Preferential assimilation of seagrass detritus by two coexisting Mediterranean sea cucumbers: Holothuria polii and Holothuria tubulosa

Journal Pre-proof Preferential assimilation of seagrass detritus by two coexisting Mediterranean sea cucumbers: Holothuria polii and Holothuria tubulosa Paola Boncagni, Arnold Rakaj, Alessandra Fianchini, Salvatrice Vizzini PII:

S0272-7714(18)30963-6

DOI:

https://doi.org/10.1016/j.ecss.2019.106464

Reference:

YECSS 106464

To appear in:

Estuarine, Coastal and Shelf Science

Received Date: 20 November 2018 Revised Date:

18 August 2019

Accepted Date: 31 October 2019

Please cite this article as: Boncagni, P., Rakaj, A., Fianchini, A., Vizzini, S., Preferential assimilation of seagrass detritus by two coexisting Mediterranean sea cucumbers: Holothuria polii and Holothuria tubulosa, Estuarine, Coastal and Shelf Science (2019), doi: https://doi.org/10.1016/j.ecss.2019.106464. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

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Preferential assimilation of seagrass detritus by two coexisting Mediterranean sea cucumbers:

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Holothuria polii and Holothuria tubulosa Paola Boncagniǂa, Arnold Rakajǂa*, Alessandra Fianchinia, Salvatrice Vizzinib,c

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ǂ These authors contributed equally to the study

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a

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Cracovia 1, 00133, Rome, Italy.

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b

Department of Earth and Marine Sciences, University of Palermo, Via Archirafi 18, 90123 Palermo, Italy

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c

CoNISMa, National Interuniversity Consortium for Marine Science, Piazzale Flaminio 9, 00196 Roma, Italy

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* Corresponding author at: Experimental Ecology and Aquaculture Laboratory, Department of Biology,

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University of Rome ‘‘Tor Vergata’’, Via Cracovia 1, 00133 Rome, Italy.

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E-mail address: [email protected] (A. Rakaj). Tel. 0039/3319859847

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Keywords: Holothuria tubulosa, Holothuria polii, Sea cucumber, Posidonia oceanica, Seagrass detritus,

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stable isotope.

Experimental Ecology and Aquaculture Laboratory, Biology Department of Tor Vergata University, Via

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ABSTRACT

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Holothuria polii and Holothuria tubulosa are two of the most commercially exploited sea cucumbers of the

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Mediterranean Sea. As deposit-feeders, they represent an important component of the benthic community.

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Hence, knowledge of their feeding behaviour is crucial for understanding their function in terms of benthic

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ecology and sediment dynamics.

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Using information obtained from stable isotope analysis, the food selectivity/assimilation, temporal variations

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in diet and trophic niche of H. polii and H. tubulosa were investigated. Analysis of carbon and nitrogen

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isotopic ratios in body wall tissue showed a preferential assimilation of seagrass detritus among multiple food

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sources, with a total contribution to the diet ranging from 63% to 74%. The temporal changes in δ13C and

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δ¹⁵N values followed the seagrass isotopic dynamics with depleted values in winter and enriched values in

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summer. Additionally, δ13C and δ¹⁵N measurements were used to outline the species' trophic niche width.

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The two species coexist, showing an overlap on the δ13C axis and a partial degree of niche segregation on

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the δ¹⁵N axis. Our results provide an overview on the feeding activity of H. polii and H. tubulosa, providing

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evidence of their crucial role on the seagrass detrital pathways for Mediterranean coastal ecosystems.

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INTRODUCTION

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Holothuria polii (Delle Chiaje, 1824) and Holothuria tubulosa (Gmelin, 1791) are among the most common

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and widely distributed sea cucumbers in the Mediterranean Sea (Francour, 1989a; Mezali et al., 2006; Rakaj

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et al., 2018, 2019). In recent years, in response to the growing demand from Asian countries, where sea

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cucumbers are considered a food delicacy (Chen, 2003, 2005; Toral-Granda et al., 2008; Purcell et al., 2012;

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Purcell, 2014; Han et al., 2016), commercial interest in these species has increased and they have become

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two of the most exploited sea cucumbers of the basin (Aydin, 2008; González-Wangüemert et al. 2014,

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2015, 2018). As a result, overfishing is threatening natural populations (Sicuro and Levine, 2011; Aydin,

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2017; González-Wangüemert et al., 2018; Moussa et Wirawati, 2018) and could prevent their recovery

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(González-Wangüemert et al., 2018). The overfishing of sea cucumbers is not only a problem of the

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overexploitation of an economic resource; it also raises ecological concerns in the Mediterranean Sea.

