Aquat Ecol DOI 10.1007/s10452-016-9599-7

Groundwater biodiversity in a chemoautotrophic cave ecosystem: how geochemistry regulates microcrustacean community structure

Diana M. P. Galassi . Barbara Fiasca . Tiziana Di Lorenzo . Alessandro Montanari . Silvano Porfirio . Simone Fattorini

Received: 24 May 2016 / Accepted: 8 September 2016 Ó Springer Science+Business Media Dordrecht 2016

Abstract The Frasassi cave system in central Italy analysis separated the copepod assemblages inhabit- hosts one of the few known examples of a ground- ing dripping pools from those of sulfidic lakes. H2S water metazoan community that is supported by concentration, pH and O2 concentration were iden- sulfur-based lithoautotrophic microbes. Despite the tified as the main factors regulating community challenging conditions represented by high concen- structure. These results indicate that the distribution trations of H2S and low concentrations of O2, this of groundwater copepods within the cave system is cave system is home to many invertebrate species. ecologically and spatially structured. Sulfidic lakes Here, we analyzed the copepods inhabiting sulfidic showed lower Simpson dominance, higher Shannon lakes and non-sulfidic dripping pools in order to diversity and higher Pielou equitability at higher H2S investigate how environmental conditions in sulfidic concentrations. The complex community structure of waters regulate the spatial distribution of the cave the copepods of the Frasassi cave system suggests microcrustacean community over time. We also that a chemosynthetically produced food source sampled copepod assemblages of sulfidic lakes under facilitated the colonization of stygobionts in sulfidic conditions of both high and low H2S concentration. groundwater due to their tolerance to the environ- Cluster analysis and canonical correspondence mental conditions.

Keywords Frasassi cave system Á Italy Á Groundwater Á Biodiversity Á Chemoautotrophy Handling Editor: Michael T. Monaghan.

Electronic supplementary material The online version of this article (doi:10.1007/s10452-016-9599-7) contains supple- mentary material, which is available to authorized users.

D. M. P. Galassi (&) Á B. Fiasca Á S. Porfirio Á A. Montanari S. Fattorini Geological Observatory of Coldigioco, Cda. Coldigioco 4, Department of Life, Health and Environmental Sciences, 62021 Apiro, Italy University of L’Aquila, via Vetoio, Coppito, 67100 L’Aquila, Italy S. Fattorini e-mail: [email protected] Departamento de Cieˆncias e Engenharia do Ambiente, CE3C – Centre for Ecology, Evolution and Environmental T. Di Lorenzo Changes/Azorean Biodiversity Group and Universidade Institute of Ecosystem Study, ISE-CNR, via Madonna del dos Ac¸ores, Angra do Heroı´smo, Ac¸ores, Portugal Piano 10, Sesto Fiorentino, 50019 Florence, Italy 123 Aquat Ecol

Introduction ectosymbiosis with sulfur-oxidizing bacteria was also postulated (Dattagupta et al. 2009), and some obser- Sulfidic caves represent rare and still very poorly vations on the ecology of ostracods (Peterson et al. known groundwater ecosystems (Engel 2007). The 2013). Although copepods are by far the most presence of toxic gases and reduced oxygen levels abundant and species-rich animal group in groundwa- makes it difficult to sample sulfidic caves, which is a ter (Galassi et al. 2014), there is no available reason of this lack of knowledge. In the case of information on the species inhabiting the Frasassi groundwater species, sampling procedures are further cave system. complicated by the temporal and spatial variability of In this paper, we analyzed the Frasassi copepod the hydrogeological and hydrochemical conditions of assemblages sampled at various sites characterized by these environments (Galdenzi et al. 2008; Flot et al. different geochemical conditions to understand the 2010), and faunal lists are available for only a very influence of environmental variables on community small number of sulfidic cave systems (Engel 2007). structure. For this purpose, we analyzed the compo- Although lithoautotrophic microbes of sulfidic caves sition of copepod assemblages across different habitat can be able to maintain relatively rich and diversified types in the cave system, including both sulfidic lakes invertebrate communities, sulfur-oxidizing chemoau- (at different H2S concentrations) and non-sulfidic totrophy also generates environmental conditions that dripping pools. allow only highly specialized species to survive. Thus, In particular: sulfidic invertebrate communities are recognized as an example of ‘‘life at extremes’’ (Lee et al. 2012), where 1. We tested whether the species composition of the the only successful species are those able to cope with groundwater assemblages is ecologically and low O2 and high H2S concentrations (Galdenzi et al. spatially structured according to the geochemical 2008; Macalady et al. 2008). conditions of different water bodies within the Large cave systems may include both sulfidic and cave system. Our hypothesis is that, if sulfide non-sulfidic groundwater bodies, thus offering the concentration is a key factor in determining opportunity of investigating how communities species composition, assemblages in the sulfidic respond to the exceptionally high selective pressures lakes should be profoundly different from those of represented by sulfidic conditions in comparison with dripping pools. Also, we hypothesized that communities of non-sulfidic environments within the species composition of the investigated lakes is same cave system. However, probably because of influenced by temporal variations in the sulfide difficulties in conducting standardized sampling in concentration (i.e., high- vs. low-sulfide concen- sulfidic caves and the rarity of these ecosystems, no tration, thereafter H and L, respectively). study attempted to determine the effects of geochem- 2. We tested whether different environmental ical parameters on their animal community structure parameters have different roles in determining (Engel 2012). species composition and abundance. Namely, we The large Frasassi cave system (central Italy) hypothesized that high-sulfide concentration represents an exceptionally well-suited model for should be an important driver of species compo- such a study. The Frasassi cave system is among the sition of lake assemblages, whereas dripping pool most studied sulfidic cave ecosystems in the world assemblages should be composed of species (Galdenzi 1990; Sarbu et al. 2000; Macalady et al. associated with higher oxygen concentration and 2006, 2008; Galdenzi et al. 2008; Jones et al. higher pH values. 2010, 2015). Its invertebrate fauna is represented by 3. We tested whether different sulfide concentrations gastropods (Bodon and Cianfanelli 2012) and pre- determine differences in species dominance, dominantly by crustaceans, especially amphipods diversity and equitability in the copepod commu- (Flot et al. 2010; Bauermeister et al. 2012, 2013; Fisˇer nity inhabiting the sulfidic lakes. We hypothe- et al. 2015) and ostracods (Peterson et al. 2013). sized that if H2S concentration is the major driver Previous research on the crustaceans of the Frasassi of species community structure, temporal varia- cave system involved evolutionary studies on amphi- tions in H2S concentration should be reflected in pods (Flot et al. 2010; Fisˇer et al. 2015), for which changes in the community structure.

