Soft-bottom Crustacean Assemblages in Mediterranean Marine Caves: The cave of Cerro Gordo (Granada, Spain) as Case Study

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1007/s10152-012-0292-5” 1 SOFT BOTTOM CRUSTACEAN ASSEMBLAGES IN MEDITERRANEAN MARINE CAVES. THE CAVE OF CERRO GORDO (GRANADA, SPAIN) AS CASE STUDY C. Navarro-Barranco1,2, J.M. Guerra-García1, L. Sánchez-Tocino2, J.C. García-Gómez1 1Laboratorio de Biología Marina, Departamento de Fisiología y Zoología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012 Sevilla, Spain. Email: [email protected] (Author for correspondence) 2Departamento de Biología Animal, Facultad de Ciencias, Universidad de Granada, 18071, Granada, Spain Corresponding author: Tel: (+34) 954556229 E-mail: [email protected] Abstract Although marine caves are priority conservation areas according the Directive 92/43/CEE of the European Community, there is a lack of studies dealing with their soft bottom communities. We selected the Cerro Gordo cave, at 15 m deep and with three different zones: a semi dark entrance 25 m long, a dark intermediate area of 35 m and the final zone at 90 m from the entrance. Sediment samples were collected by SCUBA diving at four sites: one outside the cave (as a control) and the other three inside the cave coinciding with the three zones of the cave. Six rectangular cores of 10x25 cm2 were collected in each site for macrofaunal study and three more replicates were taken for physico-chemical parameters. The granulometry showed a clear gradient from medium sands outside the cave to silt and clay in the inner zone. Measurements of the crustacean assemblages showed that the number of species and abundance were significantly higher outside the cave (30-40 species, >4000 ind m-2) than inside (5-10 species, <1000 ind m-2). Multivariate analyses showed a clear difference in species composition between outside and inside the cave. Caprellids, tanaids, cumaceans and decapods, were only found outside the cave and disappeared inside the cave, while gammarids and isopods were present both outside and inside the cave. The gammarid Siphonoecetes sabatieri and the tanaid Apseudes latreilli were the dominant species 2 outside the cave, while the gammarids Harpinia pectinata, H. crenulata and H. ala were dominant inside. The present study represents an increase in deep range and geographic distribution of Kupellonura mediterranea and Monoculodes packardi. This is the first time that soft bottom amphipod communities from submarine caves of Southern Spain are described. Keywords Crustaceans, Peracarids, Amphipods, marine caves, Mediterranean, Soft bottoms Introduction Marine cave assemblages have generated great interest in the scientific community over the last decades (Benedetti-Cecchi et al. 1996). This environment is attractive to taxonomists and ecologists for several reasons. It is a simplified and oligotrophic system, depending solely on energy inputs from the surrounding productive coastal area. There are strong discontinuities in the distribution of organisms which reflect marked gradients in the environmental conditions. These special conditions of darkness, oligotrophy and low hydrodynamism enable the presence of deeper living species in that shallow environment. Moreover, the investigation of marine caves often gives the possibility to discover new and endemic species, due to its isolation conditions (Ott and Svoboda 1976, Harmelin et al. 1985, Ros et al. 1989). For all these reasons, submarine caves are unique and vulnerable ecosystems (Sarà 1976) protected by the European Community (Habitat Directive 92/43 EEC). However, the study of marine cave communities has focused primarily on the benthic communities that inhabit hard substrate, while very little effort has been devoted to the study of soft bottom communities. Difficulties in carrying out research in such confined habitats may help to explain the lack of exhaustive studies on this topic. Underwater caves often have muddy sediment at the bottom, whose alteration and suspension are two of the greatest dangers of diving into them, since they imply an almost complete loss of visibility. All cave studies have revealed a marked horizontal zonation within the animal communities of hard bottoms inhabiting the walls (Laborel and Vacelet 1958, Sarà 1961, Riedl 1966, Pérès 1967, Cinelli et al. 1978, Balduzzi et al. 1989, Bibiloni and Gili. 1982, Harmelin et al. 1985, Gili et al. 1986, Zabala et al. 1989, Gili and Mcpherson 1987, Bibiloni et al. 