Patterns and drivers of meiofaunal assemblages in the canyons Polcevera and Bisagno of the Ligurian Sea (NW Mediterranean Sea)

Accepted Manuscript Patterns and drivers of meiofaunal assemblages in the canyons Polcevera and Bisagno of the Ligurian Sea (NW Mediterranean Sea) L. Carugati, M. Lo Martire, R. Danovaro PII: DOI: Reference:

S0079-6611(18)30093-4 https://doi.org/10.1016/j.pocean.2019.03.010 PROOCE 2094

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Progress in Oceanography

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5 April 2018 25 November 2018 27 March 2019

Please cite this article as: Carugati, L., Lo Martire, M., Danovaro, R., Patterns and drivers of meiofaunal assemblages in the canyons Polcevera and Bisagno of the Ligurian Sea (NW Mediterranean Sea), Progress in Oceanography (2019), doi: https://doi.org/10.1016/j.pocean.2019.03.010

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Patterns and drivers of meiofaunal assemblages in the canyons Polcevera and Bisagno of the Ligurian Sea (NW Mediterranean Sea)

Carugati L.1*, Lo Martire M.1, Danovaro R.1,2

1

Università Politecnica delle Marche, Dipartimento di Scienze della Vita e dell’Ambiente,

60131 Ancona, Italy, 2

Stazione Zoologica Anton Dohrn, 80121 Napoli, Italy

*Corresponding author: tel: +39 0712204328; e mail: [email protected]

Submitted to Progress in Oceanography Special Issue on “Ecology and functioning of Mediterranean submarine canyons”

Abstract Meiofaunal abundance, assemblage structure and richness of higher taxa were investigated for the first time in two submarine canyons (Polcevera and Bisagno) of the Ligurian Sea and on the adjacent open slope, in relation with the quantity and quality of sedimentary organic matter and other environmental variables, including grain size. Meiofaunal abundance and richness of higher taxa decreased with increasing water depth (from ca. 200 down to ca. 2000-m depth) in the open slope and Polcevera canyon, whereas the highest values were observed at 500 m depth in the Bisagno canyon. The comparison between canyons and the adjacent open slope, showed the lack of significant differences in meiofaunal abundance, at the same depth except for samples collected at 200 and 2000-m depth. Overall the biodiversity was higher in canyons than in the open slope. Phytopigments, utilised as a proxy of the input of primary organic matter, were up to 3 times higher in canyon than in slope sediments and, along with grain size, explained a large portion of the variability in all meiofaunal variables. Canyon and slope showed a high beta diversity (83%), mostly due to the presence of a high portion of rare taxa in the canyons. Some taxa, such as Cladocera, Cumacea, Gastrotricha, Nemertina were exclusively encountered in canyon sediments, whereas Tardigrada were encountered only in the adjacent slope. Results reported here indicated that, differences in meiofaunal assemblages between canyons and slopes are primarily driven by quantity and quality of the available food resources and by the presence of specific topographic features.

Keywords: submarine canyons, meiofauna, food sources, deep sea, Ligurian Sea

Introduction Submarine canyons can profoundly influence the environmental conditions and consequently ecological processes (Canals et al., 2009, 2013; Puig et al., 2014; Amaro et al., 2016; Fernandez-Arcaya et al., 2017). Active canyons, for instance, can be characterised by the presence of dense-shelf water cascadings (Canals et al., 2006; Pasqual et al., 2010; Tesi et al., 2010), which convey huge amounts of sediments and organic material from the continental shelf to the deep sea with important ecological implications (Canals et al., 2006; Company et al., 2008; Pusceddu et al., 2013). At the same time, canyons increase topographic complexity and can host endemic species, which contribute to enhance regional biodiversity (Tudela et al., 2003; Skliris and Djenidi, 2006; Company et al., 2008; De Leo et al., 2010; De Mol et al., 2011; Danovaro et al., 2014). More than 500 large submarine canyons have been identified in the Mediterranean Sea (Harris and Whiteway, 2011; Wurtz 2012) with a large portion located in the North Western Mediterranean. Among these, along the Ligurian margin, Polcevera and Bisagno canyons have been previously investigated for their chemical and geological features (Pierce et al., 1981; Frache et al., 1986; Corradi et al., 1987). Our knowledge about biological assemblages associated with submarine canyons and their ecology has increased significantly during the last decade (Wurtz, 2012). Now we know that a complex array of factors, including C inputs, food availability, hydrographic conditions and episodic disturbance events can shape community composition and distribution patterns (Garcia et al., 2007; Levin and Sibuet, 2012; Amaro et al., 2016). However, so far, the canyons of the Ligurian Sea remained overlooked and information on their data biology, ecology and functioning are still lacking. Metazoan meiofauna dominate the deep-sea benthos in terms of abundance and biomass (Giere, 2009; Pape et al., 2013a,b). Previous studies, conducted on continental margins all over the world, highlighted the role of grain size, food quantity and the

