Groundwater Drift Monitoring As a Tool to Assess the Spatial Distribution of Groundwater Species Into Karst Aquifers

Hydrobiologia https://doi.org/10.1007/s10750-018-3515-1

PRIMARY RESEARCH PAPER

Groundwater drift monitoring as a tool to assess the spatial distribution of groundwater species into karst aquifers

Tiziana Di Lorenzo . Donatella Cipriani . Barbara Fiasca . Sergio Rusi . Diana Maria Paola Galassi

Received: 1 December 2017 / Revised: 11 January 2018 / Accepted: 12 January 2018 Ó Springer International Publishing AG, part of Springer Nature 2018

Abstract Groundwater drift collected continuously rainfalls. According to the results of this study, at perennial outlets of karst aquifers has been exam- groundwater species do not inhabit all sectors of a ined for its potential in assessing the spatial distribu- karst aquifer. They avoid the fast-flowing conduits tion of the resident species. To this end, we have where groundwater rapidly flows, while colonizing the continuously monitored the groundwater drifted at the more inertial aquifer sectors, such as the slow-flowing Mazzoccolo spring (Western Aurunci karst aquifer, conduits. Central Italy) over 2 years. Concurrently to the biological monitoring, major ion geochemistry and Keywords Copepoda Á Groundwater Á Spring Á turbidity were investigated on the same schedule. We Piston effect Á Habitats found that the hydrochemistry did not govern the distribution pattern of the dominant taxon (Copepoda) into the Western Aurunci aquifer. In contrast, copepod drift showed clear differences in the number of Introduction individuals between the high and low water periods because of the ‘‘piston effect,’’ which is very frequent The distributional data and population size-estimates in karst and fractured aquifers due to recharge from of obligate groundwater fauna (the so-called stygobionts) in karst aquifers have been mainly based on collection in caves (e.g., Rouch, 1986; Brancelj, 2002; Handling editor: Diego Fontaneto Pipan & Brancelj, 2004; Moldovan et al., 2007; Pipan & Culver, 2007; Brancelj, 2009; Pipan & Culver, Electronic supplementary material The online version of 2013). Other access points to karst subterranean this article (https://doi.org/10.1007/s10750-018-3515-1) con- tains supplementary material, which is available to authorized habitats, such as wells, have been sampled less users.

T. Di Lorenzo (&) B. Fiasca Á D. M. P. Galassi Institute of Ecosystem Study of the CNR – National Department of Life, Health and Environmental Sciences, Research Council of Italy, Via Madonna del Piano 10, University of L’Aquila, Via Vetoio 1, 67100 Coppito, Sesto Fiorentino, 50019 Florence, Italy AQ, Italy e-mail: [email protected] S. Rusi D. Cipriani Department of Engineering and Geology, University of Istituto di Istruzione Superiore ‘‘Tulliano’’, Piazza del Chieti-Pescara, Via dei Vestini 30, 66013 Pescara, CH, Municipio 49, Arpino, 03033 Frosinone, Italy Italy 123 Hydrobiologia thoroughly (e.g., Malard et al., 1997; Galassi et al., natural springs with large fractures and openings. In 2009a, b; Hahn & Fuchs, 2009; Di Lorenzo & Galassi, many of these springs, the groundwater flow shows a 2013; Di Lorenzo et al., 2015; Iepure et al., 2017), ‘‘pulsating’’ behavior (regular alternation of high, while karst springs can boast a consolidated history of residual, and low discharge) due a phenomenon called biological data collection (e.g., Mori & Brancelj, ‘‘piston effect’’ (Nanni & Rusi, 2003; Aquilina et al., 2006, 2013; Fiasca et al., 2014; Galassi et al., 2014; 2006) that consists in a pressure transfer due to Dole-Olivier et al., 2015; Stoch et al., 2016). Data pushing freshly infiltrated waters and mobilizing from the biological survey from the past 30 years have resident groundwater. provided a clear scenario of the large-scale factors In this study, groundwater drift collected continu- influencing karst biological assemblages among ously at perennial outlets of karst aquifers has been aquifers, including evolutionary history, habitat examined for its potential of being affected by the heterogeneity, and environmental gradients (Dole- ‘‘piston effect.’’ To this end, we monitored a captured Olivier et al., 2009a, b; Hahn & Fuchs, 2009; Martin karst spring by filtering the passing-through ground- et al., 2009; Fattorini et al., 2017; Iepure et al., 2017). water over 2 years. We monitored species richness The spatial distribution of karst groundwater taxa is and abundance of copepods (Crustacea, Copepoda) highly heterogeneous and the level of endemism is along with physical and chemical parameters at the high worldwide (Culver & Sket, 2000; Proudlove, Mazzoccolo spring (Fig. 1A), the outlet of the 2001; Culver et al., 2004, 2009; Deharveng et al., Western Aurunci karst massif (Italy), monthly over 2009; Gibert & Culver, 2009; Stoch & Galassi, 2010; 2 years, and in the three different water level periods Zagmajster et al., 2014; Brancelj, 2015). Many involved in the ‘‘piston effect,’’ namely, in the high endemic species found in karst aquifers are described water level period (aquifer recharge period: from as phylogenetic relicts, belonging to old phylogenetic November to February), in the low water level (from lineages whose surface relatives went extinct from June to October), and in the residual water level (from tens of thousands to millions years ago (Delamare- March to May) periods. We selected copepods as Deboutteville & Botosaneanu, 1970; Galassi et al., target taxon because a preliminary study had shown 1999; Galassi, 2001; Danielopol & Griebler, 2008; that these crustaceans are the most abundant and Stoch & Galassi, 2010; Zagmajster et al., 2014). Most species-rich groundwater group in the Aurunci karst phylogenetic relicts are also distributional relicts, with aquifer (Di Lorenzo et al., 2005). The main aims of close counterparts traceable in disjunct geographical this study were (1) to characterize the drift of areas, or in different habitats (Holsinger, 1988). groundwater species from the aquifer in combination If historical factors have a consolidate role in with the assessment of its hydro-geochemistry, (2) to determining the distribution patterns of groundwater test whether the abundances of groundwater drift were species among different aquifers (Gibert et al., 1994 affected by the ‘‘piston effect’’ and (3) to assess the and reference therein; Galassi et al., 2009a; Iepure utility of continuous collection of the groundwater et al., 2017), the main small-scale drivers that govern drift for assessing the spatial distribution of copepod their spatial distribution within an aquifer have been species into the aquifer. far less investigated (Mori & Brancelj, 2013; Galassi et al., 2014; Fattorini et al., 2017). When a karst aquifer is physically inaccessible due to the lack of Study area extensive cave systems, the analysis of groundwater species collected at the springs is the only available Geological and hydrogeological setting tool. Periodical (monthly/seasonal) sampling via piezometers (Fiasca et al., 2014; Galassi et al., 2014; The study area is characterized by the presence of Stoch et al., 2016; Fattorini et al., 2017) has proved to carbonate formations (Cretaceous), outcropping in the be an efficient method in karst rheo-limnocrenic Aurunci Massif, that are in tectonic contact with a spring although this technique does not allow a marly–clayey terrigenous succession (Miocene–Plio- continuous monitoring. Collecting the groundwater cene), sometimes including gypsum (Servizio Geo- drift over long periods and in continuum is a technique logico d’Italia, 1967; Capelli et al., 2012). The that may be suitable for captured karst springs, or tectonic contact is partially hidden under the old 123 Hydrobiologia

