The distribution of benthic foraminifera in Bel Torrente submarine cave (Sardinia, Italy) and their environmental significance

Accepted Manuscript The distribution of benthic foraminifera in Bel Torrente submarine cave (Sardinia, Italy) and their environmental significance Elena Romano, Luisa Bergamin, Giancarlo Pierfranceschi, Claudio Provenzani, Andrea Marassich PII:

S0141-1136(17)30474-9

DOI:

10.1016/j.marenvres.2017.12.014

Reference:

MERE 4424

To appear in:

Marine Environmental Research

Received Date: 4 August 2017 Revised Date:

14 December 2017

Accepted Date: 17 December 2017

Please cite this article as: Romano, E., Bergamin, L., Pierfranceschi, G., Provenzani, C., Marassich, A., The distribution of benthic foraminifera in Bel Torrente submarine cave (Sardinia, Italy) and their environmental significance, Marine Environmental Research (2018), doi: 10.1016/ j.marenvres.2017.12.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

The distribution of benthicACCEPTED foraminiferaMANUSCRIPT in Bel Torrente submarine cave (Sardinia, Italy) and their environmental significance Elena Romano1,2, Luisa Bergamin1, Giancarlo Pierfranceschi1, Claudio Provenzani2, Andrea Marassich2 1 2

ISPRA. Institute for Environmental Protection and Research, Via V. Brancati 60 - 00144 Rome, Italy Global Underwater Explorers. 15 South Main Street - High Springs, Florida (USA)

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* Corresponding author: [email protected]

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Abstract The use of benthic foraminifera as ecological indicators in submarine caves of temperate seas have never been studied before and it represents a new approach, verified by this research. The Bel Torrente submarine cave (Gulf of Orosei, Sardinia, Italy) was surveyed by GUE (Global Underwater Explorers) scuba divers in order to georeferencing the cave before positioning the sampling stations, from the entrance to 430 m inside the cave. A total of 15 water samples were collected to investigate abiotic parameters (temperature, salinity, pH) while 15 sediment samples were collected to analyze grain size and benthic foraminifera. Benthic foraminifera, investigated for the first time in a submarine cave of temperate areas, were exclusively found from the entrance to 300 m inside the cave. Species distribution and assemblage diversity have been found to be correlated to the environmental gradient towards the inner cave, mainly due to the decreasing of temperature and salinity and the increasing of the flow energy. Water acidification seems responsible for the transition from a calcareous hyaline-dominated assemblage to an agglutinant-dominated one, occurring between 120 and 150 m from the entrance. Common taxa of the Sardinian coastal marine area are present only close to the entrance of the cave, while species found in the inner part are nearly exclusively epifaunal clinging/attached or infaunal taxa, with tolerance for wide variability of environmental parameters, such as Gavelinopsis praegeri, and opportunist infaunal taxa such as Eggerella advena. The agglutinant taxa found in the cave are conversely very rare in coastal marine assemblages of the area. This suggests a very efficient dispersal mechanism for the colonization of the caves, involving probably juvenile foraminifera at a “propagule” stage.

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Keywords: Submarine cave, Benthic foraminifera; Ecological characterization; Gulf of Orosei; Sardinia.

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1. Introduction Karst systems located on the coastal zone are characterized by the presence of several cave passages, generally due to the dissolution of limestone along structural weaknesses, occurred during Quaternary, when climatic variability determined sea-level changes. Caves may be classified as vadose, littoral, anchialine or submarine, depending on their morphology and groundwater type that, in the coastal karst area, is constituted by lower saline groundwater overlaid by lens of meteoric waters; the mixing zone between these two water masses is generally known as halocline (van Hengstum and Scott, 2011). In particular, the anchialine cave is defined by Stock et al. (1986) as “body of hyaline waters, usually with a restricted exposure to open air, always with more or less extensive subterranean connection to the sea, and showing noticeable marine as well as terrestrial influences”. Submarine caves are marine-dominated environments as regards hydrogeological and sedimentological processes while, from a biological viewpoint, they are oligotrophic systems depending on the energy input from the surrounding marine productive coastal area (Fichez, 1990). The study of marine cave communities in the Mediterranean Sea started in the middle of the last century (Pérès and Picard, 1949), primarily focusing on the benthic communities of hard substrates such as sponges, cnidarians, bryozoans, serpulids and ascidians, while very little effort has been devoted to the study of soft-bottom communities, mainly addressed to crustaceans (Bakran-Petricioli and Krŭzić, 2002; Navarro-Barranco et al., 2012). Most species found in the submarine caves are not exclusive of this environment, although a rich endemic fauna may be

