HUMAN-INDUCED ALTERATIONS OF THE MyCOBIOTA IN AN ALPINE SHOw CAVE (ITALy, Sw-ALPS) SPREMEMBE MIKOBIOTE, KI JIH V TURISTIČNI ALPSKI JAMI (ITALIJA, SZ ALPE) POVZROČI ČLOVEK Stefano MAMMOLA1,*, Simone DI PIAZZA2, Mirca ZOTTI2, Giovanni BADINO3, Marco ISAIA1,** 1 Department of Life Sciences and Systems Biology, University of Turin, Via Accademia Albertina 13, 10123 Turin, Italy. 2 Laboratory of Micology, Department of Earth, Environmental and Life Science, University of Genoa, Corso Europa 26, 16136 Genoa, Italy. 3 Department of Physics, University of Turin, Via P. Giuria 1, 10125 Turin, Italy. * corresponding author: stefanomammola@gmail.com ** corresponding author: marco.isaia@unito.it Received/Prejeto: 06.08.2015 COBISS: 1.01 ACTA CARSOLOGICA 46/1, 111–123, POSTOJNA 2017 Abstract UDC 551.44:582.28(450.215) 582.28:504.61(450.215) Stefano Mammola, Simone Di Piazza, Mirca Zotti, Gio�anni Badino, Marco Isaia: Human�induced alterations of the my� cobiota in an Alpine show ca�e (Italy, SW�Alps) Anthropogenic alterations in show caves are well documented in scientific literature. One of the main sources of perturbation derives from visitors, acting as carriers of propagules and or- ganic matter in the cave ecosystems. Such perturbation gener- ally results in a significant alteration of the natural subterranean microbiota. In this study, we characterize the mycobiota of the superficial alluvial sediment (0–5 cm) of the Bossea show cave (Nw-Italy) over two sampling seasons, aiming to investigate whether anthropogenic rather than natural factors contribute to the colonization and proliferation of alien mycobiota in the cave environment. we placed eighteen sampling plots at differ- ent distances from the touristic pathway that crosses the cave. The distance from the touristic pathway was used as proxy to sort the sampling plots in three groups according the degree of anthropogenic disturbance (high: 0–2 m; intermediate 2–40 m; low: >100 m). Moreover, in order to elucidate the potential ef- fect of other factors, we introduced in our analysis the distance from the subterranean river and the distance from the cave en- trance. In each plot, we collected two samples of alluvial sedi- ment, in winter and summer. Fungi were isolated from each sample and identified by means of an integrated morphological approach. we observed a rich and diversified fungal commu- nity – 63 taxa of Zygomycota and Ascomycota plus several un- identified yeasts – consisting of both native and alien species. Regression analysis points out a decrease of the abundance and the diversity of viable propagules at increasing distances from the touristic pathway rather than the cave entrance or the Izvleček UDK 551.44:582.28(450.215) 582.28:504.61(450.215) Stefano Mammola, Simone Di Piazza, Mirca Zotti, Gio�anni Badino, Marco Isaia: Spremembe mikobiote, ki jih � turistični alpski jami (Italija, SZ Alpe) po�zroči člo�ek Antropogene spremembe v turističnih jamah so v znanstveni literaturi dobro opisane. Glavni vir motenj so obiskovalci, ki za jamske ekosisteme delujejo kot vektorji živih propagul in organ- skih snovi. Te motnje običajno povzročijo občutno spremembo naravne mikrobiote v podzemlju. Na podlagi dveh vzorčevanj smo v študiji podrobno opisali mikobioto na površini aluvial- nih sedimentov (0–5 cm) v turistični jami Bossea (SZ Italija). Namen raziskave je bilo ugotoviti, ali h kolonizaciji in prolife- raciji nejamske mikobiote v jamskem okolju bolj prispevajo antropogeni kot naravni dejavniki. V jami smo na različnih razdaljah od turistične poti določili osemnajst mest vzorčenja. Razdaljo od turistične poti smo uporabili za razvrstitev vzorčnih mest v tri skupine glede na stopnjo človekove motnje (visoka: 0–2 m; vmesna 2–40 m; nizka: > 100 m). Da bi opre- delili morebitne vplive drugih dejavnikov, smo v naši analizi dodatno upoštevali še razdaljo od podzemne reke in od vhoda v jamo. Pozimi in poleti smo na vsaki točki odvzeli dva vzorca aluvialnega sedimenta. Iz vsakega vzorca smo izolirali glive ter jih identificirali glede na njihove morfološke značilnosti. Ugotovili smo bogato in raznoliko združbo gliv – 63 taksonov Zygomycota in Ascomycota in še nekaj neidentificiranih kva- sovk, značilnih jamskih in zunanjih vrst. Regresijska