Fig. 1 A Geographical location of the study area, with the limestone complex; (2) terrigenous complex; (3) polygenic geological–hydrogeological cross section and location of the conglomerates complex (laying on these deposits, there is a thin meteorological stations (red diamonds). B Schematic geologi- sandy layer originating from volcanic rock’s degradation that cal–hydrogeological cross section (see A for the location): (1) cannot be represented) alluvial deposits that are polygenic conglomerates ﬂows through an extensive fractured network, which (Pliocene–Pleistocene). The carbonate succession of in time become a widespread and branched karst the Aurunci Massif is a hydrogeological complex with system (hereafter: limestone complex). The marly– high inﬁltration and active groundwater circulation clayey terrigenous succession represents, in general, a (Boni et al., 1986; Capelli et al., 2012). Groundwater low hydraulic conductivity hydrogeological complex,

123 Hydrobiologia whose infiltration and groundwater circulation are November to February); (2) a low water level period negligible. These characteristics allow us to consider (L) that occurs in the dry season during the summer this hydrogeological complex as an aquitard or, period (from June to October); and (3) a recession sometimes, as an aquiclude, with respect to carbonate period (R) that occurs in the spring period (from formations (hereafter: terrigenous complex). Poly- March to May) (Online Resource 1). The rainfall genic conglomerates can be considered a high varies in the same way in all the meteorological hydraulic conductivity hydrogeological complex, stations considered (Online Resource 1). Thus, the due to their intrinsic porosity and fracturation. discharge of the Mazzoccolo spring and the rainfall Although they are marked out by excellent hydraulic regime overlap, as can be expected in a karst and properties, their limited extension reduces their quan- fractured aquifer located in the Mediterranean area titative potentiality (hereafter: polygenic conglomer- (Fiorillo et al., 2015). In order to better highlight this ates complex). overlapping, the effective rainfall (calculated by The Mazzoccolo spring (41°1501700N; 13°2700800E) subtracting evapotranspiration to precipitations in is located where the polygenic conglomerate complex each meteorological station) was examined using is in contact with the terrigenous complex. The Thornthwaite and Mather’s method (Thornthwaite & emerging groundwater comes from the limestone Mather, 1957). The results for the 2003–2004 periods aquifer (Fig. 1B). The limited extent of the outcrop are presented in the Online Resource 1. The available of the polygenic conglomerates complex, indeed, data (from different databases) have allowed us to cannot explain the high discharge values. For this extend the analyses of the rainfall and the effective reason, the spring can be classified as a barrier spring rainfall to the 2002–2014 periods (Online Resource 1). (Civita, 1972; Kresic & Stevanovic, 2009). The The rainfall and the effective rainfall (Online Mazzoccolo spring emerges at 15 m a.s.l., its dis- Resources 1, 2) distributions show high values in the charge ranges from 200 l/s to 1500 l/s (Celico, fall–winter period and very low and/or null values in 1978, 1983; Latina Province, 2010; Sappa et al., the summer period. In addition, the rainfall and 2015a), and it is exploited for drinking purpose, to feed effective rainfall values of the Vallecorsa and Lenola the Formia municipality (about 36,000 inhabitants). meteorological stations (Online Resources 1, 2), The spring capture is made up of huge draining tunnels located at the higher elevation (467 m a.s.l. and (Sappa et al., 2015b), up to 2.5 m wide and 2 m high, 470 m a.s.l., respectively), are always higher than partially built in Roman age. the Gaeta and Formia meteorological stations, located in the coastal area (32 m a.s.l. and 5 m a.s.l., respec- Aquifer recharge and spring flow rate tively). Considering what has just been shown above (Online Resource 1), it seems very clear that both the The spring discharge regime has been assessed Mazzoccolo spring discharge and the rainfall moni- analyzing the measurements made part in December tored in the recharge area of the aquifer (Vallecorsa 2002, the whole 2003, and the three first months of and Lenola) display the highest values in the fall– 2004 (Di Lorenzo et al., 2005) and those performed in winter period (H) and the lowest values in the summer 1970 (Celico, 1983). We were not allowed to take period (L), with a recession period in spring time (R). further discharge measurements for reasons of hygie- This correspondence becomes clearer if the spring nic safety. The rainfall and temperature data, both discharge is compared with the effective rainfall regarding the long period (2002–2014) and the calculated at the same meteorological stations (Online monitoring period of this study (2003–2005), have Resource 1). been obtained using the Vallecorsa, Lenola, Gaeta, and Formia meteorological station time-series (Online Resource 1), made available by the Lazio Region Civil Materials and methods Protection Department, the SIARL (2016), and the CRA (2016). Field sampling The Mazzoccolo spring discharge regime consists of (1) a high water level period (H) that occurs in the The biological sampling was carried out continuously wet season during the fall–winter period (roughly from at the spring mouth by using a drift net (mesh size: 123 Hydrobiologia

