur subsequent to cave formation on coating and eroded out in positive relief. They now remain carbonate islands may often be preserved within and as an example of inverted topography. around the cave in many forms. This information, when The eolianite exposed along the coast likely formed, at assembled correctly, can be used by the researcher to the earliest, during the last interglacial highstand (OIS 5e) develop a general geologic timeline for the area. These approximately 125,000 ka. If it were deposited during this timelines, while not absolute, are useful in the field for highstand, the paleosol would have developed during the understanding boundaries for deposition and determining following lowstand, and the removal of the paleosol by the sequence of geologic events. When compared with wave action and subsequent lowering of the bedrock timelines for other areas, the researcher can begin to surface would have occurred during the present highstand. discern the geologic history of the entire island. Thus, the presence of the paleosol-coated pits demonstrates that the eolianite bedrock here is at least 125,000 years old. ACKNOWLEDGEMENTS Pseudokarst voids such as tafoni may also be helpful in clarifying geologic timelines. The tafoni found at North The authors would like to thank the Karst Waters Point on San Salvador Island are modern features still Institute, Total SA, and Mississippi State University for forming in a recent eolianite deposited on the transgression providing the funding for this study; the Bahamian of the current highstand (Fig. 10B). This eolianite has not government for providing the research permit; Friends of been subjected to multiple sea-level events or extensive the Environment, Marsh Harbour, Abaco for providing karstification. The tafoni found on Abaco, however, are logistical support; all of the residents of Abaco Island that relict features from a previous highstand event. The Abaco made this study possible, particularly Nancy and Michael tafoni (or PITA Caves) were likely formed during the OIS Albury, Anita Knowles, Allison Ball, Diane Claridge, and 5e highstand on an eolianite that was already present. This David Knowles; Caela O’Connell and Nancy Albury for eolianite may have been deposited on the transgression of their assistance in locating and mapping the caves of the 5e highstand or during a previous highstand. As this Abaco; and Brenda Kirkland and Grady Dixon of eolianite was eroded by wave energy, the poorly-cemented Mississippi State University for all of their insights and interior was exposed to the extensive coastal weathering comments during the initial development of this manu- processes to form pseudokarst tafoni voids. Because the script. 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Journal of Cave and Karst Studies, August 2008 N 119 S. Herrando-PeÂrez, M. Baratti, and G. Messana ± Subterranean ecological research and multivariate statistics: a review (1945±2006). Journal of Cave and Karst Studies, v. 70, no. 2, p. 120±128. SUBTERRANEAN ECOLOGICAL RESEARCH AND MULTIVARIATE STATISTICS: A REVIEW (1945±2006)

SALVADOR HERRANDO-PEÂ REZ1,*,MARIELLA BARATTI2, AND GIUSEPPE MESSANA2

Abstract: Subterranean ecosystem studies using multivariate ordination and/or agglomerative classification statistical methods were reviewed in the Science Citation Index (SCI) between 1945 and 2006. Nearly 57,000 publications cited subterranean habitats or their associated biota in the SCI abstracts, however, multivariate statistics applied to strictly hypogean taxa occurred in only 65 papers from 1990 onwards. Over 90% of the multivariate applications were devoted to morphometric or geneticstudies of single species and to relationships between the environment and species assemblages. In terms of taxa and ecosystem types, stygobite and waterless cave studies featuring multivariate applications predominated, respectively. Only six different methods (Agglo- merative Clustering, Canonical Correspondence Analysis, Correspondence Analysis, Discriminant Analysis, non-metric Multidimensional Scaling, Principal Component Analysis) were used among the .30 multivariate techniques available within the biostatistical toolbox. The retrieved set of publications was sorted in a simple table by keyword according to type of biota, habitat, research topic and multivariate method, while online biostatistical resources are appended. Further comments are made on the use of statistics in the biological sciences in general.

INTRODUCTION represent samples and distances among them are propor- tional to their degree of likeliness measured by a given Multivariate statistics (i.e. ordination, classification and resemblance measure, e.g. Euclidean distances or Bray- hybrid methods) are useful in studies where one of the main Curtis similarities (see Clarke et al., 2006). As to classifi- goals may be to quantify relationships among a set of cation techniques (Legendre and Legendre, 1998), dendro- samples in which a large number of variables has been grams or trees are the display options, where branches link measured. A matrix of abundance of species assemblages and cluster groups of sample units in a sequential fashion and a matrix of measurements of environmental variables based on resemblance. Second, as part of the numerical from an array of samples spread in space and/or time is a result of a multivariate analysis, a series of coefficients are classical data set to deploy multivariate statistics in produced, which quantify the importance of each of the ecological research. For example, samples may be different variables in the ordination patterns observed; hence caves surveyed in a single region, a single cave monitored principal coefficients in eigen-analysis-based techniques several times per year, or a combination of the latter, while like Principal Component Analysis (PCA; Hotelling, 1933) the relative abundances of troglobite taxa found in one or Correspondence Analysis (CA; Hirschfeld, 1935; Hill baited trap per cave and physical variables measured and Gauch, 1980; ter Braak and Schaffers, 2004), or around each trap (e.g. humidity, temperature, etc.) may similarity contributions (Clarke, 1993) from distance-based constitute the raw data. Common research questions asked methods like non-MetricMulti-Dimensional Scaling of such matrices are whether there are patterns of species (nMDS; Shepard, 1962; Belbin, 1991), Cluster Analysis assemblages among caves and what environmental factors (CLUSTER; Sneath and Sokal, 1973) or Polar Ordination are underlying those patterns (e.g. Christman and Culver, (Bray and Curtis, 1957). 