analiza je pokazala, da se zmanjšuje abundanca in diverziteta viabilnih propagul z oddaljevanjem od turistične poti bolj kot z oddalje- vanjem od jamskega vhoda oziroma podzemeljskega vodotoka. V skladu s to ugotovitvijo se zdi, da turisti pasivno prenašajo zunanji organski material, kar lahko razloži večjo razširjenost ACTA CARSOLOGICA 46/1 – 2017112 STEFANO MAMMOLA, SIMONE DI PIAZZA, MIRCA ZOTTI, GIOVANNI BADINO, MARCO ISAIA subterranean river. Accordingly, the exogenous organic materi- als passively conveyed by tourists, possibly explains the higher proliferation of alien species in the proximity of the touristic pathway. There was also a seasonal variation in the diversity and abundance of propagules, which we interpreted in light of the peculiar microclimate of the cave. In a second step, we used Indicator Species Analysis to identify the most representative species of the different levels of anthropogenic disturbance. In particular, Aspergillus spelunceus was found to be indicator of low disturbance, whereas mucor corticola and A. asperescens were found to be indicators of intermediate and high distur- bance, respectively. Key words: anthropic disturbance, alluvial sediment, alien spe- cies, airborne fungi, environmental impact. nejamskih vrst v bližini turistične poti. Ugotovili smo tudi se- zonsko variabilnost v diverziteti in abundanci propagul, kar lahko razložimo s posebnimi mikroklimatskimi razmerami, ki vladajo v jami. V naslednjem koraku smo za identifikacijo reprezentativnih vrst, ki kažejo različne nivoje antropogenih motenj, uporabili indikatorsko vrstno analizo. Gliva Aspergillus spelunceus se je izkazala kot indikatorska vrsta za nizko stopnjo motnje, mucor corticola je bila indikator srednje, A. asperescens pa indikator visoke stopnje motnje. Ključne besede: antropogene motnje, splošni linearni mešani modeli, glive v zraku, vpliv na okolje. INTRODUCTION Over the past few decades, interest for the underground karst environments and their natural wonders has grown remarkably, not only from the speleological or scientific point of view, but also from an economic perspective. The so-called “show caves” are caves made accessible to the general public for touristic purposes, managed by a government or commercial organization. Unlike wild caves, paying visitors experience the cave via constructed trails, guided tours, artificial lighting and regular open- ing hours (wilson 2005). Since the first evidence of cave visitors in 1213 in the Postojna Cave and the early experi- ments with electric light in Australian caves in 1881, cave tourism grew considerably (Cigna 2005). The impressive numbers of visitors – up to 500,000 visitors/year/cave – and the profits deriving from such activities have recently acquired substantial importance at global scale (Interna- tional Show Cave Association 2015). Caves are delicate and sheltered ecosystems, be- cause of their peculiar habitat conditions, spatial con- finement, climatic stability and the conservation value of their biodiversity (Culver & Pipan 2009). The suscepti- bility of caves to disturbance, especially in show caves, is well documented (e.g., Fernandez-Cortes et al. 2011). It is understood that the impressive tourist flow and the as- sociated transformations contribute significantly to alter cave equilibrium, as a massive amount of additional en- ergy is introduced in the system. Energy release mostly derives from visitors – primarily in form of heat and CO2 – from artificial lights and other indirect sources, result- ing in profound climatic and energetic perturbations of the entire cave ecosystem (Cigna 1993). On top of that, human visitors carry propagules and organic matter in caves (Krajick 2001), influencing the availability of food resources with straightaway fallouts on the subterranean trophic webs (Chelius et al. 2009). The joint effect of visitors and artificial lights often results in the coloni- zation and proliferation of alien organisms, such as the so-called “lampenflora” (algae, diatoms, ferns, mosses), bacteria and fungi (Vanderwolf et al. 2013; Falasco et al. 2014). These alien organisms induce dramatic ecological changes, both from the biotic and the abiotic point of view. Changes in species