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present (Gerovasileiou and Voultsiadou, 2012). A common feature in these studies is a decrease in species ACCEPTED MANUSCRIPT richness and biomass of benthic organisms from the outermost to the innermost part of the cave, depending on the physical gradients inside the cave (light, oxygen, salinity, etc.), the trophic supply gradient and the limited capacity of the larvae for dispersion and settlement (Navarro-Barranco et al., 2012). Benthic foraminifera are a group of eukaryotic protozoa which is very abundant in the sediments; they live in most marine and transitional environments and show a clear ecological zonation with respect to environmental parameters such as salinity, oxygen and nutrient load. Moreover, they respond rapidly to environmental changes due to their life-cycle of some weeks-months. Most species have a hard shell called “test” that can be agglutinated (built with sediment collected on site), calcareous perforate (hyaline), or calcareous imperforate (porcelaneous), which may be preserved in the sedimentary record. For these features, in the last decades they have been increasingly used for environmental assessment and monitoring, to detect both natural and anthropogenically-induced environmental changes. Since they are generally abundant in transitional environments, they are supposed to be an ideal tool to identify distinct environments in submarine caves, where the variable contribution of marine and continental waters determines wide seasonal changes of the environmental parameters. Nevertheless, few researches are focused on this habitat and benthic foraminifera were exclusively studied in some tropical karst systems (Bermuda and Yucatan peninsula), where they were used both as paleoenvironmental indicators of sea level changes (van Hengstum et al., 2009; 2011) and environmental tools for ecological zonation (van Hengstum et al., 2008; van Hengstum and Scott, 2011). In particular, Van Hengstum et al. (2008) recognized 3 distinct assemblages in the Mexican cenotes (collapse sinkholes) in relation to salinity changes. Van Hengstum and Scott (2011) identified different foraminiferal assemblages in anchialine (prevalent continental influence) and submarine (prevalent marine influence) cave environments. The last one was divided into three ecozones: entrance, with the typical lagoonal assemblage like as outside the cave; circulated cave, with high diversity assemblage, testifying unstressed conditions; and isolated cave, with a low diversity assemblage to indicate environmental stress. The use of foraminifera as ecological indicators in submarine caves of temperate seas, such as Mediterranean Sea, is a new challenge that is verified by this research, because the presence of foraminifera and their reliability as environmental tools in these habitats have never been studied before. In particular, the present study aims to characterize for the first time the foraminiferal fauna of a submarine Mediterranean cave and to check the possibility of recognizing different cave environments, starting from the marine entrance and proceeding towards the inner part, on the base of faunal changes of benthic foraminifera and the variability of abiotic parameters such as pH, salinity, Dissolved Oxygen (DO), temperature and grain size. This study recognizes changes of species distribution, assemblage composition and structure as ecological response to the environmental gradients, associated with the increasing distance from the cave entrance. The distribution of foraminiferal species can define specific cave environments, taking into account the changes of environmental parameters. This research may be also considered as baseline for future sea-level monitoring of the Mediterranean Sea, because the distribution of ecological zones is regulated by mutual relationship between saline and fresh water masses. Finally, knowledge on the application of benthic foraminifera as indicators of environmental stress may benefit from studies in extreme environments such as marine caves, where they respond to the wide temporal and spatial variation of environmental parameters. This study could supply indications on the effects of environmental changes forced by anthropogenic impact in marine environment. 2. Geological and hydrological setting Bel Torrente cave is located in the Gulf of Orosei (NE Sardinia, Italy), a typical fluvial-karst landscape with crystalline Paleozoic basement, constituted by granites and metamorphic rocks, and covered by a Mesozoic sedimentary sequence (conglomerates, dolostones and limestones) (Fig. 1). The carbonate sequence has been subjected to intense karst activity and it is testified by dolines and other karst landforms; many sinkholes probably converge into a unique hypogean karst system that has its outflow most probably in the submarine spring of Bel Torrente cave (De Waele, 2002). On the coastal site, the combination of karst and littoral processes enhanced the erosion of the rocks where the mixing between fresh and salt water occurred. This

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process promoted the opening of many sea caves, especially in correspondence of joints and structurally ACCEPTED MANUSCRIPT weaker areas and, matching with fresh water outlets, determined the enlargement of important coastal karst caves that develop for kilometers (De Waele, 2005; Sanna and De Waele, 2010). Along the Gulf of Orosei several extensive cave systems have developed like as Bue Marino, Cala Luna spring, Bel Torrente spring, Fico and Utopia. Submarine caves of the Gulf of Orosei are characterized by the mixing of freshwater and marine water. In this environment waters are strictly confined and different ecological niches may be located very close (De Waele and Forti, 2003). At present, surface drainage is activated only after heavy rain periods and almost exclusively interests the canyons Codula Ilune and Codula Sisine; the first one has more than 60% of its drainage basin extended upon granites, while the second one drains part of the basaltic San Pietro plain (De Waele, 2005). Water flows in through various sinkholes in the western part of the area and flows out of the system through several submarine resurgences, among which Bel Torrente is the most important. This cave was discovered in the 1970s by speleologist J. Hasenmayer and it is one of the biggest cave systems of Sardinia. The survey started in the 1990s and about 5 km of tunnels have been surveyed, explored and mapped. The cave is generally characterized by a 5-20 m wide tunnel with an average height of 5 m and a depth down to 12 m; it is divided in a huge dry phreatic passage and a submerged active branch at 700 m from the entrance, both well-decorated above sea level with several speleothems found at up to 10 m of water depth (De Waele, 2004; De Waele et al., 2009a). The Bel Torrente spring, which drains waters from the karst canyon of Codula Sisine and from Lovettecannas, Su Canale and Su Clovu caves, has a flow rate ranging from 100 to 1000 l s-1 during overflow periods. For this reason in the first section of the cave the bottom is generally covered by powerful sandy and gravelly deposits with evident sediment structures (ripples) indicating high energy flow, while in the inner part, bottom and walls are characterized by big rocky boulders with scallops large 8-10 cm (Fancello et al., 2000; De Waele et al., 2009b). During dry conditions the water discharge is scarce (10 l s-1) and from the cave entrance up to 200-400 meters away there is the halocline at 1-2 m water depth, while after heavy rains the tunnel is entirely flooded by freshwater with flood velocities up to 2 m s-1.