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Fig. 1 Map of the Frasassi cave system (central Italy) showing the location of the sampling sites (modified after Flot et al. 2010)

Copepod species were classified according to their Materials and methods degree of dependence on groundwater into two main ecological categories: stygobites and non-stygobites. Geological setting Stygobites are species that complete their entire life cycle in groundwater, being unable to survive and The Frasassi cave system (coordinates WGS84-G: reproduce in surface-water habitats. Non-stygobites 43.402°N, 12.962°E, Fig. 1) consists of several inter- are species living in surface freshwater; they may enter connected levels, as a result of alternating erosion and groundwater both actively and passively (Galassi deposition driven by Quaternary glacial–interglacial 2001). cycles during a steady, generalized tectonic uplift of

123 Aquat Ecol the region around the Frasassi Gorge, a long and steep Different lakes within the cave system have widely canyon eroded by the Sentino River. The cave system different H2S and O2 concentrations (Macalady et al. develops in the Calcare Massiccio, a limestone 2008; Flot et al. 2010). Some small lakes, as Lago platform interspersed with a network of fractures. Verde (Grotta del Fiume, Fig. 2a, b) and Lago Claudia The plateau overlies the uppermost Triassic Burano (Grotta Grande del Vento), are fed more directly by Formation, which consists of a 2000-m-thick succes- the deep sulfidic zone of the aquifer, with a lower sion of evaporitic anhydrites, black shales and bitu- contribution of bicarbonate water formed above the minous limestones. The presence of marls and chert sulfidic stratum due to the very slow groundwater overlying the Calcare Massiccio limits rock perme- flow. Conversely, Lago della Bottiglia (Pozzo dei ability and the seepage network feeding the aquifer Cristalli; Fig. 2c) tends to be less sulfidic than Lago (Sarbu et al. 2000; Galdenzi et al. 2008; Galdenzi Verde and Lago Claudia, both in dry and wet periods 2012). (Peterson et al. 2013). Freshwater seepage and occa- The groundwater is of two types: bicarbonate and sional runoff feed not only the sulfidic lakes but also sulfidic, whose chemical composition differs accord- very small non-sulfidic pools (Fig. 2e, f), which are ing to its origin. The bicarbonate water mainly characterized by bicarbonate waters exclusively originate from the diffuse infiltration of meteoric (Fig. 3). water through the limestone, while the sulfidic water from the deep groundwater that flows through Triassic Field sampling anhydrite formation (Galdenzi and Menichetti 1995; Sarbu et al. 2000; Forti et al. 2002; Cocchioni et al. Meiofauna was sampled at five sites (Figs. 1, 2a–f): 2003; Mariani et al. 2007; Galdenzi et al. 2008; Lago Verde (LV) (Fig. 2a, b), Lago della Bottiglia Galdenzi 2012). The sulfidic water in the main aquifer (LDB) (Fig. 2c), Lago Claudia (LC), and two has a higher salinity than the bicarbonate water unnamed dripping pools close to the entrance of infiltrating from the karst surface. It is rich in sodium Grotta del Fiume (Fig. 3), namely a concretional and chloride, and contains sulfate and hydrogen dripping pool (CDP) (Fig. 2e) and a silted dripping sulfide. The H2S-rich water rising from the deep pool (SDP; Fig. 2f). In total, there were five additional fracture network interacts with dissolved O2 present in dripping pools, but they were very small and com- water percolating through the Calcare Massiccio pletely devoid of fauna. We also sampled the meio- limestone, or with atmospheric O2 in the open spaces fauna in the Lago Galdenzi and the smaller rivulets of the cave system. The H2S interacts with O2, (Fig. 2d) in the Pozzo dei Cristalli. Pozzo dei Cristalli allowing sulfur-oxidizing bacteria to produce H? to is part of Grotta del Fiume, and it is a large cave form sulfuric acid, which dissolves the limestone of section (hosting Lago della Bottiglia, the rivulet the Calcare Massiccio, thus determining the hypo- network and the small stagnant water body called genic carbonate dissolution (Galdenzi and Menichetti Lago Galdenzi), which lies about 30 m below the cave 1995; Sarbu 2000; Forti et al. 2002; Mariani et al. area hosting Lago Verde. Consequently, the upper 2007). This condition in standing water bodies level where Lago Verde is located is connected to belonging to the saturated karst induces the presence Pozzo dei Cristalli. However, we did not consider of a chemocline, which, in dry season periods, may these data in the analyses because of the small number reach the free water table, allowing the maintenance of of sampled individuals and species (a total of nine a relatively constant temperature throughout the whole individuals belonging to two species found also in water column. Conversely, during intense rainy peri- Lago Galdenzi). ods, the most superficial water in the subterranean When possible, we sampled each site six times from lakes becomes less sulfidic and less rich in chloride November 2009 to March 2011 (November, May, and sodium (Galdenzi et al. 2008; Galdenzi 2012). August, September, January and March). Samples Mixing between descending bicarbonate and rising were taken in both H and L sulfide concentrations from mineralized water determines the composition of the LDB (H2S expressed as average actual concentration groundwater in the shallow phreatic zone (Galdenzi of S2- ranging from 0.002 mg L-1 in L to -1 2012). The water table is close to the Sentino River 1.98 mg L in H) and from LV (H2S as expressed level (Galdenzi et al. 2008; Peterson et al. 2013). average actual concentration of S2- from 123 Aquat Ecol

