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Endobiotic communities of Marine Sponges in Cyprus (Levantine Sea)

Sponges host cryptic endobionts within their network of canals, with representatives from all major animal phyla. This study investigates the endobiotic community of four sponge species (Spongia officinalis, Sarcotragus spinosulus, Ircinia cf. variabilis and Ircinia oros) that were collected during scientific trawl surveys in the coastal area of Cyprus. Moreover, it examines the endobiotic community composition of S. spinosulus in relation to sponges' volume, and various environmental variables. In general, the four sponge endobiotic communities were similar; S. officinalis had a significantly different community composition to I. cf. variabilis and I. oros. The phyla identified followed the general infauna composition of sponges, with the relative abundances of the dominant phyla, Arthropoda and Annelida, ranging from 66.9 - 83.7 % and 4.8–26.5 %, respectively. The highest intensity (I) corresponded to the isopod Cymodoce truncata in S. officinalis (I = 85 individuals/sponge) and S. spinosulus (I = 27.2 individuals/sponge). A general linear model also suggested that distance from shore influenced the total endobiotic abundance of S. spinosulus. This is the first sponge endobiotic community baseline study that covers the whole coastal area of the Republic of Cyprus and is particularly important due to potential changes of Eastern Mediterranean endobiotic communities due to the invasion through the Suez Canal of non-indigenous species.


Introduction
Sponges comprise a major part of the global marine benthos (Taylor et al., 2007;Fan et al., 2012). They increase environmental heterogeneity and can act as microhabitats for vertebrates, invertebrates and a rich microbiome (Abdo, 2007;Fiore and Jutte, 2010;Pita et al., 2018). They are also contemplated as important ecological contributors in reef habitats as they perform several vital ecosystem functions for both the benthic and pelagic environments, compete for space with other benthic organisms, as well as support and enhance biodiversity through bottom-up coupling (Brown et al., 1995;Rutzler, 2003;Trussell et al., 2006;de Goeij et al., 2013;Webster and Thomas, 2016). As autogenic ecosystem engineers, they assist in the survival of productive ecosystems in low nutrient areas like the ultra-oligotrophic regions of Eastern Mediterranean (Gerovasileiou and Voultsiadou, 2012;Miller et al., 2012;Rix, 2015).
Sponge endobiotic communities have been described and investigated extensively around the world, including within regions of Southern US (i.e. Brazil and Argentina), Antarctica, Australia, the Faroe Islands and the Mediterranean Sea (Klitgaard, 1995;Ribeiro et al., 2003;Abdo, 2007;Fiore and Jutte, 2010;Schejter et al., 2012;Kersken et al., 2014;Gerovasileiou et al., 2016). Sponges found in these locations tend to primarily comprise an endobiotic community with organisms from available publications), taxonomy (24.7 %) and molecular biology (17 %) (Becerro, 2009). Koukouras et al. (1996), indicated that endobiotic sponge communities can be either similar or unique between sponge species, as well as that the volume of the sponge can affect the presence, abundance and richness of associated sponge fauna. Furthermore, endobiotic associates of the sponges A. oroides, Petrosia ficiformis, Ircinia variabilis and A. aerophoba, in the North Aegean Sea, were found not to be host specific (Koukouras et al., 1992). This leaves the question of whether the endobiotic communities of the sponge species in Cyprus are similar or different, and whether they are affected by their associated fauna, the host, the environmental variables, or a combination of these factors.
Due to the crucial role sponges have within ecosystems, the more we know about their ecology, their associated communities, and the biotic and abiotic factors that affect their ability to thrive, the better our understanding can improve on how they potentially assist the increased abundance and biodiversity of an area. This is especially important for the Eastern Mediterranean because, even though it is an ultraoligotrophic region, it once was one of the most productive sponge banks (Perez and Vacelet, 2014). Moreover, Cyprus has had a long history of sponge fishery and harvest, activities that have increasingly become more difficult due to the decreased population of commercial sponges from over-exploitation and global warming (Economou & Konteatis 1988, 1990Perez and Vacelet, 2014).
This baseline study was conducted to tackle the lack of data on sponges and their associated endobiotic communities within the coastal area of Cyprus, as well as their interaction with their environment. In this paper the endobiotic communities of four sponge species are compared as well as the relation between the endobiotic communities with the sponge species, sponge morphology and abiotic factors (depth, salinity, temperature and distance from the shore).