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3. Material and methods

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3.1. Survey and sampling On August 2014, the cave was resurveyed by GUE (Global Underwater Explorers) cave divers to have a correct positioning of the sample stations for this study. Divers positioned a permanent line from the entrance to the inner part of the cave. This line was used as reference for taking measures (length, direction and depth) and mapping of the cave. The starting point of the line was marked with GPS position. 15 cookies were positioned on the mainline, at 30 m distance one from another, marking the station numbers (Fig. 2). On each station GUE divers registered water temperature and depth by gauge (no name product, accuracy ± 1°C) and manually collected water and sediment samples for physical and faunal analyses. In particular, water sample was stored in plastic containers, while the upper 2 cm layer of the cave bottom sediment was stored in two different containers: two aliquots of 50 and 100 cm3 were respectively collected for grain size and foraminiferal analyses. Water samples, collected close to the sediment/water interface, were measured for temperature, salinity, pH and DO by means of a multiparametric probe immediately after the GUE divers were back on the boat. Collected sediment samples for faunal analysis were stained with rose Bengal/ethanol solution (2 g l-1) for the identification of living specimens at the sampling time. The study of foraminifera required a larger amount of sediment than the 50 cm3 recommended by the FOBIMO protocol (Schönfeld et al., 2012), due to the low foraminiferal density found in the cave sediments during a preliminary survey. 3.2. Grain size analysis Samples were pre-treated with a solution of hydrogen peroxide (30%) and distilled water (1:3) to remove salts and organic matter. Then, they were wet-separated into two fractions using a sieve with 63 µm mesh. The coarse fraction (> 63 µm) was sieved using ASTM series sieves, with meshes ranging from -1 to + 4 φ, while the fine fraction was not analyzed because was less abundant than 5%. The textural data were used

to identify the main grain size classes and determine the type of sediment, according to the ternary ACCEPTED MANUSCRIPT classification by Shepard (1954), modified for the inclusion of gravel. The coarse fraction was also observed under stereo microscope in order to recognize the main components of the biotic and abiotic fractions useful to recognize sedimentary sources (Romano et al., 2009).

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3.3. Benthic foraminifera Samples were washed over 63 µm sieve to eliminate staining solution and mud particles and then oven dried at 40°C. The quantitative analysis carried out on dry samples was based on the count of all specimens present in the sample or in representative aliquots containing at least 300 specimens. The living (rose Bengal stained) and dead specimens were counted separately. However, the low foraminiferal density recorded in most samples did not allow to recover a number of specimens sufficient to consider separately data from the two assemblages in order to apply statistical analysis; consequently, the sum of living + dead specimens was used to create a matrix for statistical purposes. In order to prevent the inclusion of reworked or transported specimens, only well-preserved tests, without breakages or abrasion signs, were picked, counted and classified. Moreover, according to van Hengstum and Scott (2011), one species was considered as autochthonous in one sample and included in multivariate analysis only when at least one stained specimen of that species was found. Microfaunal analysis was conducted under a stereomicroscope Leica M165C and pictures were taken by means of a Leica IC80HD camera. The classification at the genus level was made according to Loeblich and Tappan (1987), while species were determined according to some important studies on the Mediterranean area (Jorissen, 1988; Cimerman and Langer, 1991; Sgarrella and Moncharmont-Zei, 1993) and to World Modern Foraminifera Database (Hayward et al., 2011). The foraminiferal density (FN) was calculated as the number of specimens per gram of dry sediment (Schott, 1935). The species diversity, given by α-index (Fisher et al., 1943; Murray, 1991), Shannon H-index (H) (Shannon, 1948) and dominance (D) were calculated by using the statistical package PAlaeontological Statistics-PAST (Hammer et al., 2001; Hammer and Harper, 2006).

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3.4. Statistical analysis The two-way (Q-mode and R-mode) Hierarchical Cluster Analysis (HCA) was applied on a matrix with relative abundance of commonly occurring species (i.e. >5% in at least one sample), including only samples with more than 50 specimens (Barras et al., 2014). HCA used the Euclidean distance coefficient to compare samples and Ward’s method of minimum variance to assemble clusters (van Hengstum et al., 2008; 2011). This technique was applied to identify groups of samples with similar faunal content and to recognize their characterizing species (Parker and Arnold, 1999). Canonical Correspondence Analysis (CCA) was carried out on the matrix with species relative abundance, considering grain size, salinity, temperature, pH and distance from the cave entrance as environmental parameters. It was used to highlight the effects of the environmental gradient from marine to continental conditions inside the cave on species abundance (variables). CCA is a direct gradient analysis, where the gradient in environmental variables is known a priori and the species abundances are considered as a response to this gradient. CCA is considered as the best tool for ecological studies, in which matrices containing species abundance are processed to uncover factors determining community structure (Palmer, 1993). Statistical analysis was carried out by means of PAST software. 4. Results 4.1. Description of the cave Bel Torrente cave starts with a cavern area at sea level with the bottom at about 3 meters and still with an open water area (Fig. 2). The bottom of the cavern is about 4 meters shallower than the seabed and the entry area has a width of about 8 meters. As soon as the proper overhead starts, the diameter slightly reduces to about 5 m and there is a massive column in the middle that defines two distinct passages and, after that, the first halocline area at a depth of about 3-4 m is present. Here the cave gets again slightly larger and starts

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heading into the Supramonte mountains. Only 15-20 m after this point there is the first big chamber, 20 m ACCEPTED MANUSCRIPT wide and 30-40 m long, with an ample breathable air space decorated with speleothems on the top. On exiting the chamber, in a length of about 15 meters, there is a smaller section where it is still possible to surface in an air space but abruptly turns west and, following a sand slope in a relatively high but completely flooded passage, it is possible to drop into a corridor about 6-9 m wide. This wide corridor keeps going till a distance of 450 m from the entrance, more or less with the same dimensions (8 m width, 5 m height), and a sandy bottom with fallen speleothems and rocky boulders with scallops. About 400 meters into the cave there is two side passages, one per side. To the left, the passage is relatively big (5 m width, 3 m height), with a sandy bottom and very strong halocline, reconnecting to the main passage of the cave at about 500 m from the entrance through a small shaft, which goes from an air space to the main tunnel. The side passage on the right is definitely smaller and branches off in 5 different prosecutions but all of them are extremely narrow and with little to no flow. At 450 meters from the entrance, the main passage has a very defined 90 degrees turn left (SW) and there is the last halocline area before hitting only fresh water. The passage here is still the same dimensions of the initial corridor but about 550 m into the cave there is a massive breakdown area (30 m length and 15 m width) with another big air space. After the breakdown area there is a side passage to the left which leads to a dry section, not longer than 80 m, while the main passage starts going in towards sump one getting bigger and bigger in dimensions (15 m width and 9 m height).