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Indeed, H. polii and H. tubulosa represent a significant component of the benthic community in terms of

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biomass (Francour, 1989a; Bulteel et al., 1992; Coulon and Jangoux, 1993; Mezali et al., 2006) and play an

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important ecological role as ecosystem engineers. Massin and Jangoux (1976) have classified H. polii and

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H. tubulosa as “selective deposit feeders of the sediment–water interface”. Like other sea cucumbers, they

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ingest organic and inorganic matter from the upper layer of the sediment, collecting the detritus, which

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includes bacteria, protozoans and diatoms (Massin and Jangoux, 1976; Amon and Herndl, 1991; Mezali et

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al., 2003). The organic portion is digested and assimilated in the long digestive tube that occupies most of

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the coelomatic cavity, the excrement being expelled as fecal pellets (Massin and Jangoux, 1976; Feral and

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Massin, 1982). In this way, they rework the surface sediments and in doing so influence biotic interactions in

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the community (Francour 1989b; Coulon and Jangoux, 1993).

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Despite the economic and ecological value of these two species, the direct effects of fishery on H. polii and

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H. tubulosa populations, together with the indirect effects on the communities to which they belong, remain

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poorly investigated. Recently, there has been increasing interest in their trophic ecology due to pioneering

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research into artificial reproduction, which highlighted significant differences in the trophic behaviour of the

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two species at the larval stage (Rakaj et al., 2018, 2019). However, comprehensive studies on the trophic

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behaviour of adults are still lacking. Although several authors have examined the gut content of these

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species through visual inspection, identifying particles derived from algae, seagrass and animal tissues

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(Massin and Jangoux, 1976; Traer, 1980; Verlaque, 1981; Mazzella et al., 1992), this qualitative analysis

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does not make it possible to quantify and classify the bulk of the ingested material. Furthermore, only a

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portion of the ingested material may be effectively assimilated, thus limiting the effectiveness of the visual

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method (Tieszen et al., 1983; Hobson and Sealy, 1991; Grey, 2006). The use of stable isotope analysis

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overcomes these limits since it permits the evaluation of the contribution of each food source to the

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consumer’s diet (δ13C) (Michener and Kaufman, 2007; Mancinelli and Vizzini, 2015), as well as estimating

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the trophic level (δ15N) (Vander Zanden et al., 1997; Post, 2002; Mancinelli and Vizzini, 2015). In addition,

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stable isotopes determine the sources assimilated over a medium-long time span (Van Dover et al., 1992).

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To date this technique has been limited to the characterization of the isotopic signatures of H. polii and H.

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tubulosa in whole community studies (Dauby and Coulon, 1993; Vizzini and Mazzola, 2004; Lloret and

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Marín, 2009; Deudero et al., 2011; Cresson et al., 2012; Ricart et al., 2015). Only in the case of H. tubulosa

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has artificial feeding and assimilation of seagrass been investigated in laboratory conditions (Costa et al.,

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2014)so information is still lacking on the trophic ecology of these two species in the wild.

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H. polii and H. tubulosa often coexist on the sediment surface in soft-bottoms rich in organic matter (Massin

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and Jangoux, 1976; Gustato and Villari, 1980; Mezali et al., 2006). According to the competitive exclusion

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principle, the sympatric coexistence of several species can occur when niches are under-saturated due to

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some disturbance (Huston, 1979) or when there is a certain degree of segregation along the time, space or

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food axis (Schoener, 1974). The overlap between H. polii and H. tubulosa along the spatial and temporal

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axis would shift the focus onto the possible partitioning strategies along the food axis. Previous literature

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focused on particle size ingested, showing that H. polii and H. tubulosa both have a significant preference for

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the same fraction of the sediment (Mezali and Soualili, 2013); this rules out particle size selection as a

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foraging strategy for niche separation. Hence, the carbon and nitrogen isotopic signatures of consumers are

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a very useful tool for describing their trophic niche (Bearhop et al., 2004; Grey, 2006) and for evaluating

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whether food partitioning is the main mechanism to explain the sympatry between these species.