Study area
The sponges (n ¼ 43) were collected in the Republic of Cyprus using the Trawler R/ V 'MEGALOHARI', during two scientific trawler surveys: August 2015 and June 2016 MedITS (Mediterranean International Bottom Trawling Survey) and June 2016 PROTOMEDEA (Protecting Mediterranean East) project (Fig. 1). The samples were retrieved from a depth of 45e100 meters, at a towing speed of 3 knots and duration 30 minutes. The trawler specifications complied with the 'International Bottom Trawl Survey in the Mediterranean', Instruction Manual, Version 8 (Spedicato-NB, 2016). One sample was also collected by scuba diving at 43 m depth during a PROTOMEDEA survey at the Mazotos ancient ship wreck (Fig. 1).

Sponge Collection and Identification
Due to limited time available between hauls, only 43 sponges in total were collected and inspected. Scaled top and side-view photos of each sponge were taken in order to calculate maximum length, width and height (cm). These measurements were then used to calculate the approximate volume (cm 3 ) of the sponge using the most appropriate formula. A small section of sponge was cut and preserved in 70 % ethanol for later analysis and identification. The remaining sponge was sliced into thin equal strips (2 AE 0.5 cm), and the endobionts (those visible to the eye, >1 mm) were carefully removed with forceps and preserved in 70 % ethanol. The sponge and associate organism identification was achieved to the lowest taxonomic resolution using the standard identification method and an assortment of identification keys, research papers and online databases (van Soest et al., 1912, Day, 1967, Demetropoulos, 1976, Abel et al., 1986, Tornaritis, 1987, Ruffo, 1989, van Soest et al., 1994a,b, Debelius, 1999, Hooper and Van Soest, 2002, Hooper, 2003, van Soest, 2003, Fauvel, 2004, Pillai, 2009, Fiore and Jutte, 2010, Manconi et al., 2013, San Martín and Worsfold, 2015.

Environmental data
Salinity was recorded at the end of each haul using a CTD (SBE 19plus Thermosalinometer), while a MiniCTD (SBE 39 Temperature-Pressure Recorder) attached to the trawler-net, logged the depth (m) and temperature ( C). Additionally, the average distance from the shore was calculated using the start and end coordinates of each haul.

Description and comparison of different sponge associated communities
Percentage of relative abundance (R.A.) of the endobiotic organisms were calculated both at the Phylum and Family level. Prevalence (P) and Intensity (I) of each phylum were also calculated to determine their encounter rate and average number of individuals per host sponge species respectively, by using the following formulas: Species Richness, Shannon-Wiener Diversity Index (H 0 ), Pielou evenness (J 0 ) and Species Accumulation Curves were calculated per sponge species. Additionally, an analysis of similarity (ANOSIM) based on Bray-Curtis similarity was conducted in 'PastV3' (Version 3.20), to compare the endobiotic community between the sponge species, where initially an overall analysis was performed on all sponges, followed by pairwise tests to identify similarities between individual species. A similarity percentage analysis (SIMPER) was then used to identify the species contributing to the most differences between the sponges. Additionally, a Venn diagram was created to show the distribution of the unique endofauna sponge associates, versus those that were common in more than one species. A permutational multivariate analysis of variance (PERMANOVA) was also conducted in 'PastV3' to investigate the relationship between the endobiotic community of different sponge species and year.

Sarcotragus spinosulus associated community abundance
A General Linear Model (GLM) was fitted to the data in R (Version 0.98.1091), to examine if maximum depth, and average salinity, temperature and distance from the shore had any significant effect on the total endobiotic abundance of the sponge Sarcotragus spinosulus. Similarly, a GLM was also used to investigate whether volume had a significant effect on the total endobiotic abundance of S. spinosulus. Variables were only tested on S. spinosulus as it was the only species with a large enough sample pool. This was accomplished by first testing the model for normality of residuals (histogram and Q-Q plot), independence of variance, homoscedasticity and collinearity (VIF). Significance of the variables, and their interactions, was determined using the F-ratio and the P-values (significance level ¼ 0.05) calculated by ANOVA.
The relationship between the environmental variables and total endobiotic abundance of S. spinosulus was calculated using Pearson's correlation coefficient (r) in Microsoft Excel 2010. The same procedure was followed for the volume.    officinalis (J' ¼ 0.54) was around 0.5. The species that displayed the most evenness was Ircinia cf. variabilis (J' ¼ 0.62).