nutritional value of the organic matter supplied in driving meiofaunal distribution (Vincx et al., 1994; Smith and Druffel, 1998; Snelgrove and Smith, 2002;). During the last decade, meiofaunal assemblages inhabiting deep-sea canyons have been increasingly investigated (Bianchelli et al., 2010; Romano et al., 2013; Ramalho et al., 2014; Gambi and Danovaro, 2016; Roman et al., 2016). However, studies carried out on Mediterranean deep-sea canyons provided evidence of contrasting results, spanning from the lack of significant differences (e.g., in Mediterranean and Portuguese margins; Garcia et al., 2007; Bianchelli et al., 2010; Gambi and Danovaro, 2016), to the presence of differences but not consistently over time (Blanes Canyon, NW Mediterranean; Romano et al., 2013; Roman et al., 2016). Indeed, Mediterranean canyons when compared to the adjacent open slopes displayed either significantly higher or much lower meiofaunal abundances and biodiversity, making difficult to assess the relative importance of environmental and trophic factors influencing benthic assemblages in such complex systems (Ingels et al., 2009, 2013; Bianchelli et al., 2010; De Leo et al., 2010; Romano et al., 2013; Ramalho et al., 2014; Gambi and Danovaro, 2016; Roman et al., 2016). Benthic fauna, and particularly meiofauna, are also dependent upon the specific geomorphological characteristics and topographical heterogeneity of the deep seafloor (Snelgrove and Smith, 2002; Amaro et al., 2016; Zeppilli et al., 2016). The Polcevera and Bisagno canyons are an interesting case study for several reasons. Both canyons still encompass the complexities of shelf-incising submarine canyons with no clear bathymetric connection to a major river system (as defined by Harris and Whiteway, 2011). However, they differ in terms of maximum depth, length, slope and dendricity, as number of limbs. In this context, a question has been raised as to whether the interaction between food availability and canyon geomorphological characteristics could act as factors controlling meiofaunal distribution and biodiversity. To provide insights on this topic, here we investigated the variations of meiofaunal assemblages (in terms of abundance, taxa richness and taxonomic composition), grain size, quantity and quality of trophic resources

along the Polcevera and Bisagno canyons and the western open slope. We also tested the hypothesis that meiofaunal assemblages of the two canyons differ from those of the slope not only as a result of the different quantity and quality of trophic resources but also for the interactions between trophic conditions and canyon geomorphological features.

Material and Methods 2.1. Study area The Ligurian Sea is in the north-eastern part of the Western Mediterranean Sea bounded by the French (Provence, Corsica) and Italian (Gulf of Genoa) coasts, with a maximum depth of about 2850 m. The Gulf of Genoa, located between the city of Nice and Genoa, is part of the Ligurian Sea and extends, West to Eastward, from Capo Mele to the Gulf of La Spezia with a surface of about 5000 km². The Gulf of Genoa is bordered by mountains with a hydrography characterized by short streams with limited seasonal input. The Ligurian margin, characterized by a 4 km-wide continental shelf, is eroded by numerous submarine canyons. Ligurian canyons begin in shallow waters, and reach the bathyal plane approximately 30 km offshore. The canyons Polcevera and Bisagno are separated at shallower depths and then descend in parallel to converge into a single canyon at deeper depths, creating an intricated system defined as the “Canyon of Genoa”. This represents one of the largest submarine canyon in the entire Mediterranean and a crucially important fishing area for many species of commercial interest, including the red shrimp Aristeus antennatus (Orsi Relini and Relini, 1998; Tudela et al., 2003).