60 lm; Fig. 2). The net was put in place by spit-fixes APATIRSA4020, APATIRSA3030, APATIRSA4020 and rows at the beginning of each month and left there (nitrates and nitrites), ISSBFA032, respectively for 30 days. A weight was posed at the net bottom to (APAT_IRSA, 2003; ISS, 2007). Cations and anions block it. At the end of each month the net was were measured with a precision of 10%. Detection detached, and the invertebrates drifted from the limits varied from 1 to 50 lg/L according to the aquifer were retrieved and transferred to polyvinyl analyzed chemical. chloride (PVC) jars, fixed with a 3:7 mixture of water and 96% ethanol, and transported to the laboratory for further processing. Overall, 23 biological samples had Data analysis been collected from March 2003 to March 2005. The samples of July 2003 and December 2003 are lacking Environmental variables because the net had to be removed to allow spring maintenance services. In the laboratory, the biological Besides the analyses of rainfalls, temperatures, and samples were sorted using a Leica M65 microscope, discharges, discussed in the paragraph ‘‘Study area,’’ a animals counted, and copepods identified to the Draftsman’s plot was used to examine the correlation species level under an optical microscope at structure of the environmental variables. As strong 100 9 magnification using the taxonomic keys of inter-correlations (|r| [ 0.95) provide indication that Dussart (1967, 1969) and the most updated literature. not all variables may be needed in a parsimonious Eleven physical and chemical variables were model, we decided to remove, as the case may be, one measured by the Agency in charge of the Mazzoccolo of the variables of the [ 95%-correlated pairs (Clarke spring management on the same time schedule as for & Ainsworth, 1993; Anderson, 2001; Clarke & the biological sampling. Some variables were mea- Gorley, 2006). A one-way permutation analysis of sured before the water distribution system by an variance (PERMANOVA; Anderson et al., 2008) was automatic probe according to the following standard used to test probable differences among the three methods (APAT_IRSA, 2003; ISS, 2007): water level periods connected with the eleven physical ISSBBA043 (temperature), APATIRSA2110 (turbid- and chemical variables plus precipitations at Val- ity), ISSBCA023 (pH), APATIRSA2040 (hardness), lecorsa and Lenola meteorological stations measured and ISSBDA022 (electrical conductivity). Water from 2003 to 2005. The factor was named HYDRO_L samples were collected in plastic vials and analyzed consisting of three levels (H: high water level; L: low for determining chloride, iron, ionized ammonia, water level; R: recession level; perms = 9999). As the nitrites, nitrates, and fixed residue (i.e., the amount environmental variables were on different measure- of residual mineral salts after the evaporation of 1 L of ment scales, they were normalized prior to the water at 180°C) according to standard methods statistical analyses (Clarke & Gorley, 2006). Prior to

50 cm

Fig. 2 Left): Mazzoccolo spring mouth (height: 70 cm; max of 150 cm. The star represents the position of the spring mouth; width: 50 cm). Right) Drift net located in front of the spring the arrow represents the position of one of the two lateral spits. mouth (15 cm apart) by spit-ﬁx and rows. The net is a truncated The picture in the left was taken before putting the net in place. pyramid with a rectangular basis (100 cm 9 90 cm) and a trawl Both pictures were taken during a high discharge month 123 Hydrobiologia

PERMANOVA, Levene’s tests were performed to permutations of the raw data. We chose permutation check for homogeneity of dispersion. As the design tests because they are more powerful than statistical was unbalanced, unrestricted permutation of raw data tests based on normal theory assumptions when data and Type III of sum of squares were chosen (Anderson distribution departs substantially from normality et al., 2001). One-way PERMANOVAs were also run (Manly, 2006), as it was the case for the present data for each environmental variable taken singularly even after the log (x ? 1) transformation. Normality under the same setting as for the multivariate data. was tested by Shapiro–Wilks normality tests. Permu- All PERMANOVAs were run on the basis of a tational regressions were performed by the ape library Euclidean distance matrix. Pairwise post hoc t tests in R 3.0.2 (R Core Team, 2013). were applied when appropriate. The patterns of the Finally, relationships between environmental vari- environmental variables were also examined with a ables and multivariate copepod assemblages were multivariate approach using a principal coordinate modeled by a distance-based linear model (DistLM; analysis (PCO). Anderson et al., 2008). DistLM formally fits a linear model of environmental predictor variable(s) to a Biological variables response species data cloud, in the space defined by the chosen resemblance measure. A stepwise forward The exhaustiveness of the biological sampling effort selection was chosen for DistLM first, followed by a was assessed on stygobiotic species through a species BEST selection procedure. P values for testing the null richness estimation, using non-parametric estimators, hypothesis of no relationship were obtained using namely, Chao1 and Chao 2 (Chao, 1984, 1992) and appropriate permutation methods. The selection crite- Jackknife 1 and Jackknife 2 (Burnham & Overton, rion for the best set of predictor variable was R2 that is 1978, 1979; Smith & van Belle, 1984). Values were the proportion of explained variation for the model. estimated by means of 999 randomizations without All statistical analyses but species richness estima- replacement using the software EstimateS 9.1.0 (Col- tion were performed using the software E-PRIMER well, 2013). (version 6.1; Primer-E Ltd., Plymouth, UK). The The differences in copepod assemblages among the significance level a was set at 0.05. No additional three water level periods were assessed by one-way corrections for multiple comparisons were made PERMANOVAs on the base of a Bray–Curtis dissim- according to Anderson et al. (2008). ilarity matrix and under the same design as for the environmental variables. A dummy variable of one was added to all samples to allow the inclusion of Results otherwise empty cells (zero abundance) prior to the Bray–Curtis similarity coefficients computation. Prior Environmental variables to the analyses, the abundance data were log (x ? 1)- transformed. Homogeneities of dispersions were in The hydrochemical properties of groundwater samples turn checked by Levene’s tests. Pairwise post hoc collected at the spring mouth are shown in the Online t tests were applied when appropriate. The significant Resource 2. The mean, max, and min values and the patterns were shown using nMDS (non-metric Multi- standard deviations of the variables measured at the Dimensional Scaling) plots. three water level periods are shown in Table 1. Only Relationships between richness and abundance of four variables reported different values in the three the copepod species and continuous environmental water level periods, namely, temperature, turbidity, variables were examined using univariate permuta- electrical conductivity, and precipitations measured at tional regressions, i.e., regressions in which the Lenola meteorological station (Table 2). Temperature distribution of the test statistics (equation coefficients) was significantly different between the H and R under the null hypothesis was obtained by calculating periods and between the H and L but not between the R all the possible values of the test statistics under and L periods (Table 2). Differences in turbidity were rearrangements of the labels of the raw data. Specif- between the R and L periods. The electrical conduc- ically, the tests of significance of the coefficients of the tivity values in the R period were significantly lower equations (t test) were performed using 999 random than those in the H period. At Lenola gage station, the 123 Hydrobiologia precipitation values recorded in the L period were (9) was collected both in May 2003 and June 2003. In significantly lower than those recorded in the R and H addition, in June 2003, the maximum number of periods (Tables 1, 2). specimens per month (254) was collected too. SB No variable was discarded for the multivariate abundances were higher than nSB abundances in each analyses because none of them was strongly corre- month (Table 3). The most abundant SB species was lated. The PERMANOVA showed that the environ- P. reductum (806 individuals collected over the entire mental variables taken altogether were significantly survey), followed by Stammecaris orcina (Chappuis, different among the three water level periods (pseudo- 1938) (233 ind.) and Acanthocyclops agamus Kiefer,