2001). Ordination techniques may be indirect (unconstrained) Multivariate statistical methods originated from the or direct (constrained, also known as canonical) depending pioneering work by Petrie (1899) and Spearman (1904). on whether one or two data matrices, respectively, are Since then, over thirty different multivariate techniques input into the algorithm. The whole family of ordination have been described (see Gauch, 1982; Jongman et al., methods is sometimes termed as gradient analysis following 1995; Legendre and Legendre, 1998; Clarke and Warwick, ter Braak and Prentice (1988). Within the suite of canonical 2001; McCune and Grace, 2002; Leps and Smilauer, 2003; methods, ordinations can be constrained to extract a Zuur et al., 2007), mostly within the last two decades. These techniques have a number of advantages over more traditional, univariate techniques, notably in their graph- * Present address: School of Earth & Environmental Sciences, University of Adelaide, SA 5005, Australia ([email protected]) ical and numerical outputs. In ordination methods, 1 BIOESTUDIOS SAGANTA, c/ San Pedro 6, 12200 Onda - Spain complex multivariate information is summarized in a 2 Institute for the Study of Ecosystems (ISE), Via Madonna del Piano, 10, 50019 low-dimensional scatter diagram where points commonly Sesto Fiorentino (Fi) ± Italy 120 N Journal of Cave and Karst Studies, August 2008 S. HERRANDO-PEÂ REZ,M.BARATTI, AND G. MESSANA specific fraction of information from a species x sample data matrix, for instance, the amount of assemblage variation that can be explained by a set of environmental predictors (one type of constraint, i.e., the second data matrix). This is the case in Redundancy Analysis (RDA; de Wollenberg, 1977), Canonical Correspondence Analysis (CCA; ter Braak, 1986; Makarenkof & Legendre, 2004) and Canonical Correlation Analysis (CCoA; Hotelling, 1936; Anderson & Willis, 2005). On the other hand, ANOVA-like tests for differences among a priori groups of samples (a further type of constraint) in Discriminant Analysis (DA; Fisher, 1936; Legendre & Anderson, 1999; McArdle & Anderson, 2001; Anderson and Robinson, 2005; Millar et al., 2005) represent the second major kind of canonical analyses. Ordination methods have been Figure 1. Number of publications (in a log-scale) cited in the employed traditionally in a descriptive fashion, but recent Science Citation Index (1945±2006) after a search by advances have fostered ordination-based frameworks to abstract including key words standing for Subterranean carry out MANOVA inferential tests (Legendre and Habitat, Subterranean Biota (SB), Multivariate ordination Anderson, 1999; McArdle and Anderson, 1999) and and classification Methods (MM), and SB+MM. predictive modeling (e.g. Guisan et al., 1999). Comparative reviews over the properties and relative merits and (CastelloÂn de la Plana, Spain). A total of 358 key words drawbacks of the various techniques can be found in were browsed in the database in three compound sets Minchin (1987), Clarke and Green (1988), ékland (1996) linked with the standard commands AND, OR, `` '' and * and Anderson and Willis (2003). Moreover, advanced as appropriate. These sets consisted of groups of words classification methods can also quantify relationships that could retrieve from SCI any publication incorporating among two sets of variables in the so-called Multivariate multivariate methods (117 words), subterranean biota (69 Regression Trees (Breiman et al., 1984; Moore et al., 1991; words) and subterranean habitats (172 words). Subterra- Vayssieres et al., 2000; De'ath, 2002), or integrate classifi- nean habitats included caves, the soil vadose and phreatic cation and ordination techniques into two-way ordered zones, lava tubes, cenotes and blue holes, while nomencla- tables known as TWINSPAN (Hill, 1979; Gauch, 1982) ture for subterranean biota followed Malard et al. (2003). which have been particularly popular among botanists. Searches were done by abstract, after first undertaking a Although multivariate techniques are now firmly trial by title which only found three publications. The review established in many fields of ecology (particularly so for was restricted to research articles published in English. marine benthos, freshwater, and botany), their use seems restricted in subterranean ecosystem studies. To quantify RESULTS this apparent lack of implementation, we describe trends in the utilization of multivariate ordination methods and Throughout the period of 61 years surveyed, a total of agglomerative classification for the study of subterranean 56,559 publications cited subterranean ecosystems or their fauna and microbes in ecological research through a associated biota in their abstracts. Of this total, approx- literature review in the Science Citation Index (1945± imately 20% and 80% dealt with subterranean biota and 2006). Records of genetic studies (which do not subscribe habitats, respectively, and only 266 papers (0.47%) had strictly to ecology) and of submarine cave papers (which do used multivariate ordination and/or classification methods. not deal with subterranean ecosystems proper) were also The number of publications on subterranean biota and included as sample examples of other type of multivariate habitats, whether multivariate statistics were included or applications from a common raw data entry. The main not, increased with time, particularly from 1985 to 1995 fields of utilization are highlighted, a synthesis of on-line (Fig. 1). The first paper to use multivariate methods in resources for multivariate statistics is provided, and the full SCI-cited subterranean research was published in 1990. set of selected publications (n565) is appended after Many of the papers extracted from the SCI were sorting them by type of biota, habitat, research topic and devoted to flora and fauna that do not inhabit the multivariate method. subterranean realm, for instance surface vegetation in karsticregions or aquaticinvertebrates in rivers receiving METHODS water from a subterranean course. As such, all abstracts were manually scrutinized. Out of the 266 publications The literature review was undertaken in the Science using multivariate methods, only 65 papers were specifi- Citation Index (SCI, 1945±2006, inclusive) on-line at the cally related to hypogean ecosystems and they form the Department of Analytical Chemistry, University Jaume I literature compilation used hereafter (see Appendix). Journal of Cave and Karst Studies, August 2008 N 121 SUBTERRANEAN ECOLOGICAL RESEARCH AND MULTIVARIATE STATISTICS:AREVIEW (1945±2006)