Fig. 2 Pictures showing the main features of the sampling sites: Cristalli, d high-sulfidic rivulet in Pozzo dei Cristalli feeding the a Lago Verde, overview, b Lago Verde, lake banks in high- so-called Lago Galdenzi, e concretional dripping pool, f silted sulfide concentration, c Lago della Bottiglia in Pozzo dei dripping pool

7.83–8.74 mg L-1 in L to 12.15–15.20 mg L-1 in H). expressed as average actual concentration of S2- from From LC, only two samples were collected because of 1.44 to 3.76 mg L-1 both in L and H). The pelagic severe difficulties encountered in the arduous cave zone was investigated both close to lake banks and in system section of Grotta Grande del Vento (H2S open water, filtering the water from a depth of few cm

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Fig. 3 Simplified scheme representing the percolating water surface water flow by runoff, small arrows the percolating water dynamics feeding both the concretional dripping pool (the from the cave ceiling that feeds both the non-sulfidic pools and closest to Lago Verde) and the silted dripping pool mainly fed the sulfidic lakes) by surface runoff (larger arrow indicates the direction of the from the bottom along the banks to 50-cm depth in measured using a HACH DR 2000 spectrophotometer open water. Benthic samples colonized by the micro- (Hach Co., Loveland, CO, USA). Data regarding H2S bial mats were also taken. To cover the maximum concentration expressed as S2- were provided by the habitat heterogeneity, both organic and inorganic IMDEA Water Institute, Alcala de Henares (Madrid, sediments and the limestone banks devoid of sedi- Spain), and the Georg-August-Universita¨tGo¨ttingen ments were scraped with a hand net. LDB resembles a (Go¨ttingen, Germany). sort of natural borehole, with very steep banks, and with sediments represented by sand instead of silt that Statistical analyses dominated the rest of the limestone banks in the other lakes. Dripping pools were sampled with the same Overall similarity in species composition among methodology. assemblages was investigated by cluster analysis According to the literature data, the invertebrate using the Sørensen coefficient as a measure of distance fauna of the sulfidic lakes is predominantly composed and the UPGMA (unweighted pair-group method, of gastropods (Bodon and Cianfanelli 2012), amphi- arithmetic average) amalgamation rule. Cophenetic pod and ostracod crustaceans (Flot et al. 2010; Fisˇer correlation coefficients were calculated to express et al. 2015; Peterson et al. 2013). In this paper, we how faithfully the obtained trees represented the focused on crustacean copepods, which are by far the dissimilarities among assemblages. To highlight the most abundant and species-rich invertebrate group in pure turnover pattern, Simpson dissimilarity index groundwater (Galassi et al. 2014). Meiofauna was was used (Baselga 2010). Similarity among assem- extracted by filtering approximately 20-L samples blages was also investigated by using abundance data through a hand net (mesh size = 60 lm). Faunal with Bray–Curtis similarity index (Magurran 2004). samples were preserved in 80 % ethyl alcohol solu- Variation in species composition among assem- tion. Copepod individuals were later sorted, identified blages in relation to environmental variables was to species level, counted and assigned to two ecolog- analyzed with canonical correspondence analysis ical categories: obligate and non-obligate groundwater (CCA) using the CANOCO program, version 4.5A species (i.e., stygobite and non-stygobite species). (ter Braak and Smilauer 2002). Two CCAs were used. Temperature, pH, electric conductivity at 25 °C A first CCA was conducted using species presence/ and dissolved oxygen concentration were measured in absence data, and a second one with species abun- the field using a multiparametric probe (ECM dances (after log10(x ? 1) transformation). Signifi- MultiTM; Dr. Lange GmbH, Du¨sseldorf, Germany); cance of individual environmental parameters calcium content was determined by titration with (geochemical variables) was tested using a forward EDTA; sulfate and chloride concentrations were selection with 1000 Monte Carlo permutations 123 Aquat Ecol