F2,20 = 1.79, P value = 0.0167, perm = 9892). The 1938 (95 ind.). The most abundant nSB species was difference was significant between the L and H periods Bryocamptus echinatus (Mra´zek, 1893) (102 ind.). (t = 1.52; P value = 0.0188; perm = 6628) and The abundances of the remaining species, both SB and between the R and H periods (t = 1.37; nSB, were B 30 individuals (Table 3). P value = 0.0207; perm = 1708). The pattern was The highest number of specimens was collected also evident in the PCO ordination plot (cumulative during the L period (917 individuals: 796 adults and 85 variance on the first two axes = 42%; Fig. 3). The juveniles), most of them (829) being SB species. main explanatory variables of the PCO plot were 3 out During the R period, 323 individuals (284 adults and of the 4 variables that showed significant difference in 39 juveniles) were collected and only 82 individuals the univariate analyses, namely, electrical conductiv- (72 adults and 10 juveniles) were collected during the ity and precipitations that were higher in the samples H period. P. reductum was collected mainly during the collected in the H period than in those collected in the L period (76%) as well as A. agamus (65%), while S. L and R ones, and temperature that was lower in the orcina was collected with comparable abundances in samples collected in the H period than in the those the R and L periods (50% and 44%, respectively). collected in the L and R ones (Fig. 3). The abundances of SB ? nSB increased linearly, although feebly, along with an increase of temperature Biological variables (P values: intercept = 0.0122, slope = 0.0079) as well as the cumulative abundances of all SB species The copepod assemblages of the Mazzoccolo spring (P values: intercept = 0.0124, slope = 0.0080), and were dominated by stygobiotic (SB) copepods with 10 the abundances of A. agamus (P values: inter- species and 1210 individuals (1094 adults and 116 cept = 0.0316, slope = 0.0217) and P. reductum juveniles), while non-stygobiotic (nSB) copepods (P values: intercept = 0.0112, slope = 0.0071), taken occurred with 3 species and 112 individuals (94 adults singularly. On the contrary, the abundances of and 18 juveniles; Table 3; Online Resource 3). Three SB ? nSB decreased linearly with the increasing of out of 10 SB species were new to Science and endemic chloride (P values: intercept = 0.0001, to the Mazzoccolo spring. One of the collected SB slope = 0.0025), as well as the cumulative abundances species, Pseudectinosoma reductum Galassi & De of all SB species (P values: intercept = 0.0002, Laurentiis, 1997, is considered a Tertiary relict species slope = 0.0026), and the abundances of A. agamus of ancient marine origin according to Galassi et al. (P values: intercept = 0.0008, slope = 0.0091), S. (1999). orcina (P values: intercept = 0.0002, slope = 0.0029) The sampling effort was not entirely exhaustive as and P. reductum (P values: intercept = 0.0014, indicated by non-parametric estimators and by the slope = 0.0109). However, the variances explained steadiness of the uniques with increasing sampling by the models were lower than 30%. effort (Fig. 4). Actual estimates for copepod species The abundances of SB ? nSB copepod species did richness in the spring system were around 13 species not vary with the water level periods (pseudo-

(i.e., over 77% of the estimated number of stygobiotic F2,20 = 2.11, P value = 0.0682, perm = 9961). Sim- copepod species were collected during the sampling ilarly, neither the abundances of nSB copepods survey). At least one species was collected in each (pseudo-F2,20 = 1.39, P value = 0.2504, perm = sampling survey except in January 2005. The maxi- 4741), nor those of SB species (pseudo-F2,20 = 2.37, mum number of species and specimens were collected P value = 0.0516, perm = 9936) varied according to in 2003. The maximum number of species per month the water level periods. On the contrary, the 123 123

Table 1 Mean, maximum, minimum, and standard deviation (SD) values of the environmental variables measured at Mazzoccolo spring from March 2003 to March 2005in three water levels periods, namely, H (high), L (low), and R (residual) - ? - - T (°C) Turbidity pH Cond. Cl Fe NH4 NO3 NO2 Hardness Fixed residue (mg/L P. Vallecorsa P. Lenola (NTU) (lS/cm) (mg/l) (lg/l) (lg/l) (mg/l) (lg/l) (°F) at 180°C) (mm) (mm)

High level (H) Mean 13.3 0.8 7.7 335 11.1 14.8 \ 0.1 1.7 \ 0.1 17.2 205.9 146.1 154.4 SD 0.6 1.2 0.2 30 2.5 20.7 \ 0.1 1.7 \ 0.1 0.9 20.6 54.5 50.2 Maximum 14.0 3.5 7.9 388 13.8 60.0 0.1 4.9 \ 0.1 18.2 230.0 225.0 247.6 Minimum 12.2 0.1 7.5 291 6.2 1.0 \ 0.1 0.3 \ 0.1 16.2 179.0 45.0 91.2 Low level (L) Mean 14.3 0.4 7.7 312 8.3 6.7 \ 0.1 1.2 \ 0.1 17.4 181.9 77.8 67.4 SD 0.5 0.6 0.2 15 2.9 10.4 \ 0.1 0.6 \ 0.1 1.6 46.9 74.5 66.7 Maximum 14.8 1.8 7.9 336 12.1 25.0 0.1 1.8 0.1 20.0 252.0 225.2 199.4 Minimum 13.3 0.0 7.3 295 5.0 0.5 \ 0.1 \0.1 \ 0.1 15.2 84.5 3.4 2.2 Residual level (R) Mean 14.0 2.2 7.8 304 9.2 11.5 \ 0.1 1.1 \ 0.1 17.2 189.7 132.2 99.5 SD 0.5 2.1 0.1 6.1 2.7 17.5 \ 0.1 0.5 \ 0.1 0.4 15.2 81.2 56.6 Maximum 15.0 5.8 7.8 311 12.2 50.0 \ 0.1 1.7 \ 0.1 18.0 212.6 227.4 196.6 Minimum 13.4 0.2 7.6 296 6.1 0.0 \ 0.1 \0.1 \ 0.1 16.8 174.0 25.6 35.0 P. Vallecorsa and P. Lenola indicate the precipitation levels at the two meteorological stations T temperature, Cond. electrical conductivity Hydrobiologia Hydrobiologia