interactions, perturbations of nutrient cycles and structural damages to speleothems are among the most cited threats associated with caves opened to public (Sánchez-Moral et al. 1999, 2005; Fer- nandez-Cortes et al. 2011; Piano et al. 2015). Among microorganisms, fungi are exceptionally di- verse, with about 98,000 described taxa (Kirk et al. 2008). In general, the great sporulation ability and anemophily of fungi allows them to survive in a wide variety of envi- ronments (Magan 2007), including hypogean ones. with 1,029 species documented from caves (Vanderwolf et al. 2013), fungi and yeasts represent an important compo- nent of cave trophic webs (Nováková 2009), where they play a key role in recycling organic matter and making available nutrients for other organisms. In recent years, there was an increasing interest about mycobiological researches in cave environments (Rampelotto 2010), generally aimed at looking for new species (Selbmann et al. 2005), assessing diversity (Gostinčar et al. 2010; Selbmann et al. 2012), and isolating microorganisms exploitable for biotechnological purposes (Gadd 2007). An increasing number of studies have also investigated whether humans visitors has an impact on the fungal di- versity – reviewed in Vanderwolf et al. (2013). Given their peculiar role in nutrient cycling, the perturbation of the cave mycobiota can result in pro- found alteration of the cave ecosystem. Several studies have shown that different groups of fungi are involved ACTA CARSOLOGICA 46/1 – 2017 113 HUMAN-INDUCED ALTERATIONS OF THE MyCOBIOTA IN AN ALPINE SHOw CAVE (ITALy, Sw-ALPS) MATERIAL AND METHODS STUDy AREA The Bossea show cave (speleological cadastre: 108 Pi/ CN) is part of a karst system developed in a sub-basin of the Corsaglia watershed, located in the Sw-Italian Alps – mount Merdenzone, municipality of Frabosa Soprana, Province of Cuneo, Nw-Italy. The Bossea cave is includ- ed in Site of Community Importance (SCI) IT1160026, and is set in a narrow belt of limestone and dolomitic limestone imposed upon permo-carboniferous rocks – quarzites and porfiroids (Civita et al., 2005). The cave is particularly rich in water since it is crossed by a subterranean river flowing inside the cave with a flow rate ranging from 50 to 1200 l/sec. The cave has a total ground plan development of 2,800 m and an ascending structure. It opens at 836 m a.s.l. and reaches 1,040 m at its highest point. It was firstly explored in 1850 and opened to public in 1874. with 150 years of tourist frequentation, the Bossea cave is the oldest Italian cave open to public. The current touristic flow rate is es- timated to be 16,000 visitors/year (Bossea management office 2015). A gate with open bars – i.e. allowing natural airflow – at the cave entrance restricts public access (Fig. 1a). Visitors have access to the touristic section of the cave via a narrow corridor of nearly 100 m length, which is often used for contemporary art expositions. After the corridor and along the touristic pathway (Fig. 1b), visi- tors have access to a consecutive series of vast rooms – i.e., “Sala Garelli” of 100 x 60 x 40 m. The touristic path- way ends at the Ernestina Lake (Fig. 1c) at 949 m a.s.l., nearly 1 km away and 113 m higher than the entrance (Fig. 2). After the Ernestina Lake, the non-touristic sec- tion begins. A long active canyon of 3−6 m wide and up to 40 m high follows, ending in a system of submerged syphons, which have been partially explored by speleo- divers (Gregoretti 1991). in biogeochemical process within caves (Gadd 2007) and how alien species damage caves – e.g., Lescaux Cave in France (Bastian et al. 2010) and the Castañar de Ibor Cave in Spain (Jurado et al. 2010). we characterize quantitatively and qualitatively the mycobiota of the superficial alluvial sediment (sensu Sa- sowsky, 2007) of the Bossea show-cave (Nw Italy). The aim of the study is to analyze whether visitors, in con- trast to natural sources such as natural air currents and running waters, contribute to the colonization and the proliferation of the alien mycobiota. In particular, we hypothesize that the proximity to the touristic pathway – that we interpreted as a proxy for anthropogenic dis- turbance – affects positively both the diversity and the