DISCUSSION

Classification and ordination multivariate statistics have only been employed in the study of species assemblages and population structure of subterranean biota from the very beginning of the 1990's onwards. Both the total number of papers published and those that report multivariate methods follow an exponential increase, a popular trend across scientific literature often named after Price's model (FernaÂndez-Cano et al., 2004). The assimi- lation of multivariate statistics in ecological subterranean research responds to a two-fold development of this field that basically enhanced the collection of quantitative data amenable to these chiefly quantitative or semiquantitative Figure 2. Number of publications employing a total of 6 statistics. First, major subterranean research teams were multivariate methods in research papers on subterranean established in Europe and the USA in the 1980s (Gibert et biota. Publication records (n=65) were obtained from the al., 1994) and this doubtless brought about an improved Science Citation Index (1945±2006) after a search by development of more rigorous sampling techniques, as well abstract including key words standing for the subterranean as a need to explore different avenues of research. biota. See legend on top with overall subjects times the Secondly, in the last fifteen years, the explosion of number of publications/subject (records without citation of molecular analyses supplied an enormous amount of multivariate methods in abstracts are excluded). Methods quantitative information that was central to progressing are: DA= DiscriminantAnalysis PCA = Principal Compo- the systematics of subterranean fauna (Culver, 1982; nentAnalysis CLUSTER = Agglomerative Clustering CCA= Sbordoni et al., 2000; Finston et al., 2007). Canonical Correspondence Analysis CA= Correspondence The application of ordination/classification methods is Analysis nMDS= non-Metric Multi-Dimensional Scaling larger for taxa that are most conspicuous and/or frequent (nMDS). in subterranean habitats, basically insects and arachnids in waterless caves, and crustaceans in hyporheos and fresh- Approximately two thirds (41 papers) of the multivar- water caves. Up to two thirds of the multivariate cases were iate applications focused on hypogean species assemblages, applied to data from aquatic species which are generally mainly dealing with invertebrates (hyporheos and fresh- easier to sample quantitatively than their terrestrial water stygobites; 15 papers) and with bacteria from counterparts. Moreover, assemblages of aquatic taxa, both aquifers and hyporheicenvironments (12 papers). On the invertebrates and bacteria, were often described by means other hand, the majority of population studies (i.e. of ordination methods whereby indicator taxa were addressing a single species) were carried out in waterless identified in order to assess water quality of aquifers and caves where the target fauna were troglobite arthropods of phreaticand vadose environments. The six multivariate (insects and arachnids, eight papers) and trogophile methods reported in this literature review are among the vertebrates (bats and rodents, 10 papers). There were no classical ones in ecological research. In fact, CLUSTER, multivariate records for cenotes, lava tubes or blue holes. DA and PCA totalled just 50% of the case studies featuring Subject-wise, studies using multivariate methods could multivariate applications. Similar trends were found by be allocated to four categories (Fig. 2). A total of 46% (31 James and McCulloch (1990). The preferred use of DA in papers) of the multivariate applications centered upon the geneticand morphometricstudies, where differencesin relationship between species assemblages and environ- subsets of individuals from populations and communities mental variables, 27 papers were on morphology/mor- are looked for, clearly takes advantage of the ANOVA-like phometrics or genetics, or a combination of both (three approach of this method. papers), of individual species, whereas seven papers were However, despite the power of multivariate methods to published in the area of paleontology, occasionally analyze a wide variety of data in biodiversity studies, the focusing on fossil hominids (two papers). A total of six intensity of their use can be regarded as low for research different multivariate methods were recorded in this into subterranean biota, with an average of only four review, namely DA, PCA, CLUSTER, CCA, CA and publications per year surveyed in SCI. Furthermore, less nMDS, in that order of popularity (Fig. 2), whereas in 11 than 20% of the total number of the different methods abstracts the methods used were not mentioned. All the available seems to have been employed, with no records of multivariate methods except DA were employed in those techniques described from the middle of the 1990s describing environment-community relationships, while onwards (see Introduction). Such a meagre utilization may DA was utilized in half of the morphological and genetic be for different reasons. On one side, despite there being studies on single species. ample literature of quantitative studies focusing on 122 N Journal of Cave and Karst Studies, August 2008 S. HERRANDO-PEÂ REZ,M.BARATTI, AND G. MESSANA hypogean biota, the relatively low diversity, often mainly kal, 1964), together with extremely complex algorithms, recorded at small spatial scales, can turn into simplified like CCA (ter Braak, 1986), are computer-dependant and data sets that may be deemed unsuitable for multivariate could not be widely implemented until the 1970's when the analysis. This can be exacerbated by the fact that many second-generation supercomputers became openly avail- subterranean