Table 2 One-way Variable pseudo-FPvalue Perms Levene test’s P value PERMANOVAs’ and pairwise t tests’ results for PERMANOVAs each of the environmental T* 7.31 0.0039 7006 0.05 variables measured at Mazzoccolo spring from Turbidity* 3.57 0.0345 9914 0.05 ? March 2003 to March 2005 NH4 0.46 0.6283 3221 0.05 in three water levels pH 0.26 0.7853 4382 0.05 periods, namely, H (high), L (low), and R (residual) Cond.* 4.87 0.0147 9488 0.05 Cl- 2.10 0.1512 9916 0.05 Hardness 0.0312 Fe 0.51 0.6548 9815 0.05 - NO3 0.67 0.5606 9886 0.05 NO2- 0.37 0.9703 994 0.05 Fixed residue 1.07 0.3766 9902 0.05 P. Vallecorsa 2.09 0.1385 9927 0.05 P. Lenola* 4.28 0.0285 9910 0.05 Variable Groups tPvalue Perms

PAIRWISE t tests T H. R* 2.38 0.0256 114 T H. L* 3.70 0.0023 152 T R. L 1.16 0.3572 92 Turbidity H. R 1.48 0.1661 513 Turbidity H. L 0.99 0.4140 691 T temperature, Cond. Turbidity R. L* 2.51 0.0074 2268 electrical conductivity, P. Cond. H. R* 2.66 0.0157 416 precipitation, Perms number of permutations, df Cond. H. L 1.99 0.0581 505 degrees of freedom Cond. R. L 1.24 0.2395 502 * Signiﬁcant. Degrees of P. Lenola H. R 1.91 0.0891 846 freedom were 2, 20. Levene P. Lenola H. L* 2.86 0.0167 2604 test’s results are also showed P. Lenola R. L* 1.02 0.3220 2383

abundances of the three dominant SB species (P. periods (pseudo-F2,20 = 4.55, P value = 0.0136, reductum, S. orcina, and A. agamus) signiﬁcantly perm = 9671), being different between the H and L differed among the three water level periods (pseudo- periods (t = 3.18, P value = 0.0043, perm = 688).

F2,20 = 3.18, P value = 0.0245, perm = 9943), The marginal tests of DistLM highlighted that being different between the H and L periods temperature (P value = 0.0169), chloride (P val- (t = 2.45, P value = 0.0123, perm = 6618). This ues = 0.0055), and nitrates (P value = 0.0341) had significant result is shown in the nMDS plot (Fig. 5). a significant linear relationship with the species- This pattern was observed also considering adults and derived multivariate data cloud. Chloride explained juveniles separately. The number of adults of the three the highest percentage (20%) of the variability in the dominant SB species significantly differed among the data cloud. However, concerning the overall test, an 2 three water level periods (pseudo-F2,20 = 2.88, R = 0.7167 was reached only when combining 9 P value = 0.0354, perm = 9944), being different variables and excluding temperature. When just the between the H and L periods (t = 2.28, three dominant SB species were considered, the P value = 0.0145; perm = 6677). Similarly, the marginal test gave similar results as with the number of juveniles differed among the water level SB ? nSB data cloud (temperature: P value = 123 Hydrobiologia

Fig. 3 Principal Coordinate Analysis (PCO) H ordination of groundwater L samples (symbols) from the R Mazzoccolo spring. H: high 4 hydrological period; L: low hydrogeological period; R: T residual period. The H and L groups of samples were ) signiﬁcantly different n o i according to t IONIZED AMMONIA a i r

PERMANOVA’s results. a TURBIDITY v

Cond. electrical l FIXED RESIDUE a conductivity, P_LENOLA t o t precipitation values at f o Cl Lenola station, P_VALLEC HARDNESS %

9 Fe precipitation values at . -1 5 pH

Vallecorsa station, 1 (

T temperature 2 NITRITES O C P NITRATES P_LENOLA P_VALLEC COND.

-6

-6 -4 -2 0 2 4 6

PCO1 (26% of total variation)

0.0068; chloride: P value = 0.0049; nitrites: around 14°C ± 1°C. These slight fluctuations of the P value = 0.0404). However, the overall test gave a parameters values seemed to be correlated with the R2 = 0.7109 when combining 7 variables, namely, recharge variation. Physical and chemical differences chloride, fixed residue, nitrates, temperature, electrical among the three water level periods were highlighted conductivity, ionized ammonia, and pH. only between the L and H water level periods for temperature, turbidity, electrical conductivity, and precipitation values. Therefore, the ‘‘pulsating’’ Discussion behavior (regular alternation of the H–R–L periods) of the recharge has a moderate effect on the Mazzoc- Groundwater-fed springs are open windows to an colo spring physical and chemical properties. This aquifer and likely the best way to study groundwater situation can likely be due to the pressure transfer, well flow (Manga, 2001). Owing to fast and strong known as ‘‘piston effect,’’ which is very frequent in the reactions of karst systems to variable recharge condi- karst and fractured aquifers (Nanni & Rusi, 2003; tions, karst springs generally result in sudden varia- Aquilina et al., 2006). Following what has just been tions of discharge and physical and chemical supposed, during the fall–winter season (H), the parameters (Katz et al., 1998; Auckenthaler et al., aquifer is rapidly recharged through the karst network, 2002; Vesper & White, 2003). At Mazzoccolo spring, which is well developed in the Aurunci aquifer (Sappa the physical and chemical monitoring highlighted only et al., 2015a), and the piezometric level increases, a slight variation in hydrochemistry over the whole especially in the recharge area. This hydraulic head survey. The electrical conductivity varied around increase in the aquifer corresponds to an increase of 316 lS/cm ± 22 lS/cm. The temperature varied the Mazzoccolo spring discharge. Then, during the 123 Hydrobiologia Table 3 Number and abundances of species collected at Mazzoccolo spring from March 2003 to March 2005 in high water level (H), low water level (L), and residual water level (R) periods Category Mar_03 Apr_03 May_03 Jun_03 Ago_03 Sep_03 Oct_03 Nov_03