abundance of the mycobiota. Fig. 1: The bossea show cave. a) Cave entrance; b) touristic path- way; c) Ernestina lake and the waterfall; d) Alluvial sediment sampled in this study. Photo credits/by courtesy of: Francesco tomasinelli. ACTA CARSOLOGICA 46/1 – 2017114 DATA ANALySIS REGRESSION ANALySIS we analyzed data with Generalized Linear Models (GLMs) in R environment (R Development Core Team 2013). For each plot, we used as dependent variables the concentration of colony-forming unit (CFU/g*106; hereinafter CFU) and the Shannon-wiener diversity SAMPLING DESIGN In order to evaluate the effect of touristic impact on the cave mycobiota, we selected 18 squared sampling plots of 0.25 m2, placed in different sections of the cave (Fig. 2). For each sampling plot we measured the distance from the cave entrance ('DST_entrance'), the distance from the river ('DST_river'), and the distance from the touris- tic pathway ('DST_path'). According to the distance from the touristic pathway, we grouped the sampling plots in three categories: high disturbance ('hp'; 0–2 m from the pathway, 6 plots), intermediate disturbance ('mp'; 10–40 m from the pathway, 6 plots) and low disturbance ('lp'; over 100 m from the pathway, 6 plots). Sampling was per- formed in January and repeated in August 2013 reaching in total 36 samples (6 plots x 3 groups x 2 seasons). Dur- ing the first sampling in winter, each point was marked in order to relocate it in summer. Alluvial sediment (Fig. 1d) was sampled randomly within the square plot by means of sterile tools. Sediment was collected up to 5 cm depth. The sampling method ensured to collect mainly airborne fungi. Samples were stored in sterile Falcon® tubes (45 ml) and conserved in a thermal bag until the arrival at the laboratory. Fungi were isolated using the modified dilution plate protocol (Gams et al. 1987) on three media (Sig- ma-Aldrich®): MEA+C (Malt Extract Agar added with Chloramphenicol), RB (Rose Bengal agar), and SAB (Sa- bouraud agar). we obtained the initial dilution by mix- ing 1 g of each alluvial sediment sample with 10 ml of sterile water. Then we performed a serial dilution up to 1: 100,000 concentration and finally we inoculated 2 ml of diluted solution on each media in duplicate. we opted for streak 2 ml of solution after preliminary inoculation tests carried out using different portion of sample and dilution. This protocol allowed us to count, identify and isolate vital strains from the environment surveyed. The mycobiota was identified according to cultivation meth- ods by observing their micro- and macro-morphological characters, and also considering their different trophic and physiological requirements (Pitt 1979; Hoog 1978; Domsch et al. 2007). Moreover, in order to identify critical species, molecular analyses (ITS and ß-tubulin locus DNA sequence) were performed and compared to BLAST (Basic Local Alignment Search Tool) results. The isolated strains were conserved in the culture collec- tion of Mycological Laboratory of DISTAV (University of Genoa, Italy). Fig. 2: Ground plan drawing of the touristic section of the bossea show cave (modified from Capello 1954) with indications of the sam- pling plots and the assigned level of disturbance. Red points: high disturbance (hp), yellow points: intermediate disturbance (mp), blue points: low disturbance (lp, not shown on the map). STEFANO MAMMOLA, SIMONE DI PIAZZA, MIRCA ZOTTI, GIOVANNI BADINO, MARCO ISAIA ACTA CARSOLOGICA 46/1 – 2017 115 index (log 10; hereinafter SH) calculated for each plot. we included the distance of the sampling plot from the touristic pathway ('DST_path'), the distance from the entrance of the cave ('DST_entrance'), the distance from the river ('DST_river'), and the sampling season (categorical variable 'SEA' made up of two levels: win- ter and Summer) as covariates in the analysis. we inter- preted the distance from the touristic pathway as proxy for the disturbance caused by tourists visiting the cave, i.e. the higher the distance of the sampling plot from the path, the lesser the disturbance. The distance from the cave entrance was interpreted as proxy for the evaluation of the effect of anemochoric transportation (i.e. air cur- rents) and the distance from the river as