habitats are fragmented, so causing confined able to the scientific community. Finally, from their initial distributions of hypogean fauna (hence simple species graduate training and thereafter, biologists are reluctant to assemblages) with distinct taxa not overlapping but get into complex mathematics, which are too often taught occurring at sites geographically very close (Marmonier by non-biologists as an isolated subject not integrated in et al., 1993; Sket, 1999; Gibert and Deharveng, 2002). On the many-fold disciplines of biology and unrelated to lab the other side, survey efforts for unravelling diversity and field studies (see Johnson et al., 2001 for recommen- patterns of subterranean biota are largely concentrated in a dations to achieve sound statistical training and back- group of localities in North America, Europe and ground in environmental research). Additionally, statistical Australia. Proof of this resides in the fact that total instruction and utilization may sometimes be misleading. diversity of cave-dwelling fauna at global, landscape, Thus, many authors have documented an ill-use of regional and even local scales is largely ignored (Culver statistics in ecology and wildlife research regarding their and Holsinger, 1992; Culver and Sket, 2001). Thus, less application or the reporting and interpretation of results research investment necessarily entails less publication (James and McCulloch, 1990; Johnson et al., 1999; output, regardless the nature of the analytical approaches Stephens et al., 2005). Sometimes mistakes may linger on carried out. unnoticed for both naõÈve and experienced statistical users Cave and, principally, ground water ecosystems are and conceptual confusion prevails over the years, even for severely endangered in many parts of the world, and well-established concepts. For instance, Hubbard and precise management alternatives are urgently required Bayarri (2003) made a poignant observation on the (Danielopol et al., 2003) whereby subterranean biodiversity pervasive confusion between p confidence values and a can represent a key indicator of ecosystem health (Sket, error rates in hypothesis testing, yet it is striking that 1999). However, there is a general absence of keystone or Ellison (2006) confused both concepts, (ironically) after flag hypogean species, unlike other terrestrial ecosystems citing Hubbard and Bayarri (2003), in her high-ranked where the distribution of species and assemblages of journal review on Bayesian inference. Back to multivariate commonly encountered vertebrates have proven useful analysis, McCune and Grace (2002) were unable to cite for management and conservation practices, including appropriate or adequately justified applications of CA in identification of priority areas and regional patterns of the literature of community ecology, while James and subterranean biodiversity. Despite considerable effort McCulloch (1990) ``could not find a single application of being invested into describing broad biodiversity patterns multiple regression to recommend as a good example'' after in Europe (Sket, 1999; Ferreira et al., 2003) and North reviewing in the highly ranked journals of ecology and America (Peck, 1998; Christman and Culver, 2001), the systematics. paucity of quantitative data elsewhere means that data are In summary, the statistical tools are widely accessible, reported in the form of species lists and graphical but they are often misused, badly-reported and (not representation of distributions is often poor, which limit surprisingly!) generally feared by biologists. See Table 1 their impact on managers and decision-making groups. On for a syntheticlist of online resourcesfor multivariate the premise that ordinations are good communication tools statistics covering mailing lists, free and major commercial (ter Braak, 1994), bio-regionalization of subterranean software, biostatistical experts' web pages, training courses, habitats should benefit from ordination and classification and research journals within the fields of biology and of sites for which abundance, or presence-absence, data of ecology. An illustrative starting point for those interested hypogean taxa have been measured. This practice should in improving the standards of statistical reporting should be stimulated in the future. read the papers by David Anderson, Douglas Johnson and collaborators (see Anderson et al., 2001, Johnson et al., STATISTICS AND BIOLOGY:COMPLEMENTARY? 2002, and references therein) for inferential tests, and by Overall, a number of academic, sometimes practical, James and McCulloch (1990) and Paukert and Wittig aspects hamper the access of biologists to multivariate (2002) for multivariate statistics. Lastly, universities statistics. Some multivariate methods like PCA, and its hosting programs on environmental sciences should be canonical analogous RDA, were described outside the encouraged to improve (multivariate) statistical training by realm of biology, and their adoption by biologists lagged tailoring it to the needs and disciplines of students. By facing behind for years. Others are relatively new, so they have real world problems (instead of only card games, lottery not yet joined up to most textbooks and are mainly taught probabilities and the like) biologists' attitudes towards in (generally very expensive) specific training courses. statistics may improve, while their future academic and Furthermore, those multivariate methods that resort to professional profiles would be enhanced further by attaining ordination solutions by permutations, like nMDS (Krus- an early environment-focused statistical background. Journal of Cave and Karst Studies, August 2008 N 123 SUBTERRANEAN ECOLOGICAL RESEARCH AND MULTIVARIATE STATISTICS:AREVIEW (1945±2006)