1989, Fichez 1990, Benedetti-Cecchi et al 1996, Harmelin 1997, Benedetti-Cecchi et al 1998, Bell 2002, Bussotti et al. 2002, Marti et al. 3 2004a, Marti et al. 2004b, Dennito and Licciano 2006, Bussotti et al 2006, Denitto et al 2007, Moscatello and Belmonte 2007, Bussotti and Guidetti 2009). A common feature in these studies is a decrease in species richness, biomass and coverage of benthic organisms from the outermost to the innermost part of the cave. The proposed explanations for these features are the physical stress gradients inside the cave (light, oxygen, salinity, etc), trophic supply gradient and the limited capacity of the larvae for the dispersion and settlement (Harmelin 1985, Zabala et al. 1989, Fichez 1990). All these factors may also apply to soft bottom communities, so it would be expected to find similar patterns in these environments. However, until now the variation in soft substrate assemblages along a shallow submarine cave gradient has not been investigated. Peracarid crustaceans are among the most diverse and abundant organisms in soft-bottom benthic fauna (Fincham 1974; Dauvin et al. 1994; Prato and Biandiolino 2005; Lourido et al. 2008). They also play an important role in structuring benthic assemblages (Duffy and Hay 2000) and their abundance and species diversity may serve as indicators of environmental conditions (Corbera and Cardell 1995, Gómez-Gesteira and Dauvin 2000, Guerra-García and García-Gomez 2004). For anchialine caves, Iliffe (2005) indicates that this group makes up 90% of species. Furthermore, from the taxonomical point of view, many new taxa are being described from marine caves, and sediments of these caves are still considerably unexplored. It is difficult to find caves with a defined gradient of sediments from the outer to the inner zones. Consequently, the cave of Cerro Gordo, more than 100 m long, is an adequate site to study, for the first time, the soft bottom crustacean communities in a Mediterranean cave gradient. The aims of this study were: 1) Identify the crustacean species which inhabit the soft bottom of Cerro Gordo cave, and 2) determine their distribution and abundance along the environmental gradient of the cave to assess if soft bottom communities present a similar pattern to those found for the hard bottom animal communities inhabiting the walls. Material and methods Study area The study was conducted at the Cerro Gordo cave, a karstic submarine cave located in the coast of Granada (South Spain, Alboran Sea, 33º43’46’’ N, 3º45’56’’O) (Figure 1). Because of upwelling events, which bring deep and nutrient-rich water, this 4 area is characterized for its very diverse benthic communities. The cave is more than 100 m long and presents a large submerged single entrance. A marked narrowing of the cave 25 m from the entrance separates two topographic areas. The first area, extending from the entrance to the narrowing, is a wide hall, with 15 m wide and 15 m high, with an air chamber on the top. Light, although reduced, is still present at this hall. The second area is a rectilinear, blind-ending tunnel more than 75 m long that is the dark area. The depth decreases along the tunnel until it reaches an air chamber located at the end, where there are freshwater springs. Sampling stations were referred as A, B, C and D. Station A was located outside the cave, in a sandy bottom area near the entrance. Station B was located in the semi-dark area, between 15 and 20 m from the entrance. The other two sampling stations were in the dark area, one at about 60 m from the entrance and the other at the end of the cave, in an area with freshwater influence (Figure 1). Sample collection and analyses The sample collection was carried out during February 2001, using a hand-held core rectangular of 0,025m2 to a depth of 10 cm by SCUBA diving. Six replicate core samples were taken at each station for the crustacean study. Samples were washed through a 0.5 mm mesh sieve with seawater and fixed with ethanol stained with rose bengal. In the laboratory, each sample was examined using binocular microscopes. All the crustacean specimens were counted and identified to species where possible. Species diversity for each sample and the associated evenness component J´ (Pielou, 1966) were calculated applying the (log2) Shannon Wiever diversity index (H´) (Shannon & Weaver, 1963). Together with the macrofaunal samples, three more sediment cores were collected at each station for physico-chemical analysis of the sediments. All samples were