proxy for hydro- choric transportation (i.e. running water) of spores and propagules. The seasonal categorical variable was intro- duced in the regression models to take into account pos- sible seasonal effects. we carried out data exploration following Zuur et al. (2010) protocol. According to Zuur et al. (2009, 2010) the inclusion of outliers and highly correlated predic- tors in the regression analysis leads to misleading results – type I and II statistical errors. we therefore evaluated the presence of outliers in the dependent and indepen- dent variables via Cleveland dotplots and the collinear- ity among the covariates. In this respect, all continuous variables were log-transformed (log x+1) to achieve ho- mogeneity. In order to avoid the prediction of negative fitted values – meaningless from a biological point of view in terms of CFU and SH – we assumed a gamma distribution (0, ∞; e.g., Abramowitz & Stegun 1972) with a log-link function. The mixed procedure allowed us to deal with temporal dependence of the samples, given that we repeated the samplings in the same plots in the two seasons. Therefore, we included the sampling plot as random factor. The regression models were fitted via the glm. The structure of the model was y ~ DST_entrance + DST_path + DST_river + SEA. Once we fitted this initial model, we applied model selection (Johnson & Omland 2004), in order to identify the best model structure supported by observations. we performed a backward elimination, progressively exclud- ing variables according to AICc values (Zuur et al. 2009). Variables not contributing to the fit of the model – in- creasing the AICc value – were progressively dropped from the models thus avoiding over-fitting (Hawkins 2004). Model validation was carried out following Zuur et al. (2009), plotting the residuals against each covari- ate and the fitted values and checking for non-linear pat- terns in the residuals via the gam R command (Hastie 2013). INDICATOR SPECIES ANALySIS In order to determine potential species to be used as in- dicators of disturbance within the cave, we performed In- dicator Species Analysis (ISA; Dufrêne & Legendre 1997; De Cáceres & Legendre 2009). we created a community data matrix with sampling plots in rows and the CFU val- ues of each species – proxy for abundance – in columns. we created an additional categorical variable 'site group' (sensu Dufrêne & Legendre 1997) grouping the samples in the three distinct classes of disturbance (see sampling design). Indicator species analysis was performed via the multipatt command in the indicspecies (De Cáceres & Legendre 2009) R package, which uses an extension of the original Indicator Value method (Dufrêne & Legen- dre 1997) by inspecting indicator species in both indi- vidual site groups and combinations of site groups (De Cáceres et al. 2010). The statistical significance of the relationship between indicator species and groups was tested via permutation test (999 permutations) with the parameter alpha set to 0.05. RESULTS ISOLATION AND IDENTIFICATION OF FUNGAL STRAINS we collected 2,103 morphological taxonomic units (MTUs) of filamentous fungi on the 160 inoculated Petri dishes. The MTUs belong to 63 taxa included in Zygomy- cota and Ascomycota. Several yeasts and sterile mycelia were also isolated, but not identified as they fell outside the scope of our research. The latter were characterized by slow growth and they rarely developed conidiophore or sporophore. Tab. 1 reports the list of the taxonomic groups here recovered. Supporting Information 1 reports the list of the species identified on molecular base. The most recurrent genus was Penicillium, followed by mucor, trichoderma and Aspergillus. It is worth notic- ing the occurrence of teleomorphyc species, in particular Pseudeurotium bakerii. Among anamorphic fungi, the most frequent filamentous fungal species were mainly saprotrophic or non-obligate parasitic, such as Penicil- lium expansum, Aspergillus spelunceus, trichoderma har- zianum, and t. koningii. HUMAN-INDUCED ALTERATIONS OF THE MyCOBIOTA IN AN ALPINE SHOw CAVE (ITALy, Sw-ALPS) ACTA CARSOLOGICA 46/1 – 2017116 tab. 1: Checklist of the species recorded in the bossea show cave. Presence (X)/ Absence (−) of the