(Fattorini 2011). It has been suggested that CCA sand and carbonate cobbles. Both were characterized should be applied only if the first axis of a detrended by non-saline waters with low electric conductivity, correspondence analysis (DCA) is more than 2 SD high mean O2 concentration, and lower and more units (ter Braak and Prentice 1988). Thus, before variable temperature than the sulfidic lakes (Table 1). conducting the CCA, a DCA with the option ‘‘de- These geochemical conditions clearly indicate that trending-by-segments’’ (Hill and Gauch 1980) was CDP and SDP water has a surface origin, with the performed. The DCA produced a first axis of 3.111 SD additional contribution of surface runoff for the SDP, for presence/absence data and 3.372 SD for abundance where a small rivulet running from the cave ceiling data, thus indicating that CCA is appropriate. could be seen during strong rainy events (Fig. 3). To compare community structure of the same lake In total, 16 copepod species were identified, nine of in the two contrasting sulfide concentrations, diversity, which are stygobites, and three new to Science dominance and equitability were calculated by using (Table 2). Although lakes are larger than dripping Shannon diversity index, Simpson dominance and pools, the two water body types had similar values of Pielou equitability, respectively (Magurran species richness (between 3 and 8 in the lakes and 7–8 1988, 2004; Hayek and Buzas 2010). To assess in the pools). The most abundant copepod species whether these indices were significantly different were the diaptomid calanoid Eudiaptomus intermedius between H and L conditions, a bootstrap procedure (Steuer, 1897) (cumulative abundance 4396 individ- was used as implemented in PAST v.3 (Hammer et al. uals) and the cyclopoids Eucyclops intermedius 2001). In the bootstrapping, the two samples (L and H) (Damian, 1955) (cumulative abundance 865 individ- were initially pooled. Then, 1000 random pairs of uals) and Diacyclops cosanus Stella & Salvatori, 1954 samples (Li,Hi) were taken from this pool, and a (cumulative abundance 578 individuals). These three community index was calculated for each replicate species were sampled from the sulfidic lakes, pre- pair with the same number of individuals as in the dominantly from LV and were never collected from original two samples. The probability of obtaining the the dripping pools (except 21 individuals of D. observed difference by random sampling from a cosanus collected in both CDP and SDP). unique parental population was calculated as the Cluster analysis based on Sørensen similarity number of times that the absolute difference of the produced a dendrogram that clearly separates dripping indices of a replicate pair exceeded or equaled that of pools from sulfidic lakes (Fig. 4a; cophenetic corre- the original samples. P(equal) \0.05 was assumed to lation coefficient 0.939). The sulfidic lakes were in indicate a significant difference between the compared turn divided into two groups: The first composed of the communities (Fattorini 2010). two sulfide conditions (H and L) of LDB; the latter including the two conditions of LV and LC (Table 1). The Simpson dissimilarity index, which measures the Results pure turnover component, led to the same basic groupings (Fig. 4 b; cophenetic correlation coefficient The water geochemistry was different between the 0.851). sulfidic lakes and the dripping pools (Table 1). Among The use of species abundances with Bray–Curtis sulfidic lakes, LV and LC were more sulfidic than index disclosed a different pattern (Fig. 4c; cophenetic LDB, even in their L sulfide concentration, and had correlation coefficient 0.941). Dripping pools clus- higher mean electric conductivity. In LDB, variation tered apart as in the previous analyses, but lakes were in electric conductivity was lower than for the other divided into two groups according to their sulfide lakes. Oxygen concentration was generally low in all concentration (H vs. L). Thus, variation in H2S the sulfidic lakes, ranging from 0.3 to 4.3 mg L-1 concentrations had more impact on assemblage abun- (Table 1). dance structure than on species composition. The dripping pools showed different physical and The constrained ordination (CCA) biplot based on chemical characteristics of water: The CDP had the species presence–absence data (Fig. 5) resulted in bottom covered by calcium carbonate concretions, relatively high eigenvalues and cumulative percentage whereas the SDP, which is fed also by runoff, had its variances, indicative of a well-structured community bottom mainly composed by silt and to lesser extent by (Table 3). Moreover, there were strong species– 123 123

Table 1 Summary of environmental variables (mean ± SE) measured in the sampling sites analyzed in the Frasassi cave system (central Italy) -1 2? -1 2- -1 Sampling site Number of Electric conductivity Temperature (°C) pH O2 (mg L )Ca(mg L )SO4 (mg L ) samples (lScm-1 ,25°C)

LDBH 9 2670 ± 57 13 ± 0.2 7.53 ± 0.08 1.6 ± 0.4 116.3 ± 0.9 182.9 ± 3.6 (2300, 2840) (11.8, 13.7) (7.3, 8) (0.34, 4) (110, 118.9) (170, 199) LDBL 4 917 ± 55 13 ± 0.2 7.35 ± 0.13 3.0 ± 0.7 116.7 ± 0.5 177.5 ± 1 (760, 992) (12.6, 13.6) (7, 7.59) (1.8, 4.3) (115.9, 116) (176, 180) LCL 2 1300 ± 300 13.7 ± 0.1 7.33 ± 0.01 4.2 ± 0.1 106.9 ± 4.1 180.0 (1000, 1600) (13.6, 13.7) (7.32, 7.33) (4.0, 4.3) (102.8, 111) 180.0 LVH 6 2968 ± 135 13.2 ± 0.3 7.24 ± 0.08 0.9 ± 0.3 144.1 ± 1.6 191.8 ± 1.6 (2380, 3400) (12.5, 13.9) (7, 7.48) (0.24, 1.8) (141.8, 149) (190, 199) LVL 3 1789 ± 23 12.8 ± 0.1 7.15 ± 0.12 1.3 ± 0.0 136 ± 2 179.3 ± 5.8 (1751, 1830) (12.5, 13) (7, 7.4) (1.3, 1.4) (134, 140) (170, 190) CDP 6 413 ± 17 10.9 ± 0.4 7.75 ± 0.06 9.4 ± 0.4 72.6 ± 0.9 15.7 ± 0.2 (350, 450) (12, 9.42) (7.5, 7.9) (8.54, 10.57) (71, 77.2) (15, 16) SDP 7 307 ± 8 10.4 ± 0.7 7.80 ± 0.10 10.4 ± 0.5 49.0 ± 0.4 7.9 ± 0.1 (280, 334) (8, 12.5) (7.5, 8.1) (9, 12) (48, 50.5) (7.5, 8) Minimum and maximum values are also reported when available. Site acronyms as in Fig. 4 qa Ecol Aquat Aquat Ecol