Table 1. On-line resources addressing multivariate statistical methods in environmental sciences. Link Brief Description http://www.okstate.edu/artsci/botany/ordinate/ A bible of ordination methods by Mike Palmer at Oklahoma State University (USA). http://www.okstate.edu/artsci/botany/ordinate/ Mailing list on ordination methods, though a wide range of statistical ordnews.htm topics is discussed. http://www.classification-society.org International Society of Classification, including links with branches world-wide. http://www.classification-society.org/csna/lists. Mailing list on classification methods. html#class-l http://cc.oulu.fi/,jarioksa/ Personal page by Jari Oksanen at Oulu University (Finland). Free software available. http://www.stat.auckland.ac.nz/,mja Personal page by Marti Anderson at Auckland University (New Zealand). Free software available. http://www.bio.umontreal.ca/legendre/ Personal page by Pierre Legendre at Montreal University (Canada). Free software available. http://www.r-project.org/ Free software environment for statistical computing and graphics. http://www.highstat.com Training courses and BRODGAR software by Alain Zuur (Scotland). http://regent.bf.jcu.cz Training courses and CANOCO software by Jan Leps and Petr Smilauer (Check Republic). http://home.centurytel.net/,mjm/ Training courses and PCOrd software by MjM Software Design (USA). http://www.primer-e.com Training courses and PRIMER software by Bob Clarke (UK). http://aerg.canberra.edu.au/envirostats/ On-line training courses at Canberra University (Australia). http://www.statistics.com/ On-line training courses (USA). Not specifically environmental. http://biomet.oxfordjournals.org/ Biometrika (Research journal, Oxford University Press). http://www.springer.com/life+sci/ecology/ Environmental and Ecological Statistics (Research journal, Springer). journal/10651 http://www.elsevier.com/wps/find/ Environmetrics (Research journal, Wiley Inter-Science). journaldescription.cws_home/622892/ description#description http://www.springer.com/statistics/statistical Journal of Classification (Research journal, Springer). +theory+and+methods/journal/357 http://www.sciencedirect.com/science/journal/ Journal of Multivariate Analysis (Research journal, Elsevier). 0047259X

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APPENDIX Papers using multivariate classification and ordination methods for analyzing data from subterranean biota as retrieved from the Science Citation Index (1945±2006). CLASSIFICATION (numbers correspond with the list of publications further below): By biota: Marine benthos (sponges) 9,30,64 Microfauna, mainly bacteria 2,6,8,11,15,21,28,32,40,42,46,53,58 Stygobite invertebrates 1,13,16,17,20,22,25,26,33,36,37,43,44,45,47,54,56,57,60,61,63 Troglobite/Troglophile invertebrates 4,5,10,14,18,23,38,49,62 Troglophile/Trogloxene vertebrates 3,12,19,24,27,29,31,34,35,39,48,50,51,52,55,59,65 Vegetation 7,41 By habitat: Subterranean waters, mainly aquifers 2,8,15,28,32,40,42,46 Hyporheicenvironment 3,18,21,25,27,28,32,41,44,48,52,54,56±59 Freshwater caves 1,20,35,36,37,45,61,63 Marine caves 3,9,17,30,64 Waterless caves 4,5,7,10,11,12,14,16,18,19,23,24,27,29,31,34,38,39,41,48±52,55,59,62,65 By multivariate method: Canonical Correspondence Analysis (CCA) 1,9,22,26,36,54,57 Classification Analysis (CLUSTER) 9,11,13,15,17,20,31,42,64 Correspondence analysis (CA) 31,52,64 Discriminant Analysis (DA) 5,12,18,20,27,29,35,38,39,45,49,51,65 nonmetricMulti-Dimensional Scaling (nMDS) 3,6,9,14,30 Principal Component Analysis (PCA) 7,8,21,23,27,32,37,40,42,46,48±50,52,55 By subject: Biota response to environment 1,2,3,6,8,9,15,17,21,22,25,26,28,30,31,32,33,36,42,43,44,46,47,53,54,56±58,60,64 Genetics 5,10,11,12,24,37,39,62,63 Morphology/Morphometrics 4,14,16,18,20,23,24,29,35,38,45,49±51,59,61,63,65 Paleontology 7,19,27,41,48,52,55 Sampling methods 13,40

126 N Journal of Cave and Karst Studies, August 2008 S. HERRANDO-PEÂ REZ,M.BARATTI, AND G. MESSANA

PUBLICATION LIST (ordered by year of publication): 19. Postawa, T, 2004. Changes in bat fauna during the Middle and Late Holocene as exemplified by thanatocoenoses dated with C-14 AMS 1. Pipan, T; Blejec, A; Brancelj, A, 2006. Multivariate analysis of from Krakow-Czestochowa Upland caves, Poland. Acta Chiropter- copepod assemblages in epikarstic waters of some Slovenian caves. ologica 6, 269±292. Hydrobiologia 559, 213±223. 20. Prevorcnik, S; Blejec, A; Sket, B, 2004. Racial differentiation in 2. Schryver, JC; Brandt, CC; Pfiffner, SM; Palumbo, AV; Peacock, Asellus aquaticus (L.) (Crustacea: lsopoda: Asellidae). Archiv fur AD; White, DC; McKinley, JP; Long, PE, 2006. Application of Hydrobiologie 160, 193±214. nonlinear analysis methods for identifying relationships between 21. Findlay, SEG; Sinsabaugh, RL; Sobczak, WV; Hoostal, M, 2003. microbial community structure and groundwater geochemistry. Metabolic and structural response of hyporheic microbial commu- Microbial Ecology 51, 177±188. nities to variations in supply of dissolved organicmatter. Limnology 3. Carpentieri, P; Colloca, F; Ardizzone, GD, 2005. Day-night and Oceanography 48, 1608±1617. variations in the demersal nekton assemblage on the Mediterranean 22. Hunt, GW; Stanley, EH, 2003. Environmental factors influencing shelf-break. Estuarine Coastal and Shelf Science 63, 577±588. the composition and distribution of the hyporheic fauna in 4. Catala, S; Sachetto, C; Moreno, M; Rosales, R; Salazar-Schettino, Oklahoma streams: Variation across ecoregions. Archiv fur Hydro- PM; Gorla, D, 2005. Antennal phenotype of Triatoma dimidiata biologie 158, 1±23. populations and its relationship with species of Phyllosoma and 23. Machado, SF; Ferreira, RL; Martins, RP, 2003. Aspects of the Protracta complexes. Journal of Medical Entomology 42, 719±725. population ecology of Goniosoma sp (Arachnida Opiliones Gony- 5. FernaÂndez, GC; JuaÂrez, MP; Monroy, MC; Menes, M; Bustamante, leptidae) in limestone caves in southeastern Brazil. Tropical Zoology DM; Mijailovsky, S, 2005. Intraspecific variability in Triatoma 16, 13±31. dimidiata (Hemiptera: Reduviidae) populations from Guatemala 24. Maharadatunkamsi; Hisheh, S; Kitchener, DJ; Schmitt, LH, 2003. based on chemical and morphometric analyses. Journal of Medical Relationships between morphology, genetics and geography in the Entomology 42, 29±35. cave fruit bat Eonycteris spelaea (Dobson, 1871) from Indonesia. 6. Jaatinen, K; Tuittila, ES; Laine, J; Yrjala, K; Fritze, H, 2005. Biological Journal of the Linnean Society 79, 511±522. Methane-oxidizing bacteria in a Finnish raised mire complex: 25. Malard, F; Galassi, D; Lafont, M; Doledec, S; Ward, JV, 2003. Effects of site fertility and drainage. Microbial Ecology 50, 429±439. Longitudinal patterns of invertebrates in the hyporheiczone of a 7. Kaniewski, D; Renault-Miskovsky, J; de Lumley, H, 2005.Palaeo- glacial river. Freshwater Biology 48, 1709±1725. vegetation from a Homo neanderthalensis occupation in Western 26. Malard, F; Ferreira, D; DoleÂdec, S; Ward, JV, 2003. Influence of Liguria: archaeopalynology of Madonna dell'Arma (San Remo, groundwater upwelling on the distribution of the hyporheos in a Italy). Journal of Archaeological Science 32, 827±840. headwater river flood plain. Archiv fur Hydrobiologie 157, 89±116. 