Water level R R R L L L L H Acanthocyclops agamus (Kiefer, 1938) SB 0 4 11 25 8 11 7 8 Bryocamptus dentatus (Chappuis, 1937) SB 0 0 1 0 0 0 0 0 Bryocamptus echinatus (Mra´zek, 1893) nSB 1 10 7 47 0 7 7 0 Diacyclops bisetosus (Rehberg, 1880) nSB 0 0 0 9 0 0 0 0 Elaphoidella elaphoides (Chappuis, 1924) SB 0 1 1 10 2 4 1 1 Elaphoidella sp. SB 0 1 5 12 1 1 1 0 Moraria varica (Graeter, 1911) nSB 0 1 0 0 0 0 0 0 Nitocrella kunzi (Galassi & De Laurentiis, 1997) SB 0 0 2 6 1 0 0 0 Stammericaris orcina (Chappuis, 1938) SB 15 57 37 28 29 6 14 6 Parastenocaris sp. SB 0 0 0 0 0 0 0 1 Phyllognathopus inexspectatus (Galassi & De Laurentiis, 2011) SB 0 0 1 0 0 0 0 0 Pseudectinosoma reductum (Galassi & De Laurentiis, 1997) SB 2 49 68 110 204 158 68 8 Speocyclops italicus (Kiefer, 1938) SB 0 0 0 7 0 0 0 0 Abundances (SB ? nSB) 18 123 133 254 245 187 98 24 Abundances (SB) 17 112 126 198 245 180 91 24 Abundances (nSB) 1 11 7 56 0 7 7 0 N. of species (SB ? nSB) 3 7 9 9 6 6 6 5 N. of species (SB) 2 5 8 7 6 5 5 5 N. of species (nSB) 1 2 1 2 0 1 1 0 Category Jan_04 Feb_04 Mar_04 Apr_04 May_04 Jun_04 Jul_04 Ago_04

Water level H H R R R L L L Acanthocyclops agamus (Kiefer, 1938) SB 2 0 0 4 3 0 3 3 Bryocamptus dentatus (Chappuis, 1937) SB 0 0 0 0 0 0 0 0 Bryocamptus echinatus (Mra´zek, 1893) nSB 1 1 1 0 0 0 0 2 Diacyclops bisetosus (Rehberg, 1880) nSB 0 0 0 0 0 0 0 0 Elaphoidella elaphoides (Chappuis, 1924) SB 3 0 0 0 0 0 0 0 Elaphoidella sp. SB 0 0 0 0 0 0 0 5 Moraria varica (Graeter, 1911) nSB 0 0 0 0 0 0 0 0 Nitocrella kunzi (Galassi & De Laurentiis, 1997) SB 0 0 0 0 0 0 0 0 Stammericaris orcina (Chappuis, 1938) SB 7 0 2 2 3 0 4 7 Parastenocaris sp. SB 0 0 0 0 0 0 0 0 123 Phyllognathopus inexspectatus (Galassi & De Laurentiis, 2011) SB 0 0 0 0 0 0 0 0 Pseudectinosoma reductum (Galassi & De Laurentiis, 1997) SB 12 15 13 2 10 6 5 9 Speocyclops italicus (Kiefer, 1938) SB 0 0 0 0 0 0 0 0 123 Table 3 continued Category Jan_04 Feb_04 Mar_04 Apr_04 May_04 Jun_04 Jul_04 Ago_04

Abundances (SB ? nSB) 25 16 16 8 16 6 12 26 Abundances (SB) 24 15 15 8 16 6 12 24 Abundances (nSB) 1 1 1 0 0 0 0 2 N. of species (SB ? nSB) 5 2 3 3 3 1 3 5 N. of species (SB) 4 1 2 3 3 1 3 4 N. of species (nSB) 1 1 1 0 0 0 0 1

Category Sep_04 Oct_04 Nov_04 Dec_04 Jan_05 Feb_05 Mar_05 Abundances Abundances Abundances Abundances (H) (L) (R) (H ? L?R)

Water level L L H H H H R Acanthocyclops agamus (Kiefer, SB140100011622295 1938) Bryocamptus dentatus (Chappuis, SB00000000011 1937) Bryocamptus echinatus (Mra´zek, nSB 1 15 2 0 0 0 0 4 79 19 102 1893) Diacyclops bisetosus (Rehberg, 1880) nSB 0 0 0 0 0 0 0 0 9 0 9 Elaphoidella elaphoides (Chappuis, SB 0 0 0 0 0 0 0 4 17 2 23 1924) Elaphoidella sp. SB 1 1 2 0 0 0 0 2 22 6 30 Moraria varica (Graeter, 1911) nSB 0 0 0 0 0 0 0 0 0 1 1 Nitocrella kunzi (Galassi & De SB 0 1 0 0 0 0 0 0 8 2 10 Laurentiis, 1997) Stammericaris orcina (Chappuis, SB 3 11 2 0 0 0 0 15 102 116 233 1938) Parastenocaris sp. SB 0 0 0 0 0 0 0 1 0 0 1 Phyllognathopus inexspectatus SB00000000011 (Galassi & De Laurentiis, 2011) Pseudectinosoma reductum (Galassi & SB 25 26 2 3 0 2 9 42 611 153 806 De Laurentiis, 1997) Speocyclops italicus (Kiefer, 1938) SB 0 0 2 1 0 0 0 3 7 0 10 Abundances (SB ? nSB) 31 58 10 5 0 2 9 82 917 323 1322 Abundances (SB) 30 43 8 5 0 2 9 78 829 303 1210 Abundances (nSB) 1 15 2 0 0 0 0 4 88 20 112 N. of species (SB ? nSB) 5 6 5 3 0 1 1 8 9 10 Hydrobiologia N. of species (SB) 4 5 4 3 0 1 1 7 7 8 N. of species (nSB) 1 1 1 0 0 0 0 1 2 2 SB stygobiotic, nSB non-stygobiotic Hydrobiologia

18 S(est) Uniques Mean spring from the underlying aquifer (stygobionts; 16 Chao 1 Mean Galassi et al., 2014; Fattorini et al., 2017). The 14 Chao 2 Mean Jack 1 Mean biological assemblages of captured springs are much 12 Jack 2 Mean less complex. Captured springs lack crenobiotic 10 8 species due to hygiene infrastructures that isolate the 6 outlet from the surface. These springs are in total 4 darkness, thus not allowing primary productivity