Table 2 List of the copepod species collected in the sampling sites analyzed in the Frasassi cave system (central Italy) LDBH LDBL LCL LVH LVL CDP SDP

Crustacea Copepoda Copepoda Calanoida Sars G. O., 1903 Eudiaptomus intermedius (Steuer, 1897)* 85 431 2006 68 1806 0 0 Copepoda Cyclopoida Burmeister, 1835 Eucyclops intermedius (Damian, 1955)* 110 304 3 43 405 0 0 Paracyclops imminutus Kiefer, 1929 0 0 0 6 14 108 160 Macrocyclops albidus (Jurine, 1820) 0 0 0 0 0 0 15 Acanthocyclops gr. venustus (Norman & Scott T., 1906) 14 9 0 3 0 0 0 Acanthocyclops robustus robustus (Sars G. O., 1863) 0 0 0 4 0 0 0 Diacyclops cosanus Stella & Salvatori, 1954* 59 174 36 44 244 17 4 Speocyclops sp. 1* 1 7 0 0 0 0 0 Copepoda Harpacticoida Sars G. O., 1903 Attheyella (Attheyella) crassa (Sars G. O., 1863) 0 0 0 0 0 6 311 Moraria (Moraria) poppei meridionalis Chappuis, 1929 0 0 0 0 0 18 18 Moraria (Moraria) stankovitchi Chappuis, 1924* 0 0 0 0 0 49 8 Moraria sp. 1* 0 0 0 0 0 64 0 Maraenobiotus sp. 1* 2 3 0 0 0 3 0 Nitocrella stammeri Chappuis, 1938* 7 2 0 0 0 0 0 Nitocrella psammophila Chappuis, 1954* 22 27 0 0 0 0 0 Bryocamptus (Rheocamptus) zschokkei (Schmeil, 1893) 0 0 0 0 0 70 18 Total species richness 8 8 3 6 4 8 7 * Indicates stygobiotic species. Cumulative abundances per site were reported for each species. Site acronyms as in Fig. 4 environmental correlations with all four axes, which not significantly different between L and H condi- together accounted for about 98 % of the explained tions), thus suggesting that community structure variance. All geochemical variables were strongly within the same lake changed significantly according correlated with axis 1, which explained more than to sulfide concentrations (Table 4). 50 % of variance. The only variable that had a strong correlation with axis 2 was pH, which resulted the only significant variable when an automatic forward selec- Discussion tion was applied. This result was, however, a reflection of the strong intercorrelation between variables. When Only few studies have analyzed the invertebrate life in tested individually, all variables were significant at sulfidic caves, such as the Movile Cave, Romania P \ 0.05 except the electric conductivity. Species- (Sarbu 2000; Sarbu et al. 1996, 2000), the Ayyalon abundance data returned very similar results (Online Cave, Israel (Por 2007; Por et al. 2013) and the Resource 1), with a total of 96.3 % of variance Frasassi cave system (Flot et al. 2010; Bodon and explained by the four axes (Online Resource 2). All Cianfanelli 2012; Peterson et al. 2013; Fisˇer et al. variables were strongly correlated with the first axis 2015). During our sampling in the Frasassi cave (correlation in absolute values ranging from 0.715 to system, we collected 16 species of copepods, three of 0.980), which had an eigenvalue of 0.75. which are new species endemic to this cave system. Lakes that were sampled under the two sulfide The species richness appears impressive in consider- concentrations (L and H) showed higher dominance, ation of the environmental conditions determined by lower diversity and lower equitability in the L high concentrations of H2S and low O2 availability in condition (except for LV where the equitability was the sulfidic lakes of Frasassi cave system. For

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Fig. 4 Relationships between copepod communities in the conditions, LDBH—Lago della Bottiglia in high-sulfidic Frasassi cave system based on Sørensen (a), Simpson (b) and conditions, LDBL—Lago della Bottiglia in low-sulfidic condi- Bray–Curtis (c) indices and UPGMA clustering. Sørensen and tions, LVH—Lago Verde in high-sulfidic conditions, LVL— Simpson indices were calculated on presence/absence data. Lago Verde in low-sulfidic conditions, SDP—silted dripping Bray–Curtis index was applied to abundance data. CDP— pool concretional dripping pool, LCL—Lago Claudia in low-sulfidic comparison purposes, we can recall that in the Movile system developed in three levels into the carbonate Cave, a total of 18 groundwater invertebrate species platform. A total of 17 crustacean copepods were (including Platyhelminthes, Nematoda, Annelida, found in this system, seven of which were non- Mollusca, Crustacea, Insecta Heteroptera) were dis- planktonic stygobites, being always found with few covered, eight of which were stygobiotic endemics individuals in benthic and hyperbenthic habitats, with (Sarbu 2000; Sarbu et al. 1996, 2000). However, only the exception of one species of the copepod harpacti- 2 stygobiotic copepod species were recorded, namely coid genus Parastenocaris, which was found in an Eucyclops subterraneus scythicus Plesa, 1989 and epikarstic dripping pool (Galassi and De Laurentiis Parapseudoleptomesochra italica Pesce & Petkovski, 2004). Species richness in this system is comparable to 1980. In the Ayyalon Cave, the stygobiotic copepods that observed in the Frasassi cave system, but the Metacyclops longimaxillis Defaye & Por, 2010 and assemblage composition is very different, being Metacyclops subdolus Kiefer, 1938, the thermosbae- represented by stygobites of limnicoid origin, with nacean Tethysbaena ophelicola Wagner, 2012, and the the only exception represented by Pseudectinosoma decapod Typhlocaris ayyaloni Tsurnamal, 2008 were kunzi Galassi, 1997, which belongs to a genus of direct the only collected crustacean species (Por et al. 2013). marine origin (Galassi et al. 1999). The high copepod diversity of the Frasassi cave Differences in species richness among sulfidic cave system is paralleled by the relatively high number of systems have been attributed to the colonization amphipods (four species of the genus Niphargus) and pathways of their epigean relatives, the regional ostracods (21 species) more or less directly dependent stygobiotic species pool in the areas where these cave on chemoautotrophy (Peterson et al. 2013; Fisˇer et al. systems are located, and the age of the system (Engel 2015), and indicates that a chemosynthetically pro- 2007; Por et al. 2013). The Frasassi cave system is duced food source may have favored stygobiotic very large, has a long and complex geological history, colonizers to settle in sulfidic groundwater, probably and includes very different environments, all factors due to their preadaptation to tolerate these peculiar that promote diversity and make its groundwater environmental conditions. Karstic non-sulfidic cave biodiversity composite and diversified. Engel (2007) systems are widespread across the Italian peninsula, postulated a high crustacean diversity possibly asso- the best known being the Castelcivita cave system in ciated with sulfidic caves, but also pointed out that in- southern Italy (Alburni Massif), a very large cave deep surveys are needed for assessing whether 123 Aquat Ecol