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4.2. Water parameters Salinity ranges, on the whole, from 40.3 (BT3) to 12.2 (BT15). Values have scarce variability from the entrance to BT7, located 210 m from the entrance, and then they decrease with a drop to 12.2 in BT15, 450 m from the entrance (Fig. 3). pH shows scarce variability, with the highest value of 8.4 at BT3 and BT7, and the lowest value of 8.1 at BT15, indicating scarce acidification pattern from the entrance to the innermost part of the cave at the sampling moment. Temperature ranges, on the whole, from 26.1°C (BT1) to 24.4°C (BT11), with a general decreasing pattern from the outer to the inner cave, with the highest gradient between BT1 and BT4. DO shows wide variability (0.48-3.63 mg/l), from the entrance to the inner part, with a clear increasing pattern. According to Bernard and Sen Gupta (1999) the first 60 m (BT1 and BT2) are affected by disoxic conditions, while the remainder of the surveyed area may be considered as oxic (DO > 1). Negative correlation of salinity and temperature with the distance from entrance was recognized, while DO is positively correlated with distance from the entrance. Temperature and DO display negative correlation while pH and salinity are positively correlated (Tab. 1).

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4.3. Grain size Bel Torrente cave is generally characterized by coarse sediment with sand as prevailing fraction, ranging from 99.7% (BT2) to 47.1% (BT11); gravel shows maximum value in BT11 (52%) while it is absent in BT2 and BT6; the fine fraction is generally very scarce in all the samples, with the maximum of 2% in BT6, while it is totally absent in BT5 and BT11. The sediment is classified as sand, gravelly sand and, only for one sample, sandy gravel, highlighting scarce grain size variability (Fig. 4). The grain size distribution curves confirm the coarse nature of the sediments, with a mode never exceeding 1.5 φ, corresponding to medium sand (Fig. 5). The examination under stereo microscope highlights coarse or very coarse grey grains with scarce organic fraction represented by bivalves, gastropods, echinoids, annelids, and foraminifera. The inorganic fraction is mainly constituted by quartz, plagioclases, k-feldspars, biotite, volcanic lithic fragments, rare schists and carbonate fragments (Fig. 6). The last ones are more abundant in BT1 and BT2. 4.4. Benthic foraminifera The quantitative study highlights the presence of benthic foraminifera in samples from BT1 to BT11, corresponding to the section from the entrance to 330 m, with a total of 106 species, while samples from BT12 to BT15 were totally barren. In particular, 76 species are represented both by dead and living specimens. As regards absolute abundance, the dominant species in the cave are Gavelinopsis praegeri,

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Reophax dentaliniformis, Eggerella advena and Ammonia inflata, with 533, 485, 406 and 304 specimens on ACCEPTED MANUSCRIPT the whole, respectively (Plate 1). Abundant species are also Cribrostomoides jeffreysii (250), Rosalina bradyi (217), Quinqueloculina lata (142), Bolivina variabilis (140), Miliolinella subrotunda (130) and Ammoglobigerina globigeriniformis (120). Considering the relative abundance of species in a single sample, G. praegeri (up to 21.6% in BT10) is present at all stations, with the exception of BT11, showing rather steady abundance (Fig. 3). It is an epifaunal clinging species, generally living in a wide range of water depth (Sgarrella and Moncharmont-Zei 1993; Murray 2006); De Stigter et al. (1996) found G. praegeri common as living species in shelf samples of the Adriatic Sea at 146 m water depth, while it was occasionally present also deeper, down to 1200 m. Also Bolivina species (up to 14.1%, on the whole, in BT4), mainly represented by Bolivina pseudoplicata and B. variabilis, are common in the whole surveyed cave, with the exception of cave entrance. Species of this genus are distributed from inner shelf to bathyal environment; they are known as normally living in finegrained sediments with high organic matter content, possibly affected by low oxygen availability and, consequently, they are considered as opportunistic taxa (Murray, 2006). In particular, B. variabilis was abundant in the glacial fauna of pre and post sapropel S6 assemblages, and it was considered as an infaunal species, with opportunistic behavior, but not particularly resistant to low oxygen conditions (Schmiedl et al., 2003). Species diversity, both α-index and H-index, show negative correlation with the distance from the cave entrance, while D is positively correlated (Fig. 7). FN values are very variable (0.33-394) with only 3 station with FN > 10 (BT2, BT3, BT6) and do not show definite pattern with respect to the distance from the entrance. Other taxa found in Bel Torrente cave appear more localized in specific sectors as highlighted by Q-mode HCA (Fig. 8). It recognizes two main clusters, A and B, which reflect the location of the samples: the first one includes samples from BT1 to BT5 while the second one from BT6 to BT10; sample BT11, the last one containing foraminifera, is highly different from all the others. The main difference between cluster A and B is the prevalence of hyaline taxa in the first one and of agglutinated taxa in the second one (Fig. 9). The transition from A to B is marked by the increase of agglutinated taxa due to the concurrent decrease of the porcelaneous ones, while hyaline taxa maintain similar abundance.