2.2. Sampling strategy During the BioLig cruise (8th–20th May 2013) on board the R/V Minerva Uno, sediment samples were collected at 11 stations, selected along 3 bathymetric transects, 2 of which along the Polcevera and Bisagno canyons and one along the western open slope, at depths

ranging from 200 to 2000 m depth (Fig. 1; Table 1). At each sampling station, sampling activities were performed by using a NIOZ-type box corer, that, being hermetically closed, allowed to collect cores with a perfectly undisturbed sediment surface. Replicate samples from independent box-corer deployments (n = 3) were collected at all sampling stations, for meiofaunal parameters and for prokaryotic abundance and organic matter composition. Sediment samples were preserved at -20 °C for meiofaunal and organic matter analyses, or fixed in 2% formaldehyde for the analysis of total prokaryotic abundance. Despite the storage at -20 °C, all the identified organisms, including the soft-body individuals, were well-preserved. In addition, freezing did not damage the morphological features used to recognise organisms at the higher taxonomic levels to which we identified them.

2.3. Grain size, sedimentary organic matter content and biochemical composition Grain size analyses were carried out by sieving on mesh nets (Covazzi et al., this Special Issue). Sediment samples have been collected at all sampling stations for the analysis of the total phytopigments and the biochemical composition of the organic matter (proteins, carbohydrates and lipids). Briefly, chlorophyll-a and phaeopigments (used as proxies of primary organic material associated with primary producers) were analysed fluorometrically (Lorenzen and Jeffrey, 1980) and total phytopigment contents defined as the sum of chlorophyll-a and phaeopigment concentrations. Total phytopigment contents were utilized as an estimate of the organic material of algal origin, including the living (chlorophyll-a) and senescent/detrital (i.e., phaeopigments) fractions (Pusceddu et al., 2009). Sediment phytopigment concentrations were converted into carbon equivalents using a mean value of 40 mgC mg phytopigment -1 (Pusceddu et al., 2009). Protein, carbohydrate and lipid were determined spectrophotometrically, following the protocols detailed in Danovaro (2010), and their sedimentary contents (mg g dry sediment -1) expressed as bovine serum albumin, glucose and tripalmitine equivalents, respectively. Carbohydrate, protein and lipid

sedimentary contents were converted into carbon equivalents using the conversion factors of 0.40, 0.49 and 0.75 µgC µg-1, respectively, and their sum defined as biopolymeric carbon (BPC; Fabiano et al., 1995). Moreover, we used the contributions of phytopigment and protein to biopolymeric C concentrations and the values of the protein to carbohydrate ratio as descriptors of the ageing and nutritional quality of sediment organic matter (Pusceddu et al., 2009). The percentage contribution of total phytopigments to biopolymeric C is an estimate of the freshness of the organic material deposited in the sediment: since photosynthetic pigments and their degradation products are assumed to be labile compounds in a trophodynamic perspective, the lower their contribution to sediment organic C the more aged the organic material. Moreover, since the percentage fraction of organic C associated with phytopigments is also typically associated with a higher fraction of enzymatically digestible (i.e. promptly available for heterotrophs) compounds, higher values of this percentage will also be indicative of a comparatively higher nutritional quality. Since N is the most limiting factor for heterotrophic nutrition and proteins are N-rich products and degraded at faster rates than carbohydrates, the protein to biopolymeric C and the protein to carbohydrate ratios are indicative of both the ageing and the nutritional value of the organic matter (Pusceddu et al., 2009).