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In this study, we investigated an area where H. polii and H. tubulosa co-exist on uncovered sandy bottoms

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characterized by detritus inputs originating from the main carbon sources typical of Mediterranean coastal

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ecosystems such as seagrasses, macroalgae, periphyton and inland detritus. In this case, the use of stable

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isotope analysis is particularly promising, since these main sources are characterized by quite different δ13C

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values, making it possible to clearly distinguish between them (Colombini et al. 2011).

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Our specific goals were to: I) identify the main food sources supporting H. polii and H. tubulosa and infer

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their ability to select and/or preferentially assimilate a particular food source; II) detect temporal changes in

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dietary composition; III) evaluate food partitioning between H. polii and H. tubulosa since the two species

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often coexist.

96

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MATERIALS AND METHODS

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STUDY SITE The sampling was carried out at Torre Astura (Italy, Central Tyrrhenian Sea, Mediterranean Sea)

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(41°24′29′′N, 12°45′51′′E), about 1 km from the mouth of the Astura River (Fig. 1), inside a Military Area of

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the firing-range of Nettuno, the limited access to which contributes to the preservation of this area. The

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sampling site is characterized by some of the most representative fauna of the Mediterranean Sea and is

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sheltered, towards the open sea, by an underwater reef. The soft bottom is characterized by infralittoral fine

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sand and small patches of the seagrasses Cymodocea nodosa and Posidonia oceanica (Ardizzone and

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Belluscio, 1996; Iberite, 1996; Spada et al., 2001). The hard bottom is covered mainly by green and brown

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algal facies that change their composition seasonally (Iberite, 1996).

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Fig. 1. Location of the Torre Astura and sampling site

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SAMPLE COLLECTION

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each period; 102.37 ± 20.16 g; mean ± SD) and H. tubulosa (n=10 for each period; 183.17 ± 52.5 g; mean ±

113

SD) were collected, by snorkeling at depths from 2 to 6 m on fine sand bottoms outside the seagrass

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meadow and algal beds. Depending on seasonal availability, dominant food sources (macroalgae: Codium

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sp., Cystoseira sp., Sargassum sp.; periphyton; seagrasses: Posidonia oceanica, Cymodocea nodosa;

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marine sediment) (n=5 samples for each source, in each period) were collected. Additionally, due to the

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presence of the Astura River, inland sources (river sediment and riparian vegetation: Phragmites australis,

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Arundo donax), potentially reaching the marine area though the river flow (n=5 samples for each source, in

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each period), were also collected. Sea cucumbers, macroalgae, seagrasses and living tissues and leaf litter

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from the riparian plants were all collected by hand. The periphyton was sampled from sea rock surfaces

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using a brush, while the uppermost millimeters of river and the marine sediments were collected by means of

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a box corer. Upon collection, all samples were placed in a cooler with ice, and then transferred to the

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Laboratory of Experimental Ecology and Aquaculture (University of Rome, Tor Vergata). Vegetal sources

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were rinsed with distilled water to remove epiphytes and extraneous sediments, prior to analysis. Sea

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cucumber specimens were first degutted in order to collect the intestinal content; a tissue sample was then

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taken from the intermediate region of their body wall for isotopic analysis.

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The potential food sources, intestinal content and the body wall tissues were all oven dried at 60°C between

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24 and 36 h, according to their weight, before being ground using a mortar and pestle. Samples were

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acidified (2N HCl) before analysis to eliminate

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et al., 1995; Bosley and Wainright, 1999; Chanton and Lewis, 1999).