immediately stored frozen until the laboratory analysis. Granulometry was determined following the method proposed by Guitián and Carballas (1976). For the chemical analysis, the sediments were air-dried, crushed and sieved (2mm) first. Aliquots of sediment samples were also ground to < 60 µm prior to determination of major, minor and trace element concentration. Organic Matter (OM) was analysed by dichromate oxidation and titration with ferrous ammonium sulphate (Walkley and Black 1934). Kjeldahl-N was determined by the method described by Hesse (1971). Total major, minor and trace element concentration in sediments (<60 µm) were determined 5 by Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES Varian ICP 720-ES axially viewed) following aqua-regia digestion in a microwave oven (Microwave Laboratory Station Mileston ETHNOS 900). The term “total” accounts for the aqua-regia digestion, but it does not completely destroy silicates. The accuracy of the analytical methods was assessed by carrying out analyses of the BCR (Community Bureau of Reference) reference samples: BCR 320R (Channel sediment) and soil sample reference ISE 872 from the Wageningen Evaluating Programs for Analytical Laboratories, International Soil-analytical Exchange (WEPAL; ISE). Statistical analyses Biological, chemical and granulometry data were not normally distributed (Kolmogrov-Smirnov test) and did not feature homogeneity of variance (Levene test), not allowing parametric test to be performed. Thus, we opted to use Kruskall-Wallis analysis to detect differences among stations. When those differences exist, Tamhane post hoc tests were used for post hoc comparisons. The univariate statistical analyses were carried out using the SPSS 17.0 Statistic program. Cluster analyses were conducted on abundance per taxonomic group to assess the relationship between stations. Hierarchical clustering with group-average linking, based on similarity matrices (Bray-Curtis coefficient) was used. Data were previously squared root transformed to reduce the importance of extreme values. With the same aim, other two cluster analyses were conducted with the granulometry and chemical data. In such cases, the clustering with group-average linking was based on euclidean distances matrices. These analyses were done using the PRIMER package (Clarke and Gorley 2001). Results A total of 36 crustacean species was recorded from the four stations of the study. These comprised 24 amphipod species, four cumaceans, four isopods, two tanaids and two decapods (Table 1). The exterior zone was dominated by the amphipods Siphonoecetes sabatieri and Metaphoxus fultoni, the tanaid Apseudes latreilli and the decapod Diogenes pugilator. These four species comprised 74% of the specimens. The most abundant species inside the cave were three species of genus Harpinia; Harpinia ala, Harpinia crenulata and Harpinia pectinata. Harpinia pectinata, which has a shallower distribution, was the only one found in all stations, including outside the cave. 6 Harpinia crenulata was present in the three stations inside the cave, and Harpinia ala only live in the dark part of the cave. Other interesting deep species found inside the cave were the amphipod Monoculodes packardi and the isopod Kupellonura mediterranea. Amphipods were the dominant group in abundance and number of species for all stations (Figure 2). However, its number of species and individuals was lower inside the cave. The suborder Gammaridea was able to colonize the interior of the cave, while the suborder Caprellidea was only present in the exterior area. The other crustacean group that inhabits the cave sediment was the order Isopoda. Although its dominance was higher in the inner part of the cave, this group also presented a decrease in richness and abundance inside the cave. Tanaidacea, Cumacea and Decapoda were only present in the external station. Richness, abundance and diversity values showed a clear reduction pattern towards the inner part of the cave. By contrast, evenness increased slightly in station D (Figure 3). The Kruskal-Wallis analysis revealed that these differences among stations were significant for number of species, number of individuals and diversity values, but were not for the evenness. The Tamhane post hoc test results were different in each case (Table 2). The granulometry data indicated a progressive increase of the finer fractions in the inner areas in the cave (Figure 4). The Kruskal-Wallis analysis showed the existence of significant