2.4 Prokaryotic abundance in sediments For the quantification of prokaryotic abundance, stored sediment samples were treated as in Danovaro (2010). Surface sediment slurries were treated with tetrasodium pyrophosphate (100 mmol/l final concentration) and ultrasounds (1 minute of treatment followed by 30s of manual shaking, repeated 3 times), before undergoing centrifugation (800 xg, 1 min) to pellet the sediment particles. The supernatant was diluted with virus-free seawater. Diluted samples were filtered on 0.2 μm pore size 25mm filter membranes and stained with 100 μl of SYBR Green I (10,000x stock solution diluted 1:20 in TE buffer). Filters were incubated

in the dark for 20 min, washed 3 times with 3 ml of virus-free seawater and then mounted on glass microscope slides with 20 μl of antifade solution (5% phosphate-buffered saline; 5% glycerol, 0.5% ascorbic acid). Prokaryotic abundance count was carried out by epifluorescence microscopy (magnification 1000x) analyzing at least 20 optical fields or 200 prokaryotic cells. Microscope slides were stored at -20°C. Data were normalized to sediment dry weight. All analyses were carried out on 3 replicates for each site.

2.5. Meiofaunal analysis Each sample was treated with ultrasound (for 1 min x 3 times, with 30 s intervals) to detach organisms from the grain particle surface and, then, sieved through a 1000-µm and a 20-µm mesh net to retain the smallest organisms. The fraction remaining on the latter sieve was resuspended and centrifuged three times with Ludox HS40 (diluted with water to a final density of 1.18 gcm-3; Danovaro 2010). All specimens from 3 independent replicates per station were counted and sorted by taxa, under a stereomicroscope and after staining with Rose Bengal (0.5 gL-1). Meiofaunal taxa representing <1% of the total meiofaunal abundance were defined as rare taxa (Bianchelli et al. 2010). According to the literature and, therefore, for comparison purposes, meiofaunal abundance was expressed as number of individuals 10 cm-2, and their diversity expressed as richness of taxa. Overall, total richness of taxa retrieved at each station was expressed as the number of taxa retrieved from all replicates.

2.6. Statistical analyses Distance-based permutational multivariate analyses of variance (PERMANOVA; McArdle and Anderson, 2001) were used to test for differences in all investigated variables, using unrestricted permutations of the raw data. The analyses were carried out using Habitat (3 fixed levels: Polcevera and Bisagno Canyon and slope) and Depth (5 fixed levels: 200, 500,

1000, 1500, 2000m) as main sources of variance. The analyses were carried out on Euclidean distance (organic matter, meiofaunal abundance and taxa richness) or Bray–Curtis similarity matrices (meiofaunal taxonomic composition) of previously normalized (organic matter) or untransformed (faunal) data, using 999 permutations of the residuals under a reduced model. For taxonomic composition, we used Bray-Curtis distance matrix because for differences in community structure and composition, the semi-metric Bray–Curtis measure (Bray and Curtis, 1957) of ecological distance is preferred over metric measures (Anderson, 2001), like Euclidean distance (Odum, 1950; Hajdu, 1981; Faith et al., 1987; Clarke, 1993). Moreover, in order to avoid bias on significance due to data transformation, all analyses on meiofauna were ran using a non-transformed data matrix (Mc Ardle and Anderson; 2004). For those PERMANOVA tests providing significant differences among habitats at the same depth and between sampling depths within the same habitat, pairwise tests were also carried out. Because of the restricted number of unique permutations in the pairwise tests, P values were obtained from Monte Carlo samplings (Anderson and Robinson, 2003). Since PERMANOVA is sensitive to differences in multivariate dispersion among groups, we used also a test of homogeneity of dispersion (PERMDISP) to test the null hypothesis of equal dispersion among groups (Anderson et al., 2008). Bi-plots produced after a principal component analysis (PCA) were used to visualize differences among habitats in the meiofaunal taxonomic composition as all and rare taxa. Additionally, in order to quantify the beta diversity, expressed as percentage of dissimilarity in meiofaunal taxonomic composition among habitats at similar depth and between depths within each habitat, SIMPER tests were also carried out (Gray, 2000). Beta diversity may reflect two different phenomena, turnover and nestedness of assemblages, which result from two antithetic processes, namely species replacement and species loss, respectively. Thus, in order to detect whether dissimilarities in meiofaunal taxonomic composition occurred through the replacement of some taxa by others or through the loss of taxa, we also