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Stable isotope analysis was carried out at the Stable Isotope Ecology Laboratory of the University of

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Palermo. The isotopic measurements were performed with a mass spectrometer IRMS in continuous

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(Thermo-Electron Delta Plus XP) equipped with an elemental analyzer (Thermo-Electron Flash EA 1112).

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The isotopic values are expressed in δ unit notation (‰) relative to Pee Dee Belemnite limestone for δ13C

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and nitrogen in air for δ 15N following the formula:

Sample collection was carried out in summer 2015 and winter 2016. Adult specimens of H. polii (n=10 for

13C-enriched

carbonates, such as calcareous spicules (Bunn

136 δ13C ord δ15N =

sample⁄ standard − 1 × 10

137 138

where R is

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replicates of internal standards (International Atomic Energy Agency IAEA-CH-6 for C and IAEA-NO-3 for N)

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was 0.1‰ for both δ13C and δ15N.

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13C/12C

or

15N/14N

isotopic ratios. Analytical precision was based on the standard deviation of

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STATISTICAL ANALYSIS

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δ15N) to determine whether variations among sea cucumbers body wall tissue were due to the orthogonal

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factors species (H. polli and H. tubulosa) and period (summer and winter) or their interaction. A probability

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level of 0.05 was used in order to reject the null hypothesis. Prior to analysis, raw data were diagnosed for

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normality of distribution and homogeneity of variance by means of a Levene’s test and a Kolmogorov-

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Smirnov test respectively (Whitlock and Schluter, 2010). Samples were close to a normal distribution

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(Kolmogorov-Smirnov p > 0.05) with similar variances (Levene’s p > 0.05) (Table 1). Statistical analyses

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were performed with PAST 3.0 (Hammer et al. 2013).

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Trophic niche width for H. polii and H. tubulosa in δ13C–δ15N niche space, was described using SIBER

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(Stable isotope Bayesian Ellipses in R), by calculating the quantitative metrics independently for each

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population in each period (Layman et al., 2007). The measure of niche width is based on the total area of the

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convex hull (TA) bounding a sub-set of individuals of the population; this reflects the variation along both the

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δ13C range (CR) and the δ15N range (NR) signatures, the mean distance from the centroid (CD), the mean

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nearest neighbor distance to each species (MNND) and its standard deviation (SDNND). The total convex

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hull area is encompassed by the smallest convex polygon containing these individuals in the δ13C and δ15N

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niche space. The above metrics - commonly known as Layman’s metrics - are biased by the low number of

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samples and the outliers; for this reason, the evaluation of the SEAc (standard ellipse areas corrected) has

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been corrected following Jackson et al. (2011). The overlap (95% confidence intervals) between the niche

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width of the consumers was calculated as the ratio between the areas of overlap between polygons for the

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two species separately in the two periods.

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The diets of H. polii e H. tubulosa were estimated through SIAR software package V4.2, mixing model in R,

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(Parnell and Jackson, 2013) which uses the Bayesian approach to estimate the resources’ contributions to a

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consumer diet by accounting for all the uncertainties of the input data (Layman et al., 2012). Different food

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sources with distinctive isotopic values were used in mixing models to determine their relative contributions.

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We applied Isosource (Phillips and Gregg, 2003), which is a probabilistic model identifying a range of

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possible dietary inputs when the number of sources exceeds n + 1 isotopes sources. As suggested by

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Phillips et al. (2005) we used ecological criteria to aggregate, a priori, sources into isotopically distinct food

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groups, including seagrasses, macroalgae, periphyton and inland sources, in addition to marine sediment.

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Following Costa et al. (2014), isotopic fractionations used in the model were: 0.2±0.2‰ for δ13C and

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2.7±0.3‰ for δ15N.

Two-way analysis of variance (ANOVA) was performed with non-transformed stable isotope data (δ13C and

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RESULTS

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δ13C and δ15N values for H. polii varied respectively from -16.9‰ to -12.2‰ and 6.3‰ to 9.9‰; for H.

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tubulosa they varied from -17.5‰ to -12.0‰ and from 6.8‰ to 10.2‰. The results of two-way ANOVA

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analysis showed significant differences between periods, both for δ 13C and for δ15N and, between species,

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only for δ15N; also, the interaction between species and periods were significant only for δ15N (Table 1).