Number of species 2 which several crenobionts depend on. Moreover, the 0 0 5 10 15 20 25 epigean taxa collected at the outlet of a captured spring Number of samples come from the recharge area of the aquifer feeding the spring. Accordingly, the biological assemblages of the Fig. 4 Species rarefaction curves and estimators curves for Mazzoccolo spring reflected the composition and the copepods in the Mazzoccolo spring at increasing sample size. dynamics of the species dwelling in the Western S(est): species rarefaction curve of observed species richness Aurunci aquifer (stygobionts) and of the few non- (mean values estimated by mean of 999 randomizations without replacement), Uniques: curve of the mean number of species stygobiotic species that temporarily colonize it (Fiasca present in a single site. Chao1, 2 and Jack (Jackknife) 1, 2: et al., 2014; Galassi et al. 2014; Stoch et al., 2016; curves of estimated species richness using mean values of the Fattorini et al., 2017). non-parametric estimators Copepods are common components of the groundwater fauna, and greatly increase the diversity of spring season, the pressure and the spring discharge karst communities (Galassi et al., 2009b, 2014). decreases (R), reaching the lowest values during the Numerous copepod taxa (at different hierarchical summer period (L). The groundwater emerging from levels, from species to orders) known exclusively the spring is not only the water which had infiltrated in from groundwater are both phylogenetic and distri- the previous wet season (in this case, the physical and butional relicts occurring in restricted geographical chemical parameters should have shown a stronger areas and sometimes showing wide disjunct distri- fluctuation around the mean values), but a mixture of butions (Michaux, 1989; Stock, 1993; Galassi, 2001). just infiltrated water and older groundwater, as An outstanding degree of endemism occurs for observed in other karst aquifers (Liu et al., 2016). groundwater copepod species (Galassi, 2001). The The electrical conductivity variation, albeit slight, is number of endemic species is likely far underesti- due to the residence time of groundwater in the mated due to cryptic biodiversity (e.g., Galassi, limestone aquifer, which is mainly related to the - 2? 2001; Stoch, 2001; Reid, 2004). Copepods domi- bicarbonate (HCO3 ) and calcium (Ca ) contents nated in the Western Aurunci aquifer with a number coming from calcite dissolution. For this reason, the of species comparable with those collected in other groundwater feeding the Mazzoccolo spring does not - - - ? karst massifs (Mori & Brancelj, 2006, 2013; Galassi present variation in Cl ,NO3 ,NO2 ,NH4 , and et al., 2009a, 2014; Stoch et al., 2016; Iepure et al., 2? Fe content, although the electrical conductivity 2017). The non-stygobiotic species were poorly changes. The ‘‘piston effect’’ also justifies the higher represented. The occurrences of Diacyclops bisetosus value of the turbidity during the R period when the (Rehberg, 1880) and Moraria varica (Graeter, 1911) water that flushes the fine particles of the aquifer were occasional because both species were collected reaches the spring. only once, in June 2003 and April 2003, respectively. Springs are also natural laboratories for the study of Bryocamptus echinatus occurred more frequently the ecological processes occurring in an aquifer (Mori thus indicating its higher affinity for groundwater & Brancelj, 2013; Galassi et al., 2014; Stoch et al., habitats and a more stable residence in the Western 2016). Natural springs host a variety of species which Aurunci aquifer. include taxa that dwell exclusively in the spring mouth The collection of the stygobiotic taxa at the (crenobionts; Cantonati et al., 2011, 2012; Spitale Mazzoccolo spring is relevant because of the high et al., 2012), generalist species that colonize the spring number of narrow endemics (3 out of 10) with a from the surface water (epigean taxa; Hahn, 2000; particular reference to Phyllognathopus inexspectatus Bottazzi et al., 2011), and others that colonize the Galassi & De Laurentiis, 2011, closely related to the 123 Hydrobiologia

Fig. 5 Upper panel: nMDS HYDRO_CYCLE H of Bray–Curtis similarities R from log (x ? 1) L transformed abundances of Acanthocyclops agamus, Stammericaris orcina, and Pseudectinosoma reductum at the 23 samples taken at Mazzoccolo spring mouth. The other panels show superimposed circles to the nMDS plot. Circles represent species abundances. Circles’ diameter increases with increasing abundances from Pseudectinosoma reductum Table 2. 2 D Stress = 0.07 30

120

210

300

Stammericaris orcina

Acanthocyclops agamus

123 Hydrobiologia

Gondwanian species of the genus (Galassi et al., Mazzoccolo spring determining the transmissive 2011). Another striking aspect is the assemblage function of the aquifer and several slow-flowing composed of the stygobiotic species P. reductum, A. conduits, situated on each side of the drains, providing agamus, and S. orcina, which were the three most a storage function (Mangin, 1974a, b; Galassi et al., abundant copepod species collected at the Mazzoc- 2014). During the high water level period, rainfalls colo spring. Pseudectinosoma is an ancient and poorly recharge the aquifer pushing groundwater into the diversified harpacticoid genus (6 species, 5 of which saturated karst thus producing the ‘‘piston effect.’’ are stygobiotic) of direct marine origin (Galassi et al., Basically, groundwater is slowly pushed into the less 1999), with only an extant relative living in marine permeable part of the saturated zone (that is into the brackish water. With the exception of the brackish- slow-flowing conduits), while rapidly flowing in the water species P. minor, the other species of Pseudecti- fast conductive drains. During this period, the ground- nosoma show ‘spot’ distributions, endemic to circum- water that had been pushed into the slow-flowing Mediterranean stygohabitats (Galassi et al., 1999) and conduits of the Aurunci aquifer entrapped the species with one stygobiotic species from Australia (Kara- that resided there (Fig. 6a). Accordingly, the number novic, 2006). The occurrence of this genus in Australia of individuals of P. reductum, A. agamus, and S. reinforces the hypothesis of a full Tethyan track of orcina were lower in the high water level period than distribution. In particular, P. reductum, which is in the other two. During the high water level period presently known from central Italy in areas covered only few individuals of P. reductum, S. orcina, and A. by the sea until the late Miocene, might have agamus were collected at the Mazzoccolo spring originated during the Messinian regression of the because most of them were likely trapped into the Mediterranean Sea in the Italian peninsula (Galassi capacitive conduits of the aquifer (Fig. 6a). Similar to et al., 1999). The species P. reductum is known only our results, Malard et al. (1994) observed low numbers from three karst springs in central Italy, namely, of invertebrates in a French karst aquifer during the Presciano spring, Gizio spring, and Mazzoccolo high water level period. spring. P. reductum was found associated with A. When the recharge started decreasing, the ‘‘piston agamus in both the Gizio and Mazzoccolo karst effect’’ began weakening. This is called the residual springs. The cyclopoid A. agamus was originally period. The groundwater that had been previously described by Kiefer (1938) from one male from a pushed into the slow-flowing conduits started being siphon lake of the Castelcivita cave (southern Italy) drained back to the fast-flowing drains, as observed in where it is associated with Pseudectinosoma kunzi other systems (Drogue, 1980; Malard, 2001), bringing (Galassi et al., 1999). Although the cyclopoid copepod behind the fine sediments responsible for the increase A. agamus has a freshwater origin it can also be in the turbidity and transporting some of the copepod considered a relatively ancient colonizer of karst individuals residing in the capacitive system. Accord- systems. The colonization pathway of A. agamus is ingly, the abundances of P. reductum, S. orcina, and A. difficult to reconstruct, mainly because this species agamus were higher in the residual period than in the shows an enigmatic disjunct distribution in three high water level one. However, this pattern was different karst aquifers in central and southern Italy, dominated by S. orcina that was collected with the which have been separated since the Tertiary (Galassi highest abundances. During the low water period, the & De Laurentiis, 2004). In addition, it has been piston effect definitely vanished and the slow-flowing recently recorded also from groundwater habitats in conduits were completely drained, as observed in the southern end of the Alpine arch in France (Dole- other karst aquifers in France (Malard, 2001), trans- Olivier et al., 2015). S. orcina was found frequently porting a consistent number of copepod individuals associated with Pseudectinosoma species along with from the innermost sectors of the aquifer, being P. A. agamus as in the saturated karst of the Castelcivita reductum the most abundant, followed by S. orcina cave in the Alburni Massif (Galassi, 2001) and in the and finally A. agamus. Mazzoccolo spring. According to the observed pattern of occurrence As observed in other karst aquifers, the karst over 2 years, the three dominant species of the network of Western Aurunci aquifer consists of fast- Aurunci aquifer seem to reside in the slow-flowing flowing drains which drain groundwater to the conduits. It is possible that they inhabit different 123 Hydrobiologia