partitioned beta diversity into turnover and nestedness of components (Balsega 2010, 2012). To identify the potential environmental and trophic drivers of meiofaunal variables, nonparametric multivariate multiple regression analyses based on Euclidean (for abundance and taxa richness) or Bray-Curtis (for taxonomic composition) distances were carried out, using the routine DISTLM (McArdle and Anderson, 2001). Additional DISTLM analyses were performed when meiofaunal variables showed significant differences among and within habitats. The forward selection procedure and R2 as the selection criterion of the predictor variables was carried out with tests by permutation. P values were obtained using 4999 permutations of the raw data for the marginal tests (tests of individual variables), whereas for all of the conditional tests, the routine used 4999 permutations of residuals under a reduced model. To run this test meiofaunal variables were used, separately, as dependent variables whereas grain size, depth, total phytopigments, biopolymeric C, protein to carbohydrate ratio, protein to biopolymeric C, phytopigments to biopolymeric C and prokaryotic abundance were used as potential explanatory variables. To determine whether the meiofaunal assemblages of Polcevera and Bisagno canyons were influenced by interactions between environmental, trophic conditions and canyons peculiar geomorphological characteristics, we carried out another multivariate multiple regression analysis (DISTLM forward, McArdle and Anderson, 2001). Canyons geomorphological characteristics were obtained from data reported in Harris and Whiteway (2011). Beside environmental and trophic variables listed above, the following variables were included in the statistical analysis: top (head depth – depth at which the canyon commences) and low heights (termination depth – depth at which the canyon ends), height difference (measured as the difference in depth from the head to the foot of the canyon ), average length and slope, dendricity (n. of canyon limbs) and dendricity per 100,000 km2, sinuosity (Harris and Whiteway, 2011). The PERMANOVA, PCA, SIMPER and DISTLM

analyses were performed using the routines included in the PRIMER 6+ software (Clarke and Gorley, 2006; Anderson et al. 2008).

Results Geomorphological characteristics, grain size and potential food sources Polcevera and Bisagno canyons belong to the type 2 canyon according to Harris and Whiteway (2011): i.e., shelf-incising canyons with no clear bathymetric connection to a major river system. They present different maximum depth (2040 and 1805 m depth), length (46 and 29 km) and slope (5 and 6 degrees, for Polcevera and Bisagno, respectively). In addition, Polcevera canyon is characterized by higher dendricity (20 limbs per 100,000 km2), than the canyon Bisagno (18 limbs per 100,000 km2). The analysis of grain size distribution showed that sediment samples mainly consisted of silt representing on average 55% in the open slope and 54% in the canyons, followed by clay representing on average 43% along the slope, vs 42-44% of canyons. Sand accounted for ca. 2 and 3-4% in the open slope and in the two canyons, respectively. Both Polcevera and Bisagno canyons were characterized by the highest value of sand (6%) registered at 1000 and 500 m depth, respectively (Table 1). The concentration of biopolymeric carbon differed between canyons and slope, with significantly higher values registered at 200m (in both canyons) and 500 m depth (in the Bisagno canyon), compared to the slope (Fig. 2a; Table 2; Table S1; PERMANOVA, P<0.01). Along the slope, sedimentary biopolymeric carbon contents were significantly higher at 200 m than at 500 and 1500 m depth (PERMANOVA, P<0.01), whereas decreased with increasing water depth along the two canyons. This pattern was more pronounced in the Polcevera canyon, where values were significantly higher at 200 m depth than at two deepest investigated stations (Tables S1, S2; PERMANOVA, P<0.01).