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TABLE 1. Two-way ANOVAs, Levene's statistic and Kolmogorov-Smirnov statistic on isotopic values of sea

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cucumbers between species (H. polii and H. tubulosa), periods (summer and winter) and their interaction

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[n.s: p > 0.05; *: p < 0.05; **: p < 0.01; ***: p < 0.001].

183 TwoTwo-way ANOVA

δ13C

F

p

p

K-S

p

s

(Levene’s

Statistic

(K--S) (K

Statistic

)

Species

0.82

0.66

n.s

0.41

n.s

0.19

n.s

Period

24.00

19.31

***

0.62

n.s

0.45

n.s

0.49

0.39

n.s

Within

1.24

-

Species

2.08

6.50

*

1.38

n.s

0.33

n.s

Period

44.35

138.40

***

3.24

n.s

0.51

n.s

2.29

7.15

**

0.32

-

Species x Period

δ15N

MS

Levene'

Species x Period Within

184 185

Within the signature ranges of the two sympatric species, the lowest values of δ13C and the highest δ15N

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were observed in the winter period (Fig. 2). The sample size corrected Standard Ellipse Area (SEAc),

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representing the total trophic niche breadth, and Convex Hull total area (TA) were larger for H. tubulosa in

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both periods than for H. polii, with a partial niche overlap between them. In relation to Layman’s metrics (CR,

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NR, CD, MNND, SDNND), overall H. tubulosa showed higher values than H. polii in both periods (Table 2).

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When comparing the periods, the values of the metrics and the ellipse area were slightly higher for both

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species in summer. The isotopic niche overlap between H. polii and H. tubulosa ranged from 0.53 to 0.61 in

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summer and from 0.29 to 0.81 in winter.

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Fig. 2. Biplot of δ 13C and δ15N (‰); niche width is represented by the corrected Standard Ellipse Area (thick

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lines) and Convex Hull Area (thin lines) for the two species in summer and winter.

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TABLE 2. Isotopic metrics for sea cucumbers collected in summer and winter: δ13C range (CR), δ15N range

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(NR), mean distance to centroid (CD), convex hull area (TA), mean nearest neighbor distance (MNND),

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standard deviation of nearest neighbor distance (SDNND), standard ellipse area corrected (SEAc).

H. polii Summer

H. tubulosa Winter

Summer

Winter

CR

2.65

2.74

3.85

3.35

NR

1.65

1.40

2.36

1.37

CD

0.95

0.67

1.11

1.29

TA

3.23

1.86

3.95

2.84

MNND

0.51

0.36

0.52

0.29

SDNND

0.33

0.28

0.41

0.15

SEAc

1.87

0.88

2.36

2.29

200 201

The mean isotopic signatures of the body wall tissue (BWT), intestine content (IC) and the marine sediment

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(MS) were plotted in Figure 3. Along the δ13C axis, BWT diverges considerably from IC and MS, showing

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enriched values. IC and BWT show enriched values compared to MS along the δ15N axis.

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Fig. 3. Biplot of mean δ13C and δ15N values (‰; ± SD) of the intestine content and body wall tissue of sea

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cucumbers and marine sediment in the summer and winter season.

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We defined four groups of food sources in addition to the marine sediment, all of which were used in the

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mixing model (SIAR), including the main potential detritus sources of the study area. The seagrass group

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was composed of C. nodosa and P. oceanica in summer, and of only P. oceanica in winter; the macroalgae

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group was composed of Sargassum sp. and Codium sp. in summer, Cystoseira sp. and Codium sp. in

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winter; the inland sources were composed of A. donax, P. australis and river sediment. Stable carbon

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isotope signatures of potential food sources spread widely in the bi-plot area in both periods (Fig. 4a, b). The

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river sediments and the terrestrial plants showed depleted values in respect to periphyton, seagrasses and

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macroalgae. By contrast, marine sediment placed in the center of the plots showed intermediate values

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between inland and marine producers. Stable nitrogen isotope signatures did not discriminate as well as

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those of carbon values. The highest δ15N values were represented by macroalgae in winter and by

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periphyton in summer whereas the riparian plants and the river sediments exhibited the least enriched

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values in both sampling periods.