differences in the silt and clay composition among stations (W=10,38; p=0,016). Tamhane post hoc test supported the existence of three groups; an exterior zone, a semidark zone, and a third group with the samples from the dark zone of the cave. Concerning chemical analyses, univariate tests showed significant differences between stations for all variables, with the exception of organic matter, nitrogen and phosphorus. Although there were some differences between elements in the results obtained in the Tamhane post hoc tests, most of them agreed to showed significant differences between all the stations except for the last two (station C and station D) (Table 3). The trends observed along the horizontal transect also varied. For Al, As, Ba, Co, Cr, Cu, Fe, K, Li, Mn, Ni, Pb, V and Zn, there was an increase of their concentrations toward the inner part of the cave. In contrast, we obtained an opposite trend for B, Ca, Cd, Mg, Na, S and Sr (Figure 5). The cluster analyses performed for the biological, granulometry and chemical data agreed to show the existence of the three distinct groups seen before (Figure 6). The first of them included the samples from station A, with sediments dominated by medium sands and a faunistic composition clearly different from the internal 7 composition, with less than 20% of similarity. The second group was from the semidark area (station B). The samples from this zone, where the most abundant sediment fraction was the very fine sands, appeared very close in the three cluster analyses (more than 60% of similarity in their taxonomic composition). Finally, there was a third group including the samples from station C and D (the dark zone) mixed. This area had the lowest values of richness, abundance and diversity, and sediments composed mainly of silt and clay. Discussion All the analyses showed a zonation in the Cerro-Gordo cave. Three zones were clearly different in taxonomic composition as well as physicochemical characteristics: a photic or exterior area, a semidark area and a dark area. These three biocenoses are generally recognized in Mediterranean marine caves and they have been identified for both mobile and hard benthic cave communities (Riedl 1966, Pérès 1967, Bibiloni and Gili 1982, Balduzzi et al. 1989, Gili et al. 1986, Gili and Mcpherson 1987, Bibiloni et al. 1989, Fichez 1990, Marti et al. 2004a, Bussotti et al. 2006, Denitto et al. 2007, Moscatello and Belmonte 2007, Bussotti and Guidetti 2009). Nevertheless, it was unknown whether the distribution of soft substrate communities showed the same zonation so far. The data obtained for the exterior station were as expected according to previous studies of this external area (Sanchez-Moyano et al. 2005). On the other hand, the results found inside the cave were more interesting, with many rare species. Kupellonura mediterranea was described by Barnard (1925) and completed by Wägele in 1981 and Kensley in 1987. This species was found between 70 and 880 m deep and only on the coasts of Naples, Sicily and Ligurian sea. Thus, our finding shows a huge increase in the depth range and the geographic distribution of Kupellonura mediterranea. Another deep species found in the Cerro-Gordo cave was Monoculodes packardi, which had a known bathymetric distribution between 90 and 2616 m depth (Ruffo 1993). So this is, so far, the shallowest discovery of the species. The dominance shown of the order amphipoda over the rest of the crustracean orders is a common feature (Sanchez-Moyano et al. 2005). The presence of the orders isopoda and amphipoda inside the cave was not unusual because both of them have a great capacity for living in most marine habitats. Kensley (1998) indicated that deep sea (a marked oligotrophic enviroment with predominantly muddy sediment, like cave 8 environments) was the habitat where isopods were more diverse. Interestingly, caprellids, cumaceans, decapods and tanaids were absent in the inner area of the cave. All these groups have been reported from muddy sediments (see e.g. Guerra-García and García-Gomez 2004; Lourido et al. 2008) so other factors apart from the granulometry could also affect. The results showed a clear decline in richness, abundance and diversity of the crustacean soft-bottom community from the exterior to the inner dark parts of the cave. This trend has been observed for many groups like suspension and filter feeders (sponges, cnidarians, bryozoans and tunicates) (Bibiloni and Gili 1982, Gili et al. 1986, Balduzzi et al 1989, Bibiloni et al. 