8. Mouser, PJ; Rizzo, DM; Roling, WFM; Van Breukelen, BM, 2005. 27. Neves, WA; Prous, A; GonzaÂlez-JoseÂ, R; Kipnis, R; Powell, J, 2003. A multivariate statistical approach to spatial representation of Early Holocene human skeletal remains from Santana do Riacho, groundwater contamination using hydrochemistry and microbial Brazil: implications for the settlement of the New World. Journal of community profiles. Environmental Science & Technology 39, 7551± Human Evolution 45, 19±42. 7559. 28. Yan, TF; Fields, MW; Wu, LY; Zu, YG; Tiedje, JM; Zhou, JZ, 9. Preciado, I; Maldonado, M, 2005. Reassessing the spatial relation- 2003. Molecular diversity and characterization of nitrite reductase ship between sponges and macroalgae in sublittoral rocky bottoms: gene fragments (nirK and nirS) from nitrate- and uranium- a descriptive approach. Helgoland Marine Research 59, 141±150. contaminated groundwater. Environmental Microbiology 5, 13±24. 10. Schilthuizen, M; Cabanban, AS; Haase, M, 2005. Possible 29. Armstrong, KN, 2002. Morphometricdivergenceamong popula- speciation with gene flow in tropical cave snails. Journal of tions of Rhinonicteris aurantius (Chiroptera: Hipposideridae) in Zoological Systematics and Evolutionary Research 43, 133±138. northern Australia. Australian Journal of Zoology 50, 649±669. 11. Taylor, ML; Chavez-Tapia, CB; Rojas-Martinez, A; Reyes-Montes, 30. Bell, JJ, 2002. The sponge community in a semi-submerged MD; del Valle, MB; Zuniga, G. 2005. Geographical distribution of temperate sea cave: Density, diversity and richness. Marine Ecology geneticpolymorphism of the pathogen Histoplasma capsulatum (Napoli) 23, 297±311. isolated from infected bats, captured in a central zone of Mexico. Fems Immunology and Medical Microbiology 45, 451±458. 31. Furman, A; Ozgul, A, 2002. Distribution of cave-dwelling bats and conservation status of underground habitats in the Istanbul area. 12. Tiranti, SI; Dyzenchauz, FJ; Hasson, ER; Massarini, A, 2005. Evolutionary and systematic relationships among tuco-tucos of the Ecological Research 17, 69±77. Ctenomys pundti complex (Rodentia : Octodontidae): a cytogenetic 32. Haveman, SA; Pedersen, K, 2002. Distribution of culturable and morphological approach. Mammalia 69, 69±80. microorganisms in Fennoscandian Shield groundwater. Fems 13. Boulton, AJ; Dole-Olivier, MJ; Marmonier, P, 2004. Effects of Microbiology Ecology 39, 129±137. sample volume and taxonomicresolution on assessment of 33. Scarsbrook, MR; Halliday, J, 2002. Detecting patterns in hyporheic hyporheic assemblage composition sampled using a Bou-Rouch community structure: does sampling method alter the story? New pump. Archiv fur Hydrobiologie 159, 327±355. Zealand Journal of Marine and Freshwater Research 36, 443±453. 14. Bustamante, DM; Monroy, C; Menes, M; Rodas, A; Salazar- 34. Wilkinson, GS; South, JM, 2002. Life history, ecology and longevity Schettino, PM; Rojas, G; Pinto, N; Guhl, F; Dujardin, JP, 2004. in bats. Aging Cell 1, 124±131. Metric variation among geographic populations of the chagas vector 35. Burr, BM; Adams, GL; Krejca, JK; Paul, RJ; Warren, ML, 2001. Triatoma dimidiata (Hemiptera: Reduviidae: Triatominae) and Troglomorphicsculpinsof the Cottus carolinae species group in related species. Journal of Medical Entomology 41, 296±301. Perry County, Missouri: Distribution, external morphology, and 15. de Lipthay, JR; Johnsen, K; Albrechtsen, HJ; Rosenberg, P; conservation status. Environmental Biology of Fishes 31±32, 279± Aamand, J, 2004. Bacterial diversity and community structure of a 296. sub-surface aquifer exposed to realistic low herbicide concentra- 36. Jersabek, CD; Brancelj, A; Stoch, F; Schabetsberger, R, 2001. tions. Fems Microbiology Ecology 49, 59±69. Distribution and ecology of copepods in mountainous regions of the 16. Lozouet, P, 2004. The European Tertiary Neritiliidae (Mollusca, Eastern Alps. Hydrobiologia 541, 309±324. Gastropoda, Neritopsina): indicators of tropical submarine cave 37. BaltanaÂs, A; Namiotko, T; Danielopol, DL, 2000. Biogeography environments and freshwater faunas. Zoological Journal of the and disparity within the genus Cryptocandona (Crustacea, Ostra- Linnean Society 140, 447±467. coda). Vie et Milieu-Life and Environment 50, 297±310. 17. Marti, R; Uriz, MJ; Ballesteros, E; Turon, X, 2004. Benthic 38. Gracco, M; Catala, S, 2000. Inter-specific and developmental assemblages in two Mediterranean caves: species diversity and differences on the array of antennal chemoreceptors in four species coverage as a function of abiotic parameters and geographic of Triatominae (Hemiptera: Reduviidae). Memorias do Instituto distance. Journal of the Marine Biological Association of the Oswaldo Cruz 95, 67±73. United Kingdom 84, 557±572. 39. Nevo, E; Ivanitskaya, E; Filippucci, MG; Beiles, A, 2000. Speciation 18. Muster, C; Schmarda, T; Blick, T, 2004. Vicariance in a cryptic and adaptive radiation of subterranean mole rats, Spalax ehrenbergi species pair of European pseudoscorpions (Arachnida, Pseudoscor- superspecies, in Jordan. Biological Journal of the Linnean Society piones, Chthoniidae). Zoologischer Anzeiger 242, 299±311. 69, 263±281. Journal of Cave and Karst Studies, August 2008 N 127 SUBTERRANEAN ECOLOGICAL RESEARCH AND MULTIVARIATE STATISTICS:AREVIEW (1945±2006)

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Franklin, RB; Taylor, DR; Mills, AL, 1999. The distribution of and recent samples of Cryptotis (Insectivora, Soricidae) from the microbial communities in anaerobic and aerobic zones of a shallow Yucatan peninsula, Mexico. Journal of Mammalogy 76, 223±231. coastal plain aquifer. Microbial Ecology 38, 377±386. 56. Plenet, S, Hugueny, H; Gibert, J, 1994. Invertebrate community 43. Nelson, SM; Roline, RA, 1999. Relationships between metals and responses to physical and chemical factors at the river aquifer hyporheic invertebrate community structure in a river recovering interaction zone .1. Upstream from the city of Lyon. Archiv fur from metals contamination. Hydrobiologia 397, 211±226. Hydrobiologie 136, 165±189. 44. Strayer, DL; Reid, JW, 1999. Distribution of hyporheic cyclopoids 57. Richards, C; Bacon, KL, 1994. Influence of fine sediment on (Crustacea: Copepoda) in the eastern United States. 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128 N Journal of Cave and Karst Studies, August 2008 BOOK REVIEW