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Values of H. polii and H. tubulosa were more aligned with marine sources than with those from the inland in

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both periods.

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Fig. 4. Bi-plots of mean δ13C and δ15N values (‰; ±SD) of the potential food sources and holothurians in

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summer (a) and winter (b).

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The contribution of the marine sources supporting the biomass of these two species estimated from the

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Bayesian mixing model, proved to be high in both periods. In particular, the model outcomes showed that the

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sources assimilated derived for the most part from the seagrass group in both summer (H. polii: mode 68%,

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H. tubulosa: 67%) and winter (H. polii: 74%, H. tubulosa: 63%) (Fig. 5). In addition, the mixing model

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estimated a small contribution of marine sediment to the diet of both holothurians.

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Fig. 5. SIAR results show the relative trophic contribution of food sources to the diet of H. polii (c, in summer,

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d, in winter) and H. tubulosa (a, in summer; b, in winter). Box plots indicate 95%, 75%, 50% confidence

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intervals/ high-density ranges of proportions of each food item.

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DISCUSSION

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Understanding the ecological roles of exploited species, such as H. polii and H. tubulosa, is essential for

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ecosystem-based fisheries’ management (EBFM), which aims to sustain healthy marine ecosystems and the

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fishing activities they support (Purcell et al., 2013, 2016). Hence, knowing how commercially exploited

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species are linked to the ecosystem processes through their trophic behaviour is a key prerequisite for an

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EBFM approach.

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Recent studies based on artificial feeding experiments have highlighted significant differences in the

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planktotrophic larval stage of these two species. H. polii showed fast larval development and high yields also

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under high feeding regimes. H. tubulosa, on the other hand, showed slow larval development and larval

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degeneration and mortality during the auricularia stage under the same high regimes, proving that a low

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feeding regime is more suitable for this particular species (Rakaj et al., 2018, 2019).

247

In this study, using data obtained from stable isotope analysis, the food selectivity/assimilation, diet temporal

248

variability and trophic niche of H. polii and H. tubulosa adults from the Mediterranean Sea were all

249

investigated. Information on the contribution of various food items to the diet was previously unavailable for

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these deposit-feeders living in coastal areas where the detritus originates from multiple sources. Macroalgae,

251

periphyton, seagrasses and inland sources are all well represented in the sampling site and constitute the

252

main sources of detritus entering the food chain in Mediterranean coastal ecosystems.

253

Stable isotope mixing models were used to analyze the relative contribution of the food sources and infer the

254

resource selection by H. polii and H. tubulosa. These sea cucumbers feed on detritus from the top layer of

255

the sediments; however, their signatures did not coincide with the mean isotope values of the marine

256

sediments. This fact highlights their ability to select or better assimilate carbon derived from a particular

257

source (mainly seagrasses) in both periods. Considering the results, it emerges that these two species are

258

able to digest and assimilate a food source with a very low level of digestibility. In fact, the lignocellulose

259

component contained in the seagrass fibers makes digestion difficult, preventing the exploitation of seagrass

260

leaves by most consumers (Ott and Maurer, 1977).

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The enriched δ13C values observed in the body tissues (mean -14.6 ± 1.0‰) (Fig. 3) compared to the marine

262

sediment (mean -22.1 ± 0.3‰) and to the intestinal content (mean -20.7 ± 0.7‰), highlighted the ability of

263

these sea cucumbers to assimilate, above all, carbon derived from seagrass detritus - the most 13C-enriched

264

source. Seagrasses contributed to the diet of both sea cucumbers in a range of 63% to 74%, and were the

265

main component of H. polii and H. tubulosa diets, in accordance with the results of Costa et al. (2014) in

266

artificial feeding experiments. On the basis of our results, we believe that the digestibility of seagrass fibers

267

may depend, for the most part, on microbial intermediates. This hypothesis is in line with the findings of

268

Amon and Herndl (1991) in H. tubulosa and of Li et al. (2016) in Apostichopus japonicus where in both cases

269

the intestinal microbiota seems to play an important role in the digestive processes of these species. Li et al.