species in each disturbance category (hp= high disturbance; mp= intermediate disturbance; lp= low disturbance) is given for winter (W) and summer (S). Species W-hp W-mp W-lp S-hp S-mp S-lp Acremonium nepalense W. Gams − − − X − − Aspergillus asperescens Stolk X − − X − − Aspergillus puulaanensis Jurjevic, S.W. Peterson & B.W. Horn − − − − − X Aspergillus spelunceus Raper & Fennell X X X X X X Aspergillus versicolor (Vuill.) Tirab. X X X − − X Apergillus sp. − − − X − − Bionectria ochroleuca (Schwein.) Schroers & Samuels X X X X X − Cladosporium lignicola Link X X X X X X Cladosporium sphaerospermum Penz. X X X − X − Doratomyces sp. − − − X X X Fusarium culmorum (Wm.G. Sm.) Sacc. − X − − − − Geomyces sp 1 − − − − − X Geomyces sp 2 − − − X − − Gliomastix cerealis (P. Karst.) C.H. Dickinson X X − X − − Mortierella alpina Peyronel − − − X − X− Mucor cirinelloides Tiegh. X − − X − − Mucor corticola Hagem − X X − X − Mucor hiemalis Wehmer X X X − − X Mucor pyriformis f. pyriformis Scop. X X − X X − Mucor racemosus f. racemosus Fresen X X X X − − Mucor racemosus f. sphaerosporus (Hagem) Schipper X X X X X X Penicillium antarcticum A.D. Hocking & C.F. McRae − − − X X X Penicillium atramentosum Thom X X X X − X Penicillium atrovenetum G. Sm. − − − X X − Penicillium bialowiezense K.M. Zaleski − − − X − − Penicillium brevicompactum Dierckx − − − X X X Penicillium caseifulvum F. Lund, Filt. & Frisvad − − X − − − Penicillium cavernicola Frisvad & Samson X X − − − − Penicillium chrysogenum Thom X X X X X X Penicillium citreonigrum Dierckx − − − − − X Penicillium citrinum Thom X X X X X X Penicillium clavigenum Demelius X X X − − − Penicillium commune Thom X X − X − X Penicillium coprophilum (Berk. & M.A. Curtis) Seifert & Samson X X − − − − Penicillium dendriticum Pitt X X − − − − Penicillium ellipsoideospermum L. Wang & H.Z. Kong − − − − X − Penicillium expansum Link X X X X X X Penicillium glandicola (Oudem.) Seifert & Samson − X − − X − Penicillium italicum Wehmer X − − − − − Penicillium magnielliptiosporum Visagie, Seifert & Samson − − − X − − Penicillium novae-zelandiae J.F.H. Beyma − − − − − X Penicillium olsonii Bainier & Sartory − − − − X − Penicillium phoenicum D.B. Scott & Stolk − − − X X − Penicillium polonicum K.M. Zaleski X X X X − − Penicillium roseomaculatum Biourge − X X − − − Penicillium solitum Westling X − − X − X Penicillium spinulosum Thom − − X − − − Penicillium sumatrnse Svilv. − − − − X − Penicillium taxi R. Schneid. X − − − − − STEFANO MAMMOLA, SIMONE DI PIAZZA, MIRCA ZOTTI, GIOVANNI BADINO, MARCO ISAIA ACTA CARSOLOGICA 46/1 – 2017 117 The density of CFUs per gram of alluvial sediment was of the same magnitude order in all samples, with one only exception in winter (11.4 * 106 MTU g−1). Tab. 2 reports descriptive parameters of the samples collected in each category of disturbance in the two sampling sea- son. REGRESSION MODELS The most appropriate GLM structure resulting from model selection was y ~ DST_path + SEA for both Species W-hp W-mp W-lp S-hp S-mp S-lp Penicillium vulpinum (Cooke & Massee) Seifert & Samson - X X - - - Penicillium sacculum C. Booth - - X - - - Penicillium sp. 1 - X - - - X Penicillium sp. 2 X X - - - Pseudeurotium bakeri C. Booth X - X - - - Rhodotorula sp. - - - X X X Trichocladium sp. - - - - - X Trichoderma atroviridae P. Karst. X X X X X - Trichoderma hamatum (Bonord.) Bainier X - X - - - Trichoderma harzianum Rifai X X X - - - Trichoderma koningii Oudem. X X X X X - Trichoderma viridescens (A.S. Horne & H.S. Will.) Jaklitsch & Samuels X X X X - X Trichoderma sp. X X - - - - Sterile mycelia morphotype A X X X X - - Sterile mycelia morphotype B - X - X X X Sterile mycelia morphotype C - X X - - X Sterile mycelia morphotype D X - - - - - Sterile mycelia morphotype E X - - - - - Sterile mycelia morphotype F X X - - - - Sterile mycelia morphotype G X - - X - - Sterile mycelia morphotype H - - - - X - Yeast A X X X - - - Yeast B X X - X X X Yeast C - X X X X - Yeast D - - - X - X Yeast E - - - X X - tab. 2: descriptive parameters of the alluvial sediment samples (hp= high disturbance; mp= intermediate disturbance; lp= low disturbance). Season Pressure MTU (mean±SD) MTU g−1 (mean±SD) Winter lp 51.2±12.8 3.2±0.8 mp 59.8±26.1 3.7±1.6 hp 105.5±56.4 6.5±3.5 