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4.4.1. Cluster A Cluster A is divided into sub-clusters A1 and A2 (Fig. 8); the first one, on the whole, is characterized by Peneroplis pertusus (up to 17.9% in BT1), Lobatula lobatula and Rosalina floridana (up to 7.7 and 5.7%, respectively, in BT2), while Ammonia beccarii and Elphidium crispum are present exclusively at the cave entrance (Fig. 3). These taxa are typical of shallow water assemblages of the Sardinian coast (Buosi et al., 2013) and one of these, P. pertusus, bears algal symbionts (Hallock, 1984). Sub-cluster A2 is characterized by species tolerating wide range of water depth such as the agglutinant R. dentaliniformis (up to 26.7% in BT5), associated to prevailing calcareous taxa, mainly G. praegeri. R. dentaliniformis is an infaunal species which can live at water depth ranging from 46 to 168 m in the Baltic Sea (Hermelin, 1983) and at shallower water in fjord environment (Polovodova et al., 2009), while it is uncommon in the Sardinian shelf. Other common taxa are Q. lata and Quinqueloculina stelligera (up to 8.4 and 6.8% in BT4) which were considered as stress-tolerant for contamination due to heavy metals (Romano et al., 2009). Moreover, the brackish-water species Milammina fusca is only present in BT4 and BT5 samples (Murray, 2006; Fatela et al., 2007). 4.4.2. Cluster B Cluster B is constituted by sub-cluster B1, which includes only sample BT6, and sub-cluster B2, which is constituted by samples from BT7 to BT10 and strongly characterized by A. inflata (up to 31.1%), an infaunal species, represented nearly exclusively by living specimens (Fig. 3). This species has never been found in shallow-water sediments of the Sardinian coast (Cherchi et al., 2009; Buosi et al., 2012; 2013), but recovered in a range of 30-90 m water depth along the coast of Tuscany (Frezza and Carboni, 2009; Di Bella et al., 2013).

Cluster B2 is characterized by the agglutinant E. advena (up to 34.7% in BT9), Cribrostomoides spp. (C. ACCEPTED MANUSCRIPT jeffreysii up to 14.3% in BT7 and Cribrostomoides subglobosum up to 6.9% in BT9), A. globigeriniformis (up to 5.4% in BT10), and by the hyaline R. bradyi (up to 21.4% in BT9) and A. inflata (up to 11.9% in BT7). E. advena is an infaunal species, typical of high latitude basins tolerating also low-salinity conditions (Murray, 1991; 2006), considered as a stress-tolerant opportunistic species; it was supposed to benefit from refractory organic material (Schafer et al., 1975; Alve, 1995). A. globigeriniformis was found in the living assemblage, dominated by agglutinant taxa, affected by water acidification due to submarine volcanic gas emissions (Dias et al., 2010), while Cribrostomoides is considered as a shelf-slope typical taxon living in epifaunal position, free or clinging sediment grains (Murray, 2006).

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4.4.3. Cluster C The cluster C is represented only by BT11, which is dominated by living specimens of R. bradyi (57.1%), and characterized by very low species diversity and faunal abundance. R. bradyi is a typical epiphytic species which may live clinging or attached on phytal substrates, but also on detritic circalittoral sands (Sgarrella and Moncharmont Zei, 1993) and it was considered by Romano et al. (2013) as stress-tolerant taxon. The preference of this species for sediment fractions, ranging from coarse sand to gravel, has been already demonstrated by Celia Magno et al. (2012). Relatively abundant species in this sample were also Pseudotriloculina laevigata and A. globigeriniformis.

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5. Discussion This study is the first approach to investigate the possibility of using benthic foraminifera as ecological indicators in order to recognize different ecozones in submarine caves of the Mediterranean area. The few existing papers on this topic were focused only on tropical regions (i.e. Bermuda, Yucatan peninsula), describing the distribution of benthic foraminifera as a response to different groundwater masses, sediment fluxes and groundwater circulation; these Authors recognized the reliability of these environmental indicators for ecological zonation and paleoenvironmental reconstruction of sea-level changes during glacial and interglacial periods (van Hengstum et al., 2008; 2009; 2011; van Hengstum and Scott, 2011). In this study, the use of total foraminiferal assemblage (living + dead) was considered as a reliable tool for environmental characterization aimed to the ecological zonation, because it is not established on the basis of seasonal changes of water parameters, but reflects mean environmental conditions over the time of sediment deposition (Leorri et al., 2008). The consistency of its application is valid when specific methods aimed to recognize possible post-mortem processes are adopted. In order to characterize the different ecological zones, the distribution of foraminiferal assemblages was interpreted in the light of some important abiotic parameters influencing benthic foraminifera distribution, like as grain size, salinity, temperature, pH and DO. Sediment grain size and water salinity strongly influence the composition and structure of foraminiferal assemblages (Murray, 1991; Celia Magno et al., 2012), while water temperature is a main factor in the metabolism of foraminifera, controlling the synthesis of enzymes that determine the general state of the organism and its growth rate, reproduction, and the ability to precipitate the carbonate shell (Titelboim et al., 2017). Also water pH plays an important role to determine the composition of the assemblages, in particular the prevalence of agglutinated or calcareous taxa (Panieri et al., 2005; Dias et al., 2010). As regards DO, it is well known that benthic foraminiferal assemblages are deeply influenced by bottom oxygenation, so that they are used as proxies (Bernard and Sen Gupta, 1999). Although the parameters considered in this study may not be exhaustive to describe the ecological response of foraminifera, they are expected to undergo major changes along the environmental gradient from marine to continental conditions in a submarine cave and to have some influence on benthic foraminifera. 5.1. Ecological response of benthic foraminifera The recovery of benthic foraminifera, from the entrance to 330 m inside the cave, allows recognizing a zone where marine conditions prevail over the year, defined as Marine Cave (MC), and a sector, beyond 330 m, considered as under prevailing continental conditions because barren of foraminifera, and defined as Continental Cave (CC). Observing the pattern of salinity in the whole cave, a dramatic decrease was