270

(2016) observed that microbes present in the gut produce protease, amylase and cellulase; this last enzyme

271

may account for the high digestibility/assimilation of seagrass found in H. polii and H. tubulosa.

272

The high δ15N values resulting for the intestinal content compared to the marine sediment can be also

273

explained by the increase in the bacterial biomass in the digestive tract (Plante et al., 1989, 1990). Amon

274

and Herndl (1991) observed in fact that in H. tubulosa most of the bacterial growth occurred in the foregut

275

region with an increase in percentage of bacterial active cells from 4.1% in the sediment to 12.2% in the

276

foregut and a subsequent decline to 6.2% in freshly egested feces. Moreover, bacteria within the mucus

277

layer in the gut also appear to be significantly larger (more than double) in volume than sediment bacteria.

278

According to these authors, the bacteria biomass evolution along the digestive tract indicates an efficient

279

bacteria absorption; this could explain the high δ15N values also found in H. polii and H. tubulosa body wall

280

tissues (mean 8.6 ± 1.1‰). On this basis, we hypothesize that seagrass assimilation may occur in these

281

deposit feeders through the digestion of endosymbiotic bacteria in their intestine.

282

However, another feeding pathway may contribute to explain the isotopic values observed in the body wall

283

tissues of these sea cucumbers. Heterotrophic organisms are in fact abundant on the surfaces of degrading

284

seagrass detritus, as indicated from scanning electronic microscope images by Lepoint et al. (2006).

285

Therefore, these bacteria probably also constitute a further food source for these deposit feeders. This

286

hypothesis is in line with the “microbial stripping theory”, as supposed for other invertebrates (Lopez and

287

Levinton, 1987). However, microbial abundance viable in the sediment through this feeding pathway has

288

appeared to be far too low to meet energy demands, also for high selective deposit feeders (Lopez and

289

Levinton, 1987; Cammen, 1989; Harris, 1993). In this context, symbiotic gut microbes could explain the gain

290

in energy necessary to supplement their diet.

291

These results qualify H. polii and H. tubulosa for the list of the few species capable of feeding on seagrass

292

detritus in the Mediterranean Sea, alongside for e.g. copepods (Mascart et al., 2018), amphipods (Lepoint et

293

al. 2006), isopods (Sturaro et al., 2010) and polychaetes (Cigliano et al., 2003). The fact that the biomass of

294

these sea cucumbers can be very high in seagrass beds and the surrounding sandy bottoms (Francour,

295

1989a; Bulteel et al., 1992; Coulon and Jangoux, 1993; Mezali et al., 2006), together with their ability to feed

296

on seagrass detritus suggests that these species may have a key role in matter processing and in the energy

297

pathway deriving from seagrass detritus in Mediterranean coastal areas.

298

Overall, our data suggests that detritus deriving from seagrass meadows is also exported outside the

299

meadow systems where it can constitute the major carbon source for the sea cucumbers’ diet - as observed

300

in this study. From these results, we can conclude that Mediterranean seagrass beds not only constitute a

301

habitat for these two species, but more importantly, a food source through refractory detritus generation

302

which can be transferred and transformed outside the meadows, hence corroborating previous claims

303

(Danovaro, 1996; Mascart et al., 2018; Remy et al., 2018).

304

Regarding the temporal dynamics of the isotopic pattern, δ13C values in seagrasses showed wide

305

differences between periods, with depleted values in winter and enriched values in summer. This annual

306

trend is in line with the observation of several authors and is due to variability in environmental factors such

307

as irradiance and temperature (Cooper and DeNiro, 1989; Hemminga and Mateo, 1996; Vizzini et al., 2002;

308

Vizzini and Mazzola, 2003; Fourqurean et al., 2007; Vizzini, 2009). The δ15N values reveal the same

309

dynamics, depending on the chemical form and on the environmental nitrogen availability for primary

310

production (Pirc and Wollenweber, 1988; Alcoverro et al., 1995; Lepoint et al., 2000; Invers et al., 2002,

311

2004; Vizzini and Mazzola, 2003). Seasonality emerges as an important factor affecting the isotopic

312

signature of both sea cucumber species. In particular, the nitrogen and carbon isotopic enriched values

313

found in summer and depleted in winter for H. polii and H. tubulosa overlap with those of seagrasses in

314

these periods.