Sedimentary phytopigment contents were higher in the two canyons than slope, at all sampling stations (with the only exception of 200 m in the Bisagno canyon, where we found lower values compared to the slope). Slope and Polcevera samples exhibited decreasing total phytopigment concentrations with increasing water depth, whereas in the Bisagno canyon the highest value was recorded at intermediate sampling station (ca 500 m depth). The descriptors of ageing and nutritional quality of organic matter in the sediments (protein : carbohydrate ratio and the C percentage of protein to biopolymeric carbon) differed between canyons and slope, with significantly higher values registered in the two canyons than in the slope, at 200 (PERMANOVA, P<0.01) and 1000 m depth (PERMANOVA, P<0.001). At the slope, we registered the lowest value at 1000 m depth, whereas in the two canyons we found significantly lower values at the deepest stations (1500 m and 2000 m depth in the Polcevera and Bisagno canyons, respectively) than at the other sampling depths (PERMANOVA, P<0.01). The autotrophic fraction of the organic pool that represent a measure of the “freshness” of organic matter (expressed as percentage of phytopigment C to the biopolymeric carbon pool) was significantly higher in the two canyons than in the open slope at all sampling depths, except for 200 m depth (Fig. 2b). At the slope, the algal fraction of organic matter showed a clear decrease with increasing water depth. Whereas, in the Bisagno and Polcevera canyons, we found higher values at 500 and 1000 m depth, respectively, than at the other sampling stations. The carbohydrate carbon represented the major fraction of biopolymeric C accounting on average for 51% in the open slope, and from 45 to 47% in the canyons, followed by protein carbon representing on average 33% along the slope, vs 38-40% of canyons. The lipids accounted for ca. 16 and 15% in the open slope and in the two canyons, respectively (Fig. 3). PERMANOVA analysis revealed that the biochemical composition of sedimentary organic matter varied significantly among slope and canyons at all sampling

depths except for 1500 and 2000 m (Table S1). Both in the slope and canyons we observed an increase in the percentage contribution of carbohydrates to total organic matter with water depth. The abundance of benthic prokaryotes was significantly higher in the Bisagno canyon than at slope and Polcevera canyon, at 200 (PERMANOVA, P<0.01) and 500 m depth (PERMANOVA, P<0.001). PERMANOVA analysis showed the lack of statistical differences between sampling depths within each habitat, except for significant lower values found at 500 than 1000 m along the slope and at 200 than 500 m in the Bisagno canyon (Tables S1, S2¸ PERMANOVA, P<0.05).

Faunal diversity and assemblage structure Total meiofauna abundances did not differ between canyons and the open slope, except at the deepest sampling depths, where the number of individuals were significantly higher in the slope than in the Bisagno canyon (Fig. 4a; Table S3, PERMANOVA, P<0.01). At the slope, the number of individuals did not significantly vary along the bathymetric gradient (PERMANOVA, ns). The minimum slope abundance recorded was 109 ± 66 ind. 10 cm−2 at 500 m and the maximum was 650 ± 222 ind. 10 cm−2 at 200 m depth. In the Polcevera canyon, the number of individuals were higher at 200 m depth (643 ± 230 ind. 10 cm−2) than at the deepest station (138 ± 63 ind. 10 cm−2 at 2000 m depth). Along the Bisagno canyon we reported a significantly higher meiofaunal abundances at 500 (552 ± 173 ind. 10 cm−2) than at 2000 m depth (68 ± 10 ind. 10 cm−2) (Tables S3, S4; PERMANOVA, P<0.05). Overall, 14 meiofaunal higher taxa were identified (Fig. 4b). Considering all sampling stations, a total of 12 and 11 taxa were found in the Bisagno and Polcevera canyons, respectively, while, 10 taxa were recorded in the western open slope. Nemertina and Gastrotricha were exclusive of the Bisagno canyon, whereas Cladocera were found only along the Polcevera canyon. Cumacea were present in both the canyons and not along the slope, whereas Tardigrada were exclusive of slope sediments. The maximum number of taxa