BOOK REVIEW

below¼ independent of recharge from the overlying or immediately adjacent surface.'' Caves, in Klimchouk's view, are of three types: (1) epigenic (he prefers ``hyper- genic'' as a parallel to hypogenic), (2) hypogenic, and (3) coastal and oceanic. At first I cheered this three-part classification, as it clearly identified the island caves I work in, but they are never again discussed in the book. Klimchouk's main concept is that in a large regional aquifer, a significant portion is buried deeply and is confined. These basins are so large that lateral water flow into and out of them is inefficient, and the high hydraulic heads created by the confined conditions produce slow vertical ascending flow across the confining units. Most basins are recharged by water infiltrating from the surface, but in remote areas far from the rising water. The ascending flow is focused on topographic lows where the confining units are thinnest, or where structural or lithologic discontinuities have facilitated vertical flow. Soluble rocks in this sequence, whether carbonate or sulfate (halides are not discussed), initially have very low permeability. However, as water flows upward across them in what the author calls ``transverse flow,'' they become highly permeable. The author considers this a major re- organization of the flow, but it is hard to see why that would be so, as the input from below the cave unit, and the Hypogene Speleogenesis: Hydrogeological and Morphoge- output through the overlying confining unit, needs to be netic Perspective slow. I use the term ``cave unit'' here, while the author uses ''cave formation,'' as in ``geologic formation.'' But the Alexander Klimchouk, 2007. Carlsbad, N.M., National word ``formation'' is confusing either as a noun (forma- Cave and Karst Research Institute, Special Paper No. 1, tion, as in speleothem) or as a verb (formation, as in 106 p., 8.5 3 11 inches. ISBN 978-0-9795422-0-6, soft- development). Once the cave unit has reached a perme- bound, $35. ability equal to that of the confining unit above, with openings still well below cave-passage size, the cave unit This book was written mainly during Dr. Klimchouk's would offer no further hydrologic advantage to the stay as a Visiting Scholar at the National Cave and Karst regional flow system. Subsequent dissolutional enlarge- Research Institute in 2006±2007. It is an intense compila- ment would be merely a consequence of slow fluid flow tion of his ideas on hypogenic speleogenesis. Its main through the cave unit. Still, can such caves form? I would thrust is that dissolution in deepartesian or confined basins say yes, as such basins form over long periods of time and creates many caves, and that they are under-appreciated by can provide stable flow systems for millions of years, so the speleological community. As someone who has had to even if dissolution is very slow, it can still form use non-traditional ideas to understand cave development macroscopic voids. The author invokes thermal gradients on carbonate islands and coasts, I agree with his assertion or mixing of chemically disparate waters to accomplish that traditional stream-cave models do not successfully dissolution at the cave scale. As the author notes, the explain all dissolutional cave development. I also agree growth of openings is non-competitive (i.e., all openings with him that explorational bias has created a cave grow without any single one capturing all the water), so database and speleogenetic theories that are skewed toward enlargement of vugs and joints could occur anywhere stream caves. The author also chides the speleological ascending water moves through the cave unit. The primary community for its definitions that assume that karst is cave outcome would be isolated chambers and fissures, restricted to carbonate rocks. In preference to Art Palmer's collections of chambers and fissures, or maze caves, as definition of hypogenic caves as those formed where the determined by the degree of lateral connectivity or of solutional capacity is derived from deep-seated processes, focusing of the ascending flow. As this flow is very slow the author favors that of Derek Ford: ``the formation of and non-turbulent, dissolutional bedrock forms related to caves by water that recharges the soluble formation from convecting water flow are possible, and even expected. This Journal of Cave and Karst Studies, August 2008 N 129 BOOK REVIEW convection can be thermal, driven by solute density, or understand the mechanism. Dr. Klimchouk describes, and both. Because I feel that the author's definition of displays in many well-presented photos, a list of features he hypogenic is too restrictive, I call these caves ``confined claims are diagnostic of confined cave development. These caves'' instead of his ``hypogenic caves,'' to describe them include maze patterns, cupolas, former infeeders in the by their environment of origin. floors and outlets in the ceilings, and wall grooves, all So far, so good. But the author is often vague about the assumed to form by rising water. Their presence in caves water chemistry and dissolutional mechanisms, becoming now located in unconfined or vadose environments is a specific only when describing confined caves now relict at result, in his opinion, of inheritance from a confined cave the earth's surface. His ascension model does not work if origin, with the later overprint by unconfined and/or lateral transport from recharge zones does not continually vadose conditions. Dr. Klimchouk has used the idea of replace the rising water. Compaction and magmatic overprinting to explain why some caves do not show sources cannot by themselves supply enough ascending sufficient evidence to support his model. This is a valid water over the long periods of time needed to accomplish approach, as, by definition, these confined caves cannot be the described