315

Stable isotope analyses are also a powerful tool for studying the ecological niche extension (Newsome et al.,

316

2007). In our case-study, the coexistence of these deposit-feeders, in a stable ecosystem, may be possible

317

only if there is a degree of segregation of their ecological niches in terms of foraging behavior or digestive

318

physiology (Hutchinson and Macarthur, 1959; Schoener, 1974). The isotopic signature of body wall tissue

319

was considered representative of the trophic niche of sea cucumber during the months preceding sampling

320

(Layman et al. 2007). From our results, the isotopic niche metrics (SEAc and CD) indicate that H. tubulosa

321

has a wider isotopic niche than H. polii; although the two species showed a similar redundancy level (MNND

322

and SDNND metrics).

323

When comparing the isotopic niches by measuring their overlap with a range 0-1 (where 0 indicates no

324

overlap and 1 indicates a complete overlap), the result fails to distinguish a complete resource partitioning

325

between H. polii and H. tubulosa along the food axis. Indeed, during summer, the overlap of the niches

326

ranged between 0.53 and 0.61 whereas, during winter, it ranged between 0.29 and 0.81. The wide winter

327

variation reflects a larger niche extension gap between the two species in this season. Furthermore, no

328

significant difference emerged between H. polii and H. tubulosa in the δ13C values. Apparently, resource

329

segregation does not prove interspecific competition for food sources in terms of δ13C. Instead, the partial

330

degree of segregation observed depends mainly on the δ¹⁵N values (Tab.1), which could indicate possible

331

differences in digestive physiology in terms of intestinal microbiota (Sponheimer et al. 2003); or a

332

specialization in order to utilize the same resource (seagrass detritus), albeit within different microhabitats

333

e.g. in terms of sediment layers (with can present specific bacterial communities). Unfortunately, literature is

334

lacking on this topic and therefore further research is needed to investigate the trophic processes in these

335

deposit feeders.

336 337

CONCLUSION

338

The present study is the first to outline the trophic ecology of the Mediterranean sea cucumbers H. polii and

339

H. tubulosa by means of isotopic analysis, and to evaluate the role of different trophic resources in their diet.

340

Stable isotope results indicate that when various sources are present, as was the case in the study area, a

341

selection/preferential assimilation of seagrass detritus by H. polii and H. tubulosa occurs. The δ13C and δ15N

342

values in sea cucumbers follow the seasonal isotopic dynamics of the seagrasses also outside the seagrass

343

beds. This finding signifies that the feeding activity of these species plays a crucial role in the organic matter

344

cycling derived from the seagrass meadows in Mediterranean coastal ecosystems. Future research is

345

therefore needed to investigate the feeding microhabitats and to better understand the role of the intestinal

346

microbiota in the digestive processes of the species analyzed in this study.

347 348

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Sampling site Astura Tower

N

500 m

11

10

δ15N‰

9

8

7

H. polii summer

H. polii winter H. tubulosa summer

H. tubulosa winter

6 -18

-17

-16

-15

δ13C‰

-14

-13

-12

11

Intestine Content

10

Body Wall Tissue

9

8

7

6

5

Marine Sediment H. polii summer H. polii winter H. tubulosa summer H. tubulosa winter Marine sed. summer Marine sed. winter

4

3

-22.5

-21.0

-19.5

-18.0

-16.5 13

C‰

-15.0

-13.5

-12.0

Summer

Mar. Sediment

Periphyton

Macroalgae Inland sources Marine Sediment

‰

Seagrass

O

H. tubulosa H. polii

‰

Winter

Mar. Sediment

Macroalgae Periphyton Seagrass Inland sources

‰

Marine Sediment

O

H. tubulosa H. polii

‰

Periphyton

Periphyton

Periphyton

Periphyton