1989, Harmelin 1997, Benedetti-Cechi et al 1998, Corriero 2000, Bell 2002, Martí et al 2004a, Bussotti et al 2006), big decapods (Gili and Macpherson 1987), meiofauna (Todaro et al 2006), planktonic organisms (Garrabou and Flos 1995, Moscatello and Belmonte 2007), polichaets (Denitto and Licciano 2006), fishes (Bussotti et al. 2002, Bussotti et al. 2003), etc. However, this trend has not been reported for the macroinfauna so far. There are very few investigations dealing with macroinfauna of submarine caves. Moreover, if we want to understand how the environmental gradients in marine caves affect the crustacean community, it is difficult to extract conclusions from the comparison of our results with those obtained in these other studies. Monteiro-Marques (1981), made a taxonomical description of macroinfauna assemblages from some submarine caves from southern France. Nevertheless, his samples were washed through a 1 mm mesh sieve, so most of crustaceans were not taken into account. The work of Akoumianaki and Hughes (2004) took place in the Grotta Azzurra, on the coast of Italy. They studied the distribution of macroinfauna inside the cave and no inward decrease of macroinfauna abundance or diversity was observed. The explanation for this was the existence of sulphur springs at the end of the cave, which provided an additional source of food which has a positive effect on the colonization of macroinfauna inside the resource limited cave environment. Thus, to our knowledge, the present study is the first evidence of a decrease in soft bottom macroinfauna diversity in Mediterranean marine caves, when compared to communities outside the cave. 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Sci Mar 73(2):387-397 Wägele JW (1981) Study of the Hyssuridae (Crustacea: Isopoda: Anthuridea) from the Mediterranean and the Red Sea. Isr J Zool 30(1-2):47-87 Walkley A, Black IA (1934) An examination of Degtjareff method for determining soil organic matter and a proposed modification of the chromic acid titration method. Soil Sci 37:29-37 18 Zabala M, Riera T, Gili JM, Barange M, Lobo A, Peñuelas J (1989) Water flow, trophic depletion, and benthic macrofauna impoverishment in a submarine cave from the western mediterranean. Mar Ecol 10(3):271-287. Fig. 1. Location and longitudinal section of Cerro Gordo cave, showing the samples stations. Fig. 2. Dominance (%) of different groups calculated in function of number of species (A) and individuals (B) Fig. 3. Mean values ± Standard deviation of number of species/sample (S), individuals/m2 (N), eveness (J’) and diversity (H’) in each station. Fig. 4. Percentage of each granulometric fraction in the sediment per sample. OM= Organic matter Fig. 5. Mean concentration ± Standard deviation of each chemical variable per station. Fig. 6. Cluster analysis of samples using the abundance of each species (A), the chemical data (B) and the granulometric data (C) 19 020 40 60 80 100 D C B A CUMACEA TANAIDACEA DECAPODA ISOPODA AMPHIPODA 020 40 60 80 100 D C B A CUMACEA TANAIDACEA DECAPODA ISOPODA AMPHIPODA A) B) Stations Stations Dominance (%) Dominance (%) 20 0% 20% 40% 60% 80% 100% A1 A2 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3 Silt and clay Very fine and fine sands Medium sands Very gross and gross sands Samples Percentage(%) 21 A B C D 0 0,5 1 OM Station % OM A B C D 0 0,04 0,08 N Station % N A B C D 0 300 600 P Station P (mg/kg) A B C D 0 10000 20000 30000 Al Station Al (mg/kg) A B C D 0 4 8As Station As (mg/kg) A B C D 0 10 20 30 B Station B (mg/kg) A B C D 0 50 100 Ba Station Ba (mg/kg) A B C D 0 100000 200000 300000 Ca Station Ca (mg/kg) A B C D 0 0,06 0,12 Cd Station Cd (mg/kg) A B C D 0 10 20 Co Station Co (mg/kg) A B C D 0 20 40 Cr Station Cr (mg/kg) A B C D 0 10 20 Cu Station Cu (mg/kg) A B C D 0 20000 40000 Fe Station Fe (mg/kg) A B C D 0 3000 K Station K (mg/kg) A B C D 0 20 40 Li Station Li (mg/kg) A B C D 0 12000 24000 Mg Station Mg (mg/kg) A B C D 0 200 400 Mn Station Mn (mg/kg) A B C D 0 2000 4000 Na Station Na (mg/kg) A B C D 0 20 40 Ni Station Ni (mg/kg) A B C D 0 20 40 Pb Station Pb (mg/kg) A B C D 0 2000 4000 S Station S (mg/kg) A B C D 0 600 1200 Sr Station Sr (mg/kg) A B C D 0 50 100 V Station V (mg/kg) A B C D 0 50 100 Zn Station Zn (mg/kg) 22 100806040200Bray CurtisSimilarity (%) A4 A1 A6 A2 A3 A5 D5 D2 D3 D6 C5 C6 C2 C3 C4 D1 C1 B1 B3 B2 B6 B4 B5 050000100000150000200000 Euclidean Distance A1 A2 A3 C1 D3 D1 D2 C2 C3 B2 B1 B3 A2 020406080 Euclidean Distance A1 A3 B1 B2 B3 D1 D2 C2 C1 C3 D3 A) B) C)