Summer lp 35.8±9.3 2.2±0.5 mp 51.5±3.6 3.2±0.2 hp 66.8±12.9 4.7±0.8 Shannon-wiener diversity (SH) and abundance (CFU). The variable distance from entrance (DST_entrance) and distance from the river (DST_river) were dropped during model selection since they did not contributed in improving the model performance. There was a de- crease in Shannon diversity with increasing distances from the touristic pathway, although this trend only ap- proached statistical significance (DST: Estimated β±SD: –0.0208±0.011, p=0.06). Diversity was also found to be significantly higher in the winter season (SEA, test rela- tive to "Summer": Estimate β±SD: 0.1136±0.042, p<0.01; Fig. 3). we found the same trends when modeling abun- dance (CFU) versus the considered covariates. Specifi- cally, we observed a significant decrease of abundance with increasing distance from the touristic pathway (DST: Estimated β±SD: –0.1400±0.032, p<0.001) and significant higher abundance values in winter (SEA, test relative to "Summer": Estimated β±SD: 0.3055±0.098, p<0.01; Fig. 4). INDICATOR SPECIES ANALySIS The indicator species analysis pointed out 3 singletons (sensu De Cáceres et al. 2010), representative of the HUMAN-INDUCED ALTERATIONS OF THE MyCOBIOTA IN AN ALPINE SHOw CAVE (ITALy, Sw-ALPS) ACTA CARSOLOGICA 46/1 – 2017118 3 classes of disturbance. Aspergillus asperescens was found to be the most suitable indicator of those plots charac- terized by high level of disturbance (hp; IndVal=0.577, p<0.05). The medium disturbance (mp) site group was Fig. 3: Predicted values (black lines) and 95 % confidence in- tervals (grey surfaces) of the ef- fect of distance from the touristic pathway (m, log-transformed) in the two sampling seasons on the diversity of the mycobiota of the alluvial sediment (Shannon-Wie- ner index) in the bossea show cave. Results derived from the generalized linear models (GLm) analysis. Fig. 4: Predicted values (black lines) and 95 % confidence in- tervals (grey surfaces) of the ef- fect of distance from the touristic pathway (m, log-transformed) in the two sampling seasons on the abundance of the mycobiota of the alluvial sediment (density of colony-forming unit, CFU/g * 106) in the bossea show cave. Re- sults derived from the generalized linear models (GLm) analysis. characterized by mucor corticola Hagem (IndVal= 0.608; p<0.05), whereas Aspergillus spelunceus was found to be indicator of low disturbance (lp; IndVal=0.787; p<0.05). STEFANO MAMMOLA, SIMONE DI PIAZZA, MIRCA ZOTTI, GIOVANNI BADINO, MARCO ISAIA ACTA CARSOLOGICA 46/1 – 2017 119 Anemochoric, hydrochoric and biochoric transportation are the natural vectors enhancing cave colonization by fungi and yeasts (Northup et al. 1994). If compared with non-sheltered natural habitats, diversity and biomass of fungi in caves are generally low (Vanderwolf et al. 2013). However, caves exploited for touristic purposes represent a remarkable exception to this general trend (e.g., Mosca & Campanino 1962; Pulido-Bosch et al. 1997). In fact, thanks to tourist flow, external fungal spores and yeasts access the cave ecosystems, affecting the abundance and the diversity of the subterranean microbiota (Chelius et al. 2009). In recent years, several authors considered the impact of tourists on hypogean microbial communities and how fungi may contribute to introduce and facilitate the proliferation of alien microorganisms inside the cave (e.g., Bastian et al. 2009, Jurado et al. 2010, wang et al. 2010; Fernandez-Cortes et al. 2011). In this work, we used a similar approach, focus- ing more specifically on alluvial sediment of the Bossea show cave. we found that alluvial sediment samples are characterized by a rich and diversified mycobiotic com- munity (Tab. 1). The mycobiotic communities of Bossea have never been investigated before, precluding the com- parison of our result with previous studies. However, in agreement with other studies conducted in similar habitats (Nováková, 2009; Fernandez-Cortes et al. 2011; wang et al. 2010), the most recurrent genera of filamen- tous fungi were Penicillium, mucor, trichoderma, and Aspergillus and the most common species were Penicil- lium expansum, P. brevicompactum, and mucor hiemalis. Most probably, the remarkable abundance of