Fig. 6 Diagram showing the fast-ﬂowing drain and the slow-ﬂowing conduits with the micro-habitats where Acanthocyclops agamus, Pseudectinosoma reductum, and Stammericaris orcina reside, during a high water level, b residual period, and c low water level in the Western Aurunci karst aquifer feeding the Mazzoccolo spring. The diagram represents a schematic vertical section of a sector of the aquifer (not in scale)

sectors of these conduits. Our hypothesis is that S. the involvement of micro-habitats where fine sedi- orcina likely inhabits the few unconsolidated sediments were accumulated, is consistent with the ments that accumulate in the slow-flowing conduits ecology of this species. P. reductum and A. agamus that are nearest the fast-flowing drains, which is in likely reside in the slow-flowing conduits that are those habitats that are drained first at the beginning of farther from the drains (Fig. 6b). P. reductum inhabits the residual period (Fig. 6b). S. orcina is rather a the micro-fractures and A. agamus lives in that part of burrower than a swimmer. It is known to live in sandy the saturated aquifer that is characterized by vuggy sediments of the small dripping pools of Castelcivita porosity (i.e., secondary porosity generated by disso- Cave and Pertosa Cave (southern Italy) and it was lution of features in carbonate rocks leaving large never collected from the saturated karst in these caves holes, vugs; Ford & Williams, 1989). Vuggy porosity (Galassi, 2001). It has been found also in the consists in lenticular round-shaped voids (vugs), interstitial habitat of the Bracciano Lake (central ranging from few millimeters to 2 cm in size. Vugs Italy; Cottarelli & Drigo, 1972). The increased are only partly connected with the slow-flowing turbidity during the residual period, which indicated conduits (Ford & Williams, 1989). A. agamus could

123 Hydrobiologia reside in these habitats from which groundwater is kind of food, such as live preys, in groundwater feebly mobilized (Fig. 6c). These considerations communities. This explains in part the success of about the micro-habitats should be taken as specula- scavengers and detritivores over predators in the tive and further tested. groundwater systems. However, further studies are The biotic and environmental variables that were maybe necessary to completely discard the hypothesis measured at the Mazzoccolo spring were faintly cor- that biotic factors have a role in explaining part of the related. DistLM analysis proved that temperature, observed pattern of occurrences. chloride, and nitrates, taken singularly, were the major discriminants of the occurrence pattern of the three dominant species at the Mazzoccolo spring. However, Conclusions altogether they did not explain more than 23% of the variance of the multivariate biological data. Accord- Continuous groundwater drift monitoring by perma- ingly, the univariate linear models showed that nent nets at captured karst springs is a unique temperature and chloride were feebly correlated opportunity to study the spatial distribution of the (\ 30%) to the occurrences of P. reductum, S. orcina, groundwater species inhabiting physically inaccessi- and A. agamus, taken singularly. Chloride together ble karst aquifers. The Mazzoccolo spring copepod with sulfate and bicarbonate ? carbonate is the major drift showed clear differences in number of individ- ion that is contained in surface water and groundwater, uals between the high and low water periods because originating from the water–rock interaction. The of the well-known ‘‘piston effect,’’ which is very origin of chloride in the Mazzoccolo spring is to be frequent in karst and fractured aquifers due to recharge attributed to natural origin. These results highlighted from rainfalls. The lowest abundances of the three that the patterns of occurrence of these three species dominant copepod species occurred during the high were not, or only slightly, related to the variations in water level when groundwater is slowly pushed into hydrochemistry during the three water level periods at the slow-flowing conduits while rapidly flowing in the Mazzoccolo spring. fast conductive drains. Conversely, the highest abun- In this study, biotic factors such as population dances were collected during the low water period, strategies, reproduction time, competition, and the when the ‘‘piston effect’’ has stopped, and the slow- presence of predators have not been considered as flowing conduits were completely drained, transport- factors shaping the observed patterns of occurrence of ing a consistent number of copepod individuals from the three dominant species at the outlet of the Aurunci the innermost sectors of the aquifer. According to the karst aquifer. Differently from their epigean relatives, results of this study, groundwater species seem not to stygobiotic copepods reproduce continuously over a inhabit all sectors of a karst aquifer. They avoid the year and live much longer (Galassi, 2001). In respect fast-flowing conduits while colonizing more inertial to surface habitats, the variety of resources is reduced aquifer sectors, such as the slow-flowing conduits. in groundwater, the major energy input is external to the system, with photosynthesis missing and Acknowledgements We are indebted to Acqualatina S.p.A chemoautotrophy quantitatively unimportant (Culver, that kindly provided the physical and chemical data. The project was partially funded by the European Community (LIFE12 1994; Galassi et al., 2017). The impoverishment and BIO/IT/000231 AQUALIFE). We thank Roberta Polimanti and monotony of available food resources, coupled with Emidio Di Lorenzo for the language revision and two the aphotic nature of the groundwater habitats, have anonymous reviewers who provided suggestions to improve resulted in a strong selection for omnivory, a broad- the manuscript. ening of diet, and a reducing of food competition (Culver, 1994). The high morphological variation in References mouthparts among closely related species found in the same site (as syntopic) in a rheo-limnocrene spring has Anderson, M. J., 2001. A new method for non-parametric suggested that trophic diversification may be impor- multivariate analysis of variance. Austral Ecology 26: tant to avoid competition among groundwater cope- 32–46. Anderson, M. J., R. N. Gorley & K. R. Clarke, 2008. PER- pods (Galassi, 2001). The broadening of diet have MANOVA? for Primer: guide to software and statistical resulted in reduced efficiency in obtaining a particular methods. PRIMER-E, Plymouth. 123 Hydrobiologia

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