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recorded only at BT15 station (12.2), while only moderate difference was recognized between MC (average ACCEPTED MANUSCRIPT salinity 38.8, standard deviation 2.3) and CC (average salinity 28.2, standard deviation 10.8). This is in good agreement with the hydrological features of the cave that is characterized, from the entrance to about 330 m, by a clear separation of marine and freshwater, with a halocline located at 1-2 m water depth. Consequently, the habitat of benthic foraminifera is influenced by the marine water mass which determines environmental stability for the major part of the year. However, considering that brackish water foraminiferal assemblages may live at salinities as low as 2 (Murray, 1991), the lack of living and dead foraminifera from BT12 to BT15 may not be explained by steady low-salinity conditions. It should be considered that sediment sampling was carried out in August, far from the rainy season, which lasts from October to April and accounts for 80% of the annual total precipitation (Delitala et al., 2000). At the sampling moment, the decreasing pattern of salinity with increasing distance from the entrance is little marked, with values similar to those of the cave entrance, around 40, up to 210 m. Lower salinities may be recorded in this environment in different times of the year due to higher freshwater flood, and consequently, it may be supposed that the recorded high salinity values are due to the prevailing of marine conditions in the cave during late summer. Stable environmental conditions promote the development of highly diverse assemblages, while severe fluctuations of environmental parameters are the cause of low-diversity faunas (Schmiedl et al., 2003). Consequently, the negative correlation of species diversity (α-index and H-index) with the distance from the cave entrance, and the positive correlation of D recorded in the MC sector, testifies increasing environmental stress proceeding towards the inner cave (Fig. 7). In spite of low spatial variability of salinity, species diversity appears positively correlated with salinity, while D demonstrates negative correlation (Fig. 10); differently, they do not show any correlations with pH values. This means that salinity plays a role in the structure of foraminiferal assemblage, while the variability of water acidity does not directly influences assemblage diversity and heterogeneity.

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5.2. Environmental features of foraminiferal ecozones It was demonstrated that cluster analysis, applied to relative abundance of benthic foraminifera, is an effective method to recognize different environmental settings inside submarine caves of tropical areas, where salinity was found to be the main environmental factor influencing foraminifera distribution (van Hengstum et al., 2008; 2009; van Hengstum and Scott, 2011). Statistical analysis carried out on commonly occurring species from MC, helped to recognize different group of samples, corresponding to distinct ecozones (Scott et al., 2007). Each foraminiferal assemblage associated to the ecozone may be interpreted by ecological viewpoint in order to identify the environmental conditions characterizing the ecozone. A major zonation of the MC was defined by different foraminiferal assemblages: Entrance (BT1-BT5), Transitional (BT6-BT10), and Distal Cave (BT11) ecozones. The arrangement of distinct clusters of samples, in accordance with their location with respect to the cave entrance, demonstrates that the distribution of foraminiferal species into the cave follows a specific gradient and that transport does not significantly displace the dead assemblage. The whole Entrance ecozone is dominated by the sessile species G. praegeri, a common species in highenergy environment with coarse sediment of northern Europe (Murray, 2006). Then, the high water flow episodically occurring in the cave tunnel seems to be an important factor determining the high abundance of hyaline epifaunal clinging taxa. Nevertheless, the common occurrence of typical species of shallow-water sandy bottom of the Sardinian coastal area (P. pertusus, L. lobatula, E. crispum, A. beccarii) at stations BT1BT3, identifies a sub-ecozone with very similar conditions to the normal marine ones (Cherchi et al., 2009; Buosi et al., 2012; 2013); at BT4-BT5 stations, Bolivina species, generally abundant in fine sediments enriched in organic matter, became very common. Also van Hengstum and Scott (2011) found abundant Bolivina, associated to fine-grained sediments with possibly disoxic conditions, in the cave sector closer to the sea. Differently, high percentages of Bolivina species were recovered in coarse sediments of high energy environments of the Ria de Vigo on the Portuguese coast (Diz et al., 2004). Probably, the infaunal microhabitat of these species preserves them from the environmental disturbance due to high speed flow and favors them, as well as the attached epifaunal taxa, with respect to free living epifaunal species. Moreover, some Bolivina were recognized as tolerant species for low-salinity environment; B. pseudoplicata was found

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in marsh environment of Portuguese rivers at salinities ranging between 12 and 25 and it was dominant in the ACCEPTED MANUSCRIPT tidal marsh at salinity of 25 (Fatela et al., 2007). Also the common occurrence of the infaunal agglutinant R. dentaliniformis may be interpreted not only in the light of water acidification as described above, but also of increasing environmental instability due to high energy. These indications concur to recognize an inner subecozone, where foraminifera start to be affected by environmental stress due to high environmental energy due to the flood of continental water in the tunnels, showing the beginning of a progressive separation from the normal marine conditions. Another stress component may be connected to episodic oxygen deficiency conditions in summer period, determined by oxygen consumption for the decomposition of organic material. These conditions offer further support to the presence of Bolivina in the cave environment. The transition from Entrance to Transitional ecozone is characterized by the shift from hyaline to agglutinant-prevailing assemblage, due to a considerable relative increase of agglutinant taxa to detriment of the porcelaneous ones (Fig. 9). They are generally absent in the Sardinian shallow water assemblages while they are common in shelf environment of high latitude (Murray, 2006). This change of assemblage composition may be interpreted taking into account the water acidification occurring in correspondence of temperature and salinity decrease. A study of benthic foraminiferal assemblages in an area affected by water acidification due to volcanic gas emission recognized a similar change from normal marine conditions to lower pH values (Dias et al., 2010). In this paper, the assemblage with total disappearance of porcelaneous species and prevailing agglutinant ones corresponded to pH of 7.8, while 100% agglutinant species were recorded at pH 7.6. In the MC section, no significant pH changes were recorded from Entrance to Transitional ecozone during the sampling period but, because the total assemblage reflects the yearly average environmental conditions, it may be supposed that the faunal shift is due to lower pH values existing during the rainy season. Consequently, the water acidification may only be supposed to be a significant factor influencing assemblage composition, but a seasonal monitoring of this parameter is necessary to confirm this hypothesis. The infaunal or epifaunal clinging lifestyle of species characterizing the ecozone may be attributed to the response of foraminifera to high energy environment. The opportunistic character of these taxa and the lower species diversity suggest environmental instability due to rapid changes of environmental parameters. On the whole, these conditions identify the Transitional ecozone. The high percentages of living A. inflata, which characterize only BT6, let us suppose peculiar conditions at the sampling moment, prevailing over the average conditions described above. Also in BT 11 specific conditions were recognized during the sediment sampling, due to the high dominance of living R. bradyi, which characterizes the Distal Cave ecozone. The exclusively presence of living foraminifera, let us suppose that seasonal water flows periodically renew all the sediment, transporting the dead fauna away. Consequently, it may be deduced that, also in the Distal Cave ecozone, the distribution of foraminifera seems mainly conditioned by high environmental energy periods, also confirmed by a strong presence of coarse sediments. 5.3. Canonical Correspondence Analysis The CCA supplies information on the response of foraminiferal species to environmental parameters such as distance from the cave entrance, sediment fraction percentages (gravel, sand, and pelite), salinity, DO, temperature and pH (Fig. 11). The clear separation of Entrance from Transitional ecozone is evident also from this analysis, because the first one corresponds to negative values of the first axis, while the second one to positive values. The distance from the entrance is nearly coincident with the first axis and stations belonging to Entrance ecozone are tidily aligned along it together with their characterizing species, to testify the high importance of environmental gradient from the entrance to the inner cave. Environmental factors, directly correlated with the distance from the entrance, strongly condition the separation from Entrance to Transitional ecozone and the species distribution inside the first one. The ecological interpretation, based on species autoecology, let us suppose that these factors all contribute to environmental instability, which increases towards the inner part of the cave. Because temperature, salinity and DO plot on the first axis it is demonstrated that they contribute to the species distribution, while pH seems to have minor influence. However, it should be taken into account that salinity, temperature, DO and pH are strongly influenced by