As regards the invasion history of the copepod fauna of the Frasassi cave system, the assemblages of dripping pools included mostly non-stygobiotic spe- cies that enter the system from the surface waters, whereas they are very rare in the sulfidic lakes, probably because most of them are unable to cope with the environmental conditions of these water bodies. Sulfidic lake assemblages are largely composed of stygobiotic species. Even Eudiaptomus intermedius,a copepod commonly found in surface lakes and ponds across Italy (Marrone et al. 2011), occurs in the Frasassi cave system with a population that shows distinct stygomorphic traits (including a reduced body size, depigmentation, anophthalmy and the presence of an egg sac with 2–4 large eggs; Fig. 6), although genetically not isolated from surface conspecific populations (Marrone et al. 2011). This phenomenon suggests that such stygomorphic traits can evolve in subterranean populations even in the absence of a significant genetic isolation (Marrone et al. 2011; Klaus et al. 2016). This species showed the highest abundance in the low-sulfidic periods in both LV and LC, where it was collected with ovigerous females, adult males, nauplii and all the copepodid stages. These findings, coupled with the lack of fast infiltra- tion pathways and surface water bodies (Galdenzi et al. 2008, 2012) from which it might be washed out, suggest that this species is really able to reproduce in Fig. 5 CCA biplot showing the effect of the geochemical the sulfidic karst. It was always accompanied by the characteristics in determining the species composition of stygobiotic cyclopoid Diacyclops cosanus, another groundwater copepod communities in the Frasassi cave system planktonic species commonly found out of cave based on presence/absence data. The relative importance of systems, for example, in both true freshwater and individual variables is expressed by the length of the respective vectors. a plot showing site assemblages, b plot showing brackish subterranean habitats, and which is also able species. CDP concretion dripping pool, LCL Lago Claudia in to survive in saline waters, with high electric conduc- low-sulfidic condition, LDBH Lago della Bottiglia in high- tivity (up to 3000 lScm-1), high chloride and sulfate LDBL sulfidic condition, Lago della Bottiglia in low-sulfidic concentrations, and in alluvial coastal aquifers, where condition, LVH Lago Verde in high-sulfidic condition, LVL Lago Verde in low-sulfidic condition, SDP silted dripping pool. marine intrusion occurs due to groundwater exploita- Attc—Attheyella crassa, Acar—Acanthocyclops robustus robus- tion (Di Lorenzo and Galassi 2013). The remaining tus, Acav—Acanthocyclops gr. venustus, Bryz—Bryocamptus copepod species were predominantly benthic or zschokkei, Diac—Diacyclops cosanus, Euci—Eucyclops inter- Eucyclops intermedius medius, Eudi—Eudiaptomus intermedius, Maca—Macrocy- hyperbenthic, such as , a sty- clops albidus, Morp—Moraria poppei meridionalis, Mor1— gobiotic species found with high abundance in the Moraria sp. 1, Mors—Moraria stankovitchi, Mar1—Maraeno- sulfidic lakes. biotus sp. 1, Nitp—Nitocrella psammophila, Nits—Nitocrella In accordance with our hypothesis that the compo- stammeri, Pari—Paracyclops imminutus, Spe1—Speocyclops sition of the groundwater fauna should be ecologically sp. 1 and spatially structured according to different geo- recorded species are really living in the sulfidic portion chemical conditions, cluster analysis showed a clear of these aquifers. Our study demonstrates that several partition between sulfidic habitats and non-sulfidic copepod species are really associated with sulfidic dripping pools, whose copepod fauna was different waters. from sulfidic water bodies in terms of both species 123 Aquat Ecol

Table 3 Results (F statistics) of CCA for species presence–absence data

Variable ka Individual tests Forward selection Weighted correlations PFPFAxis 1 Axis 2 Axis 3 Axis 4