speleogenesis. While the author is correct in explored until they have been exposed at the earth's surface saying that confined caves have suffered from an explora- and are decoupled from their original confined environment. tional bias, he at the same time infers that his described In making that transition, they would likely be overprinted caves end at the limit of current exploration. His ascending by unconfined and eventually vadose processes. He com- flow argument requires that the caves be laterally restricted, pletely reinterprets the caves of the Guadalupe Mountains in and not connect over a large lateral scale. However, these this manner. However, to prove that overprinting can mask confined caves include many of the longest caves in the the features diagnostic of a former confined-aquifer cave, world, such as the gypsum mazes of Ukraine, which have one should examine caves that formed where confined large areal extents, even if their great length is a result of a conditions never existed. If such caves show none of the multitude of closely-spaced passages. The presentation of diagnostic features, then the Klimchouk argument would Lechuguilla Cave (his Figure 17) as a model for his cave gain credibility. Otherwise it would be badly weakened. theory is instructive. Even with the 23 vertical exaggeration, This brings us back to the neglected third cave category, the input and output points on the profile are widely that of coastal and oceanic caves. Where they are separated laterally. Given, however, that the author is introduced on page 5, the author states that they are making his case for large regional aquifers perhaps hundreds treated separately in a later section. But they are not of kilometers in lateral extent, this lateral development is treated at all in the rest of the text. This omission is critical, minor. In an interesting contradiction, the author on page 94 as these coastal caves display all of the critical dissolutional highlights the usefulness of his confined caves as hydrocar- features that the author describes as unique to his confined bon reservoirs. Given that his model of confined cave cave model, which include cupolas, infeeders, ceiling genesis requires slow leakage of water through the overlying channels, partitions, etc. They are not only present in confining beds, would not oil and gas also leak away? coastal caves, they are abundant. These caves have been The major failing of the book is the re-interpretation of argued, by myself and co-authors, to be hypogenic, in the some existing theories of cave development. Art Palmer's Palmer sense, as they form by rejuvenation of solutional maze cave theories take an especially hard beating. The aggressiveness by mixing of waters at depth. They contain author proclaims, through some shaky arguments, that the features described by Klimchouk because they too form mazes formed by floodwater or by diffuse infiltration in slow-moving water where processes such as solute-driven through an overlying caprock do not exist, and that caves convection also operate. But because these carbonates have described as such are merely enlarged from hypogenic never been confined or deeply buried, they negate the precursors. Palmer considers all mazes in soluble rocks to overprinting argument and demonstrate that the features developwhere solutional enlargement is non-competitive considered diagnostic of deepconfinement by the author (i.e., with all flow paths enlarging at comparable rates). In are not limited to that mode of speleogenesis. He mentions floodwater conditions, all possible flow paths receive all the that coastal caves ``¼are treated separately because of the water they can handle, regardless of each path's efficiency, specific conditions for speleogenesis determined by the hence there is no competition. The same is true for water dissolution of porous, poorly indurated carbonates by entering a soluble unit through fractures in an overlying mixing of waters of contrasting chemistry at the halocline.'' insoluble unit. It is also true for a variety of mixing and But coastal caves formed by mixing dissolution are not hydrothermal models, including the confined cave model restricted to eogenetic rocks, as made clear in the paper by postulated by Klimchouk. But his use of the map of Skull Vacher and Mylroie (2002), which Klimchouk cites in his Cave, New York (a cave he has never visited) to debunk book. One could also argue that, in the case of carbonates, the floodwater model is especially egregious. Had he ever Klimchouk's hypogenic caves are merely the result of visited the cave and seen floodwater debris 25 m above the rejuvenation of coastal and oceanic caves taken into the floor, he would realize that the cave does flood. If he had mesogenetic zone by burial, and that his confined examined the outlet choked with glacial sediment, he would carbonate caves are an overprint of those caves. 130 N Journal of Cave and Karst Studies, August 2008 BOOK REVIEW

Thus, the single biggest flaw in the book is the author's This book is interesting, controversial, and dogmatic. tendency to dismiss ideas that are not in complete Dr. Klimchouk has presented an argument about cave agreement with his own. This is especially discomforting, origin under confined conditions that has great potential, as he has introduced some interesting ideas and concepts. but it is an over-reach of an excellent idea. Instead of fitting He devalues his own arguments with incorrect attacks on his model into a continuum of karst processes, he attempts the work of others. Because of the broad scope of this to minimize and cast out (or ignore) other ideas. However, work, and the many examples provided from around the if one can separate the wheat from the chaff, this book world, the book initially appears to have a large mass of contains many useful ideas about karst processes in evidence behind it. But when one realizes that some of the confined aquifers. examples given, which are personally known to this reviewer, have been completely misinterpreted, then the Reviewed by John E. Mylroie, Department of Geosciences, Mississippi reader wonders how much to trust the other interpreta- State University, Mississippi State, MS 39762 ([email protected]. tions. edu).

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