seasonal changes and values recorded at the sampling moment, may be only partially reflected by the total ACCEPTED MANUSCRIPT assemblage, which was considered for this analysis. The hypothesis that water energy may be another element of environmental stress which increases towards the inner cave is confirmed by the position of gravel, which plots on the positive side of the first axis, while sand plots on the negative one. The coarser bottom sediment of the inner stations testifies average conditions of higher energy in the inner cave.

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5.4. Foraminifera dispersal The prevalence, in the whole MC, of taxa not generally present along the Sardinian coasts and, in particular of agglutinant species, very rare in shallow water environment of the Mediterranean basin (Murray, 1991), was recorded. Conversely, most of these taxa are common in shelf areas of the northern seas and this raises the question of their dispersion. Previous studies on the dispersal of benthic foraminifera demonstrated that very small (< 32 µm) young foraminifera, called propagules, are widely dispersed and can remain in a cryptic stage also up to 2 years, until they encounter favorable conditions for their growth (Alve and Golstein, 2003; 2010). The same mechanism may be effective for the colonization of submarine caves, where environmental conditions are very different from those of the shallow water marine environment of the area. A wider dispersion mechanism from distant geographic regions, through currents, should be supposed for cave colonization.

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5.5. Future perspectives The present research is only a first environmental characterization of a submarine temperate cave but successive environmental monitoring is planned for Bel Torrente and other neighboring caves in order to better recognize temporal environmental variability and to understand its extent. The study of confined environments affected by wide spatial and temporal variability of environmental parameters such as submarine caves, as well as, for example, methane seeps (Panieri et al., 2005) or hydrothermal vents (Dias et al., 2012; Uthicke et al., 2013) may be helpful for the comprehension of general mechanisms which regulate major marine systems. In particular, the identification of the effects of temperature and pH on foraminiferal assemblages in cave environment may supply information on the effects of climate changes and ocean acidification mainly due to anthropogenic impact. The hypothesis of faunal turnover from calcareous to agglutinant-prevailing assemblages as a response to water acidification in the cave could be of some interest to predict major changes of benthic foraminiferal faunas in temperate marine basins as a consequence of carbonate undersaturation, with possible implications for carbon sequestration (Feely et al., 2004).

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6. Conclusion This study highlighted that benthic foraminifera may live in cave environment, not only close to the entrance, but even at considerable distance from it, and respond to changes of environmental parameters. Like as recognized in cave environments for other taxonomic groups, they demonstrated decrease in species richness and density from the outermost to the innermost part of the cave, depending on physical gradient. For this reason they may be used as a tool to recognize distinct ecological zones in the cave environment. Although this study was conducted during dry season, when a stable layer of marine water covers the cave bottom in the sector populated by foraminifera, wide change of water parameters are supposed to occur during rainy season, when considerable freshwater floods occur in the tunnel. Our results pointed out that salinity changes affect the assemblage structure in terms of diversity and heterogeneity, while water acidification is only supposed to be responsible for the faunal shift from hyaline to agglutinant-dominated one. Seasonal monitoring of water parameters would be necessary to support the idea that pH changes are the main factor promoting the increase of agglutinated species. The epifaunal clinging or infaunal lifestyle of common species testify that environmental energy, due to episodic water floods, is a main factor influencing assemblage composition and the higher flow intensity in the inner stations is confirmed by coarser sediment grain size. On the whole, an increasing environmental stress towards the inner cave may be attributed to seasonal changes of environmental parameters, which generally affects foraminiferal assemblages, and it is testified by the dominance of species recognized as opportunistic or, at least, with tolerance of wider environmental conditions than typical shallow water taxa of