(11) was found at 200 and 500 m depth in the Polcevera and Bisagno canyons, respectively. Along the slope and Polcevera canyon we registered a significant decrease in the number of taxa with water depth, whereas in the Bisagno canyon the number of taxa was highest at intermediate sampling depth (500 m). The meiofaunal assemblages in the Ligurian canyons and open slope were dominated by nematodes (84-96%) followed by copepods (0.2-14%). All other taxa represented <1% of the total meiofaunal assemblage. Relative nematode abundance was slightly higher in the Bisagno canyon compared to the Polcevera canyon and slope. The reduced dominance of nematodes at 200 m depth along the Polcevera canyon was countered by a relative increase of copepod and nauplii abundances. The abundance of copepods was higher in the Polcevera than Bisagno canyon at the shallowest sampling depth (Fig. 5a,b; Table S5). Meiofaunal taxonomic composition did not change significantly between the two habitat types except at 200 and 2000 m depth. In accordance with the PERMANOVA analyses, the results of the PCA ordination plots confirmed the lack of consistent differences between canyons and slope (Fig. 6). The assemblage structure of meiofaunal higher taxa was significantly different along the bathymetric gradient in the two canyons, whereas no statistical differences were found among sampling depths along the slope. Comparing different habitats at similar depth, we found the highest percentage of dissimilarity between the slope and the Bisagno canyon, at 500 m depth (66%). We also found a high percentage of dissimilarity (44%) in meiofaunal taxonomic composition between the two canyons. Comparing different sampling depths within the same bathymetric transect, we found that beta diversity varied from 19 to 69% along the open slope, from 25 to 64% in the Polcevera canyon and from 46 to 75% in the Bisagno canyon. Our results showed that beta diversity among habitats was mostly attributable to nestedness (i.e., taxa loss) than turnover (i.e., replacement of taxa), with the only exception found at 200 and 1000m depth among slope and Polcevera canyon, where beta diversity corresponded almost exclusively to turnover.

Similarly, taxa loss accounted more than replacement of taxa for beta diversity found within each habitat, except between 200 and 1000 m depth along the slope (where 40% was attributable to replacement and 30% to taxa loss) and between 200 and 50 m depth along the Bisagno canyon (where 33% was attributable to replacement and 22% to taxa loss). The results of the PERMANOVA analysis for meiofaunal composition of rare taxa revealed a significant effect of the factors habitat (PERMANOVA, P<0.01) and depth (PERMANOVA, P<0.001) (Table 3). Canyons and slope samples separated in the PCA, where first two PC axes explained 61.2% of the variation. The first PC axis was manly defined by Polychaeta (0,586) and negatively by Tardigrada (-0,089). The second PC axis was mainly defined by Oligochaeta (0,778) and negatively by Kinorhyncha (-0,547) (Fig. 7a). The highest percentage of dissimilarity (100%) in the meiofaunal composition of rare taxa was found at 1500 m depth between the slope and the Polcevera canyon. Within each habitat, the percentage of dissimilarity ranged from 83 to 100% along the open slope, from 64 to 88% along the Polcevera canyon and from 63 to 94% along the Bisagno canyon. Trophic variables along with grain size significantly explained more than 50% of the observed variation in meiofaunal variables, with the main contributor being total phytopigments, significantly explaining 37, 46 and 27% of the variance of meiofaunal abundance, richness and taxonomic composition, respectively. Grain size, and in particular the percentage of silt, explained 12 and 6% of the variation of meiofaunal abundance and taxonomic composition, respectively. Gravel contributed to explain 9% of the variation of taxa richness (Table 4). The DISTLM analyses performed to test the influence of the interactions between environmental, trophic and geomorphological characteristics on meiofauna among the two canyons, revealed that grain size, trophic conditions (i.e., phytopigments, percentage of protein to biopolymeric C) and geomorphology, (i.e., maximum depth of canyon)

cumulatively explained a large fraction of the variation of meiofaunal assemblages between the two canyons (Table 5).