Total endobiotic abundance of Sarcotragus spinosulus, in relation to environmental variables
Distance from the shore (GLM; F 1,9 ¼ 5.68, p ¼ 0.041; Table 4) significantly affected the endobiotic abundance of S. spinosulus, while depth, salinity and  Table 4). Moreover, total endobiotic abundance had a very weak negative correlation with maximum depth (r 2 ¼ -0.10), and a weak relationship with salinity (r 2 ¼ -0.40; Fig. 5A & B). In contrast, average temperature and distance from the shore displayed a weak (r 2 ¼ 0.31), and a very weak positive (r 2 ¼ 0.13) relationship with total abundance respectively ( Fig. 5C & D).
The variable interaction effect (Table 4) presented an interaction between depth and salinity (GLM, F 1,9 ¼ 8.95, p ¼ 0.015), and one between salinity and temperature (GLM, F 1,9 ¼ 7.49, p ¼ 0.023).  3.5. Total sponge endobiotic abundance in relation to sponge volume A significant relationship between sponge volume and total endobiont abundance (GLM: F 1,25 ¼ 14.3, p ¼ 0.0009; Table 5), but not species (GLM: F 3,25 ¼ 2.77, p ¼ 0.063) was found. Additionally, the GLM produced a positive correlation (r 2 ¼ 0.48) between total endobiotic abundance and volume. The endobiotic phyla abundance increased proportionally to the percentage of the sponge that they already inhabited i.e. at a volume of 591.29 cm 3 the highest abundance was obtained by the Arthropoda phylum (n ¼ 10), followed by the Annelida (n ¼ 5); at a volume of 9599.44 cm 3 the highest abundance was obtained again by the Arthropoda (n ¼ 89), followed by the Annelida (n ¼ 13) (Table 6).