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the Sardinian coast. The environmental instability over the year may explain also the absence of ACCEPTED MANUSCRIPT foraminifera, at the sampling moment, in the inner sector where sediment, salinity, temperature, DO and pH conditions do not indicate prohibitive conditions. Although no endemic cave taxa were recorded, the most of the common species in the cave are typical of high latitude marine basins to indicate a possible influence of lower seawater temperature inside the cave. Their presence inside a submarine Mediterranean cave suggests an effective transport mechanism for wide distances, which may be interpreted according to the propagules dispersal theory. This study is the first step for the knowledge of benthic foraminifera ecology in submarine caves of temperate seas and may be considered as a baseline for the ecological assessment of other Mediterranean caves, useful to highlight the effects of anthropogenic impact, especially in terms of sea-level changes and water contamination. Information on their response to spatial environmental variability was proved by applying statistics to faunal and environmental data. Some deductions on the response of foraminiferal species to temporal environmental variability were indirectly obtained, but seasonal monitoring of these environments is necessary to confirm the hypotheses made in this study. Cave environments, due to wide temporal and spatial variability of environmental parameters, may be considered as a laboratory to study mechanisms that regulate the response of benthic foraminifera to environmental variability at a global scale.

References

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7. Acknowledgment This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. The authors are grateful to Maria Celia Magno and Francesco Venti for grain size analyses and image editing. They also greatly appreciated the work done by Global Underwater Explorers and their divers (Anton van Rosmalen, Dorota Czerny, Ineke Van Daele, Jan Duikt, Jonas Patteet, Katja Muermans, Marco Colman, Matthias Trappeniers, Onno van Eijk, Peter Brandt, Ricardo Constantino, Sander Jansson), during the project. Thanks are also due two anonymous reviewers because their comments were useful for a reconsideration of some aspects of the study and their suggestions helped to improve the quality of the manuscript and to Elena Mumelter for the revision of English.

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Fig. 1. Geological scheme of the Orosei Gulf (modified from INQUA SEQS 2012 Meeting - Fieldtrip Guidebook). Fig. 2. Map of Bel Torrente cave with sampling stations. Fig. 3. Values of abiotic parameters measured at the sampling moment (Temperature, Salinity, pH, DO) and depth of sampling stations. Relative abundance of main foraminiferal species and of different ecozones recognized by means of Hierarchical Cluster Analysis. Fig. 4. Sediment classification according to Shepard (1954), modified for presence of gravel.

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Fig. 5. Grain size analysis. Frequency curves of samples classified as “sand”. Fig. 6. Different sediment types in Bel Torrente cave under stereo microscope.

Fig. 7. Values of α-index, H-index and D-dominance in the sampling stations. Correlation of faunal parameters with distance from the entrance is highlighted. Blue, green and orange bars identify samples of cluster A, B and C, respectively.

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Fig. 8. Two-way (Q-mode and R-mode) Hierarchical Cluster Analysis carried out on the foraminiferal quantitative results. Fig. 9. Abundance of hyaline, porcelaneous and agglutinant specimens in sample clusters recognized by means of cluster analysis.

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Fig. 10. Scatter plot of salinity vs. α-index, H-index and D-dominance. Positive correlation was recognized. Fig. 11. Canonical Correspondence Analysis (CCA) on the foraminiferal quantitative results. Grain size fractions (gravel, silt and pelite), distance from the entrance (dist), temperature (T), salinity and pH were considered as environmental parameters.

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Plate 1. Common species in Bel Torrente cave. 1) Ammoglobigerina globigeriniformis (rose Bengal stained specimen), ventral view, sample BT5; 2) Ammoglobigerina globigeriniformis (rose Bengal stained specimen), dorsal view, sample BT5; 3) Gavelinopsis praegeri (rose Bengal stained specimen), dorsal view, sample BT10; 4) Gavelinopsis praegeri (rose Bengal stained specimen), ventral view, sample BT10; 5) Lepidodeuterammina ochracea (rose Bengal stained specimen) dorsal view, sample BT7; 6) Gavelinopsis praegeri (rose Bengal stained specimen), ventral view, sample BT10; 7) Bolivina pseudoplicata (rose Bengal stained specimen) side view, sample BT4; 8) Bolivina variabilis (rose Bengal stained specimen) side view, sample BT3; 9) Rosalina bradyi (rose Bengal stained specimen) dorsal view, sample BT12; 10) Rosalina bradyi (rose Bengal stained specimen) ventral view, sample BT12; 11) Quinqueloculina stelligera, side view, sample BT2; 12) Reophax dentaliniformis side view, sample BT3; 13) Eggerella advena, side view, sample BT3; 14) Eggerella advena (rose Bengal stained specimen), side view, sample BT6; 15) Peneroplis pertusus (rose Bengal stained specimen), side view, sample BT1; 16) Cribrostomoides jeffreysii (rose Bengal stained specimen) side view, sample BT7; 17) Ammonia inflata (rose Bengal stained specimen), ventral view, sample BT6; 18) Ammonia inflata (rose Bengal stained specimen), dorsal view, sample BT6. Tab. 1. Correlation matrix (Pearson index R) of distance from the entrance (dist), salinity, temperature (T) and pH. R values are reported in the lower section, while p values are in the upper one. Values identifying significant correlations (p < 0.01) are in bold. Distance Distance

Depth

T

DO

Salinity

pH

0

0,16471

6,70E-05

4,76E-06

0,00464

0,03591

Depth

0,37806

0

0,4801

0,37559

0,71584

0,69313

T

-0,8473

-0,1977

0

6,07E-06

0,09542

0,19226

DO

0,90036

0,24661

-0,8964

0

0,02971

0,07052

Salinity

-0,6873

-0,1027

0,44628

-0,5606

0

3,13E-07

pH

-0,5443

-0,1112

0,3564

-0,4795

0,93531

0

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− Benthic foraminifera in Mediterranean submarine caves were studied for the first time − Ecological zones were recognized in the cave environment − Change of environmental parameters influence species diversity and distribution − Species in the cave are not common in the Sardinian marine coastal area

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− An effective dispersal mechanism is supposed for cave colonization