Electric conductivity 0.38 0.376 1.16 0.096 2.08 -0.697 0.390 0.226 -0.212 pH 0.49 0.004 4.59 0.045 3.02 0.771 -0.547 0.014 -0.113 Ca2? 0.55 0.362 1.19 0.017 3.75 -0.867 0.434 -0.013 -0.139

O2 0.59 0.382 1.31 0.013 4.18 0.927 -0.292 -0.096 0.018 2- SO4 0.61 1.000 0.00 0.024 4.45 -0.944 0.281 0.073 0.113 Temperature 0.62 0.665 0.68 0.007 4.59 -0.954 0.277 -0.015 0.067 Eigenvalues 0.652 0.325 0.154 0.135 Cumulative % variance 50.4 75.5 87.4 97.8 ka indicates the increase in eigenvalue (additional fit). P indicates the significance level of the conditional effects based on Monte Carlo tests (999 random permutations), and F is the test statistic. Variables are the same as in Table 1

Table 4 Values of Simpson dominance, Shannon diversity and Pielou equitability for two lakes [Lago della Bottiglia (LDB) and Lago Verde (LV)] at high- and low-sulfide concentration LDB LV High Low P High Low P

Dominance 0.262 0.338 0.001 0.300 0.572 0.001 Diversity 1.520 1.245 0.001 1.346 0.783 0.001 Equitability 0.731 0.599 0.001 0.751 0.565 0.184 P values indicate the probabilities obtained from bootstrap analysis

composition and relative abundances. As hypothe- sized, we also found that the community structure of sulfidic lakes is influenced by the sulfide concentration

(H vs. L), which suggests that H2S concentration is a key factor regulating species composition, whereas the immigration of individuals from non-sulfidic water should be discounted, as shown by the absence of stygoxenes [with the only exception of few individuals of Paracyclops imminutus Kiefer, 1929 and Acantho- cyclops robustus robustus (Sars G. O., 1863)]. The CCA biplot reinforced the information derived by the cluster analyses and recovered three main groups of sites: (1) LDBH ? LDBL, (2) LC ? LV and (3) CDP ? SDP. The species that mostly accounted for the dissimilarity between LDB and LV ? LC are all Fig. 6 The calanoid Eudiaptomus intermedius (Steuer 1897), stygobiotic species: Speocyclops sp.1, Acanthocyclops the most abundant species in the planktonic habitat of the gr. venustus, Maraenobiotus sp. 1, Nitocrella psam- sulfidic lakes in the Frasassi cave system (automated 3-D mophila and N. stammeri, which were found in the reconstruction). It is possible to see through the body vesicles containing fatty deposits to favor flotation and a black frontal benthic samples from sediment layers of LDB but not median organ with presumably neurosecretory function in LC and LV. Conversely, LC and LV shared the 123 Aquat Ecol highest abundances of Eudiaptomus intermedius, 0.24 mg L-1, corresponding to a true suboxic condi- followed by Eucyclops intermedius and Diacyclops tion). The same arguments may explain the presence of cosanus. LDB harbored a different species assemblage only seven individuals of Eudiaptomus intermedius and at both high- and low-sulfide concentrations, because two individuals of Diacyclops cosanus in Lago Galden- it is the less sulfidic lake among those analyzed, as a zi, and the total absence of copepods in the flowing 2- consequence of its higher depth (so that the sulfidic rivulets feeding this pool, where H2S (expressed as S ) layer is much far distant from the water table). reaches values of 12.4–14.4 mg L-1, accompanied by Moreover, differences in geochemistry between L suboxic oxygen concentration, and electric conductivity and H conditions are not as great as for LV, which also of 3070 lScm-1. Temperature was also recovered as supports our claim that the sulfide concentration is the an important factor for lake assemblages. Temperature most important environmental parameter regulating was constant in the sulfidic lakes (*13 °C) and community composition. LC resembles LV in its L significantly higher than in dripping pools (*10 °C sulfide concentrations; thus, its copepod assemblage even if more variable over the year). closely follows that of LVL. Compared with dripping pools, sulfidic lakes are We hypothesized that the main chemical factor more stable environments, but with much higher influencing lake assemblages should be the sulfide concentration of H2S and hence lower pH and oxygen concentration, whereas dripping pool assemblages concentration. Differences in geochemical characteris- should be more influenced by oxygen and pH. CCA tics between lakes and dripping pools derive from the confirmed our hypothesis, showing that dripping pool origin of their water. Dripping pools collect surface assemblages are characterized by high oxygen concen- waters percolating from the cave ceiling or by runoff tration and pH values, whereas lake assemblages are (Meleg et al. 2015) and are thus characterized by waters strongly influenced by sulfide concentrations. The mean with low concentration of dissolved ions, and hence