Discussion
The four sponge species follow the general faunal composition (Annelida, Arthropoda, Chordata, Echinodermata and Mollusca) that usually forms the infaunal  community of sponges worldwide (Ribeiro et al., 2003;Abdo, 2007;Fiore and Jutte, 2010;Gerovasileiou et al., 2016). The same synthesis at the phylum level was also found for the communities associated with two species of Haliclona sp. in Southwest Australia, and the sponges Agelas oroides and Aplysina aerophoba in the North Aegean Sea (Abdo, 2007;Gerovasileiou et al., 2016). Moreover, the sponge Mycale microsigmatosa from Southeast Brazil, had an associated endobiotic community that comprised species mainly from the phyla Crustacea, Polychaeta, Mollusca, Cnidarian and Echinodermata (Ribeiro et al., 2003).
The dominant phylum (R.A. > 50%) within all sponge species was Arthropoda, and more specifically the obligate sponge-dweller shrimp Synalpheus gambarelloides (Duffy et al., 2000). Similarly, S. gambarelloides (R.A. ¼ 86.6%) was the dominant organism in the sponge Sarcotragus muscarum in a study conducted in Turkey -Levantine Basin ( € Ozcan and Kata gan, 2011). The high abundance of S. gambarelloides could be explained by its bigger size, which possibly provides it with a competitive advantage of obtaining more space and nutrients ( € Ozcan and Kata gan, 2011). The same concept can probably also be applied to the isopod Cymodoce truncata, where in the absence of the Synalpheus shrimp, its intensity was observed to be three times higher.
The Arthropoda had the most common infaunal associates within the four sponge species, with the hairy crab Pilumnus hirtellus being identified in all of them (Table 1). The presence of P. hirtellus in numerous sponge species was confirmed by several studies, including an investigation conducted in the Gulf of Morbihan, France on the sponge Celtodoryx girardae (Perez et al., 2006).
Annelida are almost always second or third most dominant phylum in sponges (Ribeiro et al., 2003). In Spongia officinalis, Sarcotragus spinosulus and Ircinia cf. variabilis, Annelida was the second most abundant phylum. This was also observed in two studies conducted in the North Aegean Sea using the sponges I. variabilis and S. officinalis (Koukouras et al., 1985(Koukouras et al., , 1992. The Annelida also varied greatly in their frequency and abundance within the sponges, with the exception of Hediste diversicolor which obtained the highest dominance and prevalence within all sponge species. Similarly, H. diversicolor was identified as dominant in sponge communities in the Bay of Izmir, Aegean Sea (Çinar and Ergen, 2001).
Moreover, the bivalve Barbatia barbata, which was present in all sponge species except S. officinalis was also identified in Aplysina (Verongia) aerophoba, North Aegean Sea, while the other two species, Lima lima (Bivalvia) and Raphitoma contigua (Gastropoda), haven't been associated with any other sponge so far (Voultsiadou-Koukoura et al., 1987). The Ophiurida Amphiura chiajei was also present in the sponges A. aerophoba and Axinella cannabina, but there are no studies indicating the starfish Marthasterias glacialis as an endobiotic sponge associate (Koukouras et al., 1996). There is however some evidence demonstrating a feeding relationship between M. glacialis and various sponge associates, such as the bivalve Arca noae, which is a host mollusc for the sponge Crambe crambe (Marin and Belluga, 2005). It is possible that M. glacialis was lodged into the sponge by the force of the haul. Similarly, it's possible that the Spicara smaris were also accidentally lodged into I. oros during the trawl, because even though fish have been identified within sponges before, juvenile S. smaris usually inhabit soft bottoms or Posidonia meadows (Tunesi et al., 2005;Gerovasileiou et al., 2016).
The differences between the prevalence and the intensity of H. diversicolor, S. gambarelloides and C. truncata could be a consequence of several biotic and abiotic factors that affect either the sponge (host) or the associates (endobionts) directly, including environmental, biological or morphological variables (Iyaji et al., 2009;Khidr et al., 2012;Sherrard-Smith et al., 2012). An interesting case to point out is the hairy crab P. hirtellus where no matter its prevalence within sponges, it seems to have a constant intensity of about 2.7 individuals per host sponge. This could be a result of the life history of the endobiont itself. Examples of where the sponge morphology might be playing a role in the number of individuals per host sponge are the endobionts C. truncata, S. gambarelloides and H. diversicolor. This is because these species are relatively large organisms compared to the rest of the sponge associates, and in the sponge species identified they always seem to follow the same trend: highest numbers of individuals were identified within the sponge S. officinalis, followed by S. spinosulus, I. cf. variabilis and then I. oros, ( Table 1).
Many of the infauna associates were common between more than one sponge species (Table 1), and all four sponges shared at least two endobiont species. Many of the endobionts were also shared with various other sponge associate communities i.e. the species, Lepidasthenia elegans (Polychaeta), Athanas nitescens (Decapoda), Colomastix pusilla (Amphipoda), C. truncata (Isopoda), Dexamine spiniventris (Amphipoda) and B. barbata (Bivalvia), were also identified in the sponge Aplysina (Verongia) aerophoba in the North Aegean Sea (Voultsiadou-Koukoura et al., 1987). A recent investigation into the endobiotic community of the sponge Sarcotragus foetidus in Greece and Cyprus, revealed common endobiotic species of Annelida (6 species), Arthropoda (5 species) and Mollusca (1 species) with the sponge samples from the current investigation (Pavloudi et al., 2016). To be more precise, it had the most mutual species from the Annelida phylum with the sponge I. cf. variabilis, from the Arthropoda with S. spinosulus, and from the Mollusca with I. oros.
Distance from the shore was the only variable that affected the total endobiotic abundance of S. spinosulus, and after further analysis the following pattern arose: at closer distances there was lower species richness with higher abundance, but as distance increased the abundance of those species decreased and species richness across all phyla increased.
Depth is a known factor influencing sponges and the abundance of endosymbionts (Ribeiro et al., 2003). In the current study depth did not significantly affect the endobiotic abundance of S. spinosulus, however, that might be because of the depth range present within our study area; no shallow zones present. Moreover, since there is no known direct association between depth and salinity, the interaction is most likely driven by a third unknown variable that could be triggered by a number of factors including currents, oxygen availability or turbidity (Bell, 2007).
The sponge volume also affected the total endobiotic community abundance. As the volume of the sponge increased and there was more available space (i.e. niche) for the organisms to use, the total endobiotic abundance increased too.

Conclusions
The sponge endobiotic communities identified in Cyprus seem to have similarities in their general structure with sponge associated infauna reported from other areas. Moreover, Synalpheus shrimp dominated the samples it inhabited, probably because of its bigger size and competitive advantage over other endobionts.
From all four sponge species, Sarcotragus spinosulus is most likely the species that can endure the most external pressures. Depth did not significantly affect the endobiotic abundance of S. spinosulus, but that might be due to the depth range used in the study, rather than the significance of depth. Bigger sponges were able to facilitate higher community abundances, while at the same time the number of organisms increased proportionally to their relative abundance within the sponge specimens. Based on the present investigation and the arrival of the nonindigenous species (NIS) through the Suez Canal, we suggest that the endobiotic research areas that need prompt attention include the distribution and abundance of phyla within sponges, and how these interact with each other and their host.
The potential effects of these NIS, which more often than not establish successfully in the Levantine Sea, a well-known hot spot of bioinvasions, need to be documented (Katsanevakis et al., 2014;Giakoumi et al., 2016;Galil et al. 2017Galil et al. , 2018Rilov et al., 2017).

Declarations
Author contribution statement