such spe- cies is related to their wide ecological requirements. For instance, Penicillum expansum and P. brevicompactum are cosmopolitan widespread species, which can ger- minate and grow at moderately cold temperature like the one found in Bossea – close to 9 °C; see Piano et al. (2015): p. 4, fig. 2 for a microclimatic characterization of the cave. Diversity and abundance of fungi was found to be unevenly distributed within the cave. Regression analy- sis points toward a decrease of the abundance and diver- sity of vital propagules at increasing distances from the touristic pathway (Figs. 3-4), thus highlighting an effect of tourists in conveying microorganisms inside the cave. It is well document that tourist may release a significant amount of exogenous organic materials into the cave at every visit, in form of clothes lint, hair, skin and other kind of organic debris stuck to shoe-soles (Krajick 2001; Culver & Pipan 2009), offering suitable substrates for fungi (Dickson & Kirk 1976; Khizhmyak et al. 2003). As observed by Northup et al. (2000), this excess of organic matter often supports the proliferation of alien species to the cave mycoflora that likely compete and overwhelm the slow-growing native species. Moreover, tourists are regarded as one of the main sources of conidia and spores within the cave (Krajick, 2001), which are depos- ited on the surface of the alluvial sediment of the cave by air currents and gravity. However, we acknowledge that other factors may play an additional role in conveying fungi within the cave (Vanderwolf et al. 2013), i.e. the anemochoric, hydrochoric and zoochoric transportation (Northup et al. 1994). In the specific case of Bossea, the air flow- ing from the entrance and the subterranean river that cross the cave may be reasonably regarded as carriers of propagules and spores, respectively for anemochoric and hydrochoric transportation. Cave-dwelling animals – such as arthropods and bats – may also play a role in this sense. Concerning anemochoric transportation, it is worth to mention that during model validation we excluded the distance from the entrance of the cave as a significant covariate – i.e. the variable was dropped during model selection. It is reasonable to hypothesize that an effect of the local air current could have resulted in a higher presence of fungi in the nearby of the cave entrance. This trend is in contrast with several studies conducted so far (Hsu & Agoramoorthy 2001; Urzi et al. 2010; Kuzmina et al. 2012; Mulec et al. 2012), in which an effect of the dis- tance from the entrance on diversity and biomass of mi- croorganism was observed. In our case, we may hypoth- esize that such effect was called off by the stronger effect of the anthropogenic distance. Similar considerations apply to the hydrochoric transportation, as the distance from the river was dropped during model selection. with respect to the biochoric transportation, it is worth to mention that the density of bat and insects are very low in this particular cave. Specifically, neither large bat colonies nor guano depositions are present in the cave. Only isolated individuals of Rhinolophus ferrume- quinum (Rhinolophidae) and myotis sp. (Vespertilioni- dae) are occasionally present in the cave. Moreover, we deliberately avoided areas where the presence of insects has been documented (see Morisi, 1992). As already hypothesized by other authors (wang et al. 2010, 2011), we further observed that the diversi- ty and abundance of propagules are season dependent. Specifically, both diversity and density of colony-form- ing unit was higher in winter samples. we tentatively relate this trend to the local meteo- rology of the cave. From a meteorological point of view, the Bossea cave is regarded as an “hot-air” trap (sensu DISCUSSION HUMAN-INDUCED ALTERATIONS OF THE MyCOBIOTA IN AN ALPINE SHOw CAVE (ITALy, Sw-ALPS) ACTA CARSOLOGICA 46/1 – 2017120 REFERENCES Abramowitz, M. & I.A. Stegun, 1972: Gamma and Re- lated Functions.- In: Abramowitz, M. et al. (eds.) handbook of mathematical Functions with formu- las, Graphs, and mathematical tables. U.S. Depart- ment of Commerce, National Bureau of Standards, pp. 76–83, Dover. 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