O2 concentration in dripping pools was about 2–10 with low electric conductivity, higher mean oxygen times higher than that recorded in the surface layers of concentration and lower and more variable tempera- sulfidic lakes, which explains the importance of this ture. Thus, species composition of freshwater pool parameter for copepod species composition in dripping assemblages responds to high oxygen concentration pools. However, the mean O2 concentration in sulfidic and pH values. Except for P. imminutus and D. cosanus, lakes was at the boundary between dysoxic (0.3–3.0 the species inhabiting the dripping pools were never mg L-1) and oxic ([3.0 mg L-1) conditions, and found in the sulfidic lakes, even in their L condition. sometimes also suboxic (\0.3mg L-1) (Tyson and Both SDP and CDP are not connected to the sulfidic Pearson 1991; Malard and Hervant 1999). The ground- saturated karst, but with the fracture network of the water organisms are exposed to all the three conditions Calcare Massiccio. However, they have different mentioned above. Although all sulfidic lakes clustered hydrogeological characteristics. The SDP is filled by in the CCA analysis apart from dripping pools, they dripping water that has a shorter residence time have different geochemical characteristics, which are compared with the CDP, as indicated by the more responsible for differences in species composition. LV variable temperature observed during our sampling and LC were more sulfidic than LDB, even in their L campaign, the lower electric conductivity and the high condition, and have higher electric conductivity. The abundance of a surface eurytopic species, Attheyella sole species able to cope with high-sulfide concentra- crassa, which was found with 311 individuals (whereas tions, along with low O2 concentration, were the only six individuals were found in the CDP). planktonic species, which were, however, found only Even if there is no clear-cut dichotomy between in the topmost 50 cm of water column in free open species composition in high- and low-sulfide concen- water. Copepods were never found to cross the tration periods in the lakes, species abundances were chemocline, being also unable to survive at the contact sensitive to the geochemical separation, being much with the microbial mats. Thus, copepods seem to avoid lower in the high-sulfide concentration periods, which high-sulfidic habitats with H2S concentration as in LV, supports our hypothesis that different sulfide concen- and saline waters (with electric conductivity values trations determine differences in community structure. ranging around 3000 lScm-1), where also very low This is revealed by Bray–Curtis abundance-based oxygen concentrations were measured (below cluster analysis and the community structure indices, 123 Aquat Ecol which emphasized a decrease in dominance and an oxidizing bacteria (Engel et al. 2003, 2004;Macalady increase in equitability at high-sulfide concentrations. A et al. 2006, 2008; Jones et al. 2015) and the challenging possible explanation is that increased sulfide concen- environmental conditions for invertebrate species living trations probably induce a strong population decline of in extreme environments colonized by these bacteria, the species that dominated copepod assemblages at low- may rise further questions on the trophic relationships sulfide concentrations, thus allowing a more linking the primary producers to the other components equitable species-abundance distribution, and hence of the food chain in this peculiar groundwater ecosys- higher diversity and equitability. A possible alternative tem. Ectosymbiosis was thought as a mean to overcome or complementary explanation is that, if copepods use the chemical constraints to live in the sulfidic karst (i.e., the biofilm of sulfur-oxidizing bacteria as a food, a for the amphipods Niphargus ictus Karaman, 1985 and higher equitability at higher sulfide concentration may Niphargus frasassianus Karaman, Borowski and Datta- also be due to increased food resources reducing gupta, 2010), but the true advantage of this interaction is competition. This second hypothesis appears less prob- still matter of contention. Bauermeister et al. (2013) able, because we never found copepods at the contact demonstrated that Thiothrix spp. ectosymbionts of N. with the microbial mats (where pH is very low), which ictus and N. frasassianus are probably not essential for suggests that they are not able to take a direct advantage preventing sulfide poisoning of their hosts in cave from the biofilm of the sulfur-oxidizing bacteria. waters, and likely they have evolved detoxifying However, a definitive conclusion about the contribution metabolic routes independently. This recent discovery of sulfur-oxidizing bacteria to the diet of copepods could gives rise to the need for examining whether these be reached only by comparing copepod trophic niches in bacteria provide the Niphargus with other advantages. sulfidic and non-sulfidic water bodies by analyzing No less important, it remains unresolved which position stable isotope ratios, which is out of the scope of this in the trophic chain is taken by the copepods, which are research, but which might represent a stimulating the most abundant invertebrate group in the Frasassi working hypothesis for future studies. cave system, although with less species than ostracods, In conclusion, the relatively high biodiversity and e.g., if they feed directly on bacteria or on the particulate complex community structure of the copepods inhab- organic matter, indirectly derived by microbial mat iting the Frasassi cave system indicate that a chemosyn- decomposition. thetically produced food source favored several copepod species to settle in sulfidic groundwater, Acknowledgments The Project was partially funded by the probably due to their ability to tolerate these peculiar European Community (LIFE12 BIO/IT/000231 AQUALIFE). We are indebted to Simone Cerioni of the Speleological Group environmental conditions. Extremely high-sulfide con- of Genga, Maurizio Mainiero of the Marche Speleological centrations, corresponding to those measured in the Group of Ancona, Samuele Carnevali of the Gruppo deeper waters of the Frasassi lakes, where the microbial Speleologico of Fabriano, and Maxwell Montanari of the mats thrive, are known to alter the metabolism of most OGC for technical support in the sampling campaigns. Financial support is also acknowledged for speleological invertebrates (Somero et al. 1989;Grieshaberand equipment and laboratory materials for preliminary analyses Vo¨lkel 1998). However, short-term tolerance to low and sample preparation provided by the Observatorio Geologico oxygen concentration has been claimed by Malard and di Coldigioco (OGC). We are also grateful to the Georg-August- Hervant (1999) for some groundwater amphipod and Universita¨tGo¨ttingen, Go¨ttingen, Germany (Sharmishtha Dattagupta, Linn Groeneveld, Nicolas Cerveau, Mahesh isopod species, and adaptive molecular mechanisms Desai, Jean-Franc¸ois Flot, Jan Bauermeister) and the IMDEA were observed by Lawniczak et al. (2013)inthe Water Institute, Alcala de Henares, Madrid, Spain (Sanda amphipod Niphargus. Reduced metabolic rates of Iepure) for providing us with chemical data. We are also grateful groundwater taxa, compared to those of surface water to two anonymous referees for their helpful suggestions. relatives, have long been inferred to be an adaptive trait where there is a low or discontinuous food supply and unpredictable shifts between dysoxic and oxic condi- References tions (Di Lorenzo et al. 2014). 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