Discussion The comparison between the results reported here with those previously reported for other canyons of the NW Mediterranean Sea (Cap de Creus/Sete and Blanes Canyons), revealed a similar meiofaunal abundance and richness of higher taxa (Bianchelli et al., 2010; Romano et al., 2013). In addition, the analysis of meiofaunal assemblages confirmed the presence of a different structure along the two canyons comparing shallower and deeper depths and between canyons and open slope, at 200 and 2000 m depth.

Depth-related patterns of meiofaunal variables The decrease of meiofaunal abundance with increasing water depth is a repeatedly reported pattern in deep-sea biology investigations and has been linked to the bathymetric decrease in organic matter supply (Gremare et al., 2002; García et al., 2007; García and Thomsen, 2008). However, this pattern can be altered by the presence of peculiar topographic and hydrodynamic features characterizing submarine canyons (Ingles et al., 2009, 2011; Amaro et al., 2016; Roman et al., 2016). The complex topography of the canyons can modify current speed and direction, enhancing sediment-transport processes, sedimentation rates and in turn influencing the diversity and functioning of both pelagic and benthic ecosystems (Puig et al., 2014; Amaro et al., 2016; Fernandez-Arcaya et al., 2017). In the present study, we found a decreasing pattern of meiofaunal abundance with increasing water depth, but with significant exceptions. In fact, while both the Polcevera canyon and the open slope displayed this classical pattern, the Bisagno canyon did not. A similar contrasting pattern was also observed for the richness of higher taxa, which picked at intermediate water depth in the Bisagno canyon. This pattern was apparently due to the

specific ecological features of this canyon, where we found coarser sediments and a higher availability and freshness of organic matter (i.e., sedimentary contents of total phytopigments and their percentage contribution to biopolymeric carbon) at 500 m depth, in correspondence of the highest number of taxa. The multivariate multiple regression analysis confirmed that total phytopigments explained most of the variance in meiofaunal taxa richness (72, 58 and 41% along the slope, Polcevera and Bisagno canyons, respectively; Table S6). Meiofauna standing stocks, indeed, are closely linked not only to the food quantity, but also to the quality of the organic matter supplied (Snelgrove and Smith, 2002). The algal material reaching the deep-sea floor is known to represent an important trophic resources for meiofauna, especially for benthic copepods (Hicks, 1983). This could be one possible explanation for the observed increased abundance of copepods at the shallowest depth in the Polcevera canyon, namely the direct utilization of fresh material by harpacticoids (Tselepides and Lampadariou, 2004). Besides trophic availability, grain size contributed to explain the variation of meiofaunal assemblages along the bathymetric transect within each habitat, confirming the important role of this factor in driving the distribution of benthic assemblages in the deep sea and, particularly, in deep-sea canyons (Giere et al., 2009; Roman et al., 2016; Thistel et al., 2017). In addition, along the Polcevera canyon, the variation of meiofaunal taxonomic composition was explained by prokaryotic abundance, highlighting the important role of this component as food for meiofaunal assemblages. Indeed, an important portion of deep-sea meiofaunal organisms, especially deep-sea nematodes, is represented by bacterivorous and microbial feeders (Gambi and Danovaro, 2006, 2016; Ingels et al., 2009; Danovaro et al., 2013; Gambi et al., 2014).

Meiofaunal assemblages: canyons vs slope Previous studies that compared meiofaunal variables between canyons and the open slopes, provided contrasting results, from the lack of significant differences (Garcia et al., 2007; Bianchelli et al., 2010; Gambi and Danovaro, 2016), to the evidence of higher or lower meiofaunal abundance and diversity in canyon than in the open slope (Koho et al., 2008; Romano et al., 2013; Roman et al., 2016). In our study, the comparison between canyons and the adjacent slope, revealed the lack of significant differences in meiofaunal abundance (at the same depths except for samples collected at 200 and 2000-m depth). Overall, the number of taxa was higher in canyons than open slope, conversely to what previously reported for other deep-sea Mediterranean canyons (Bianchelli et al., 2010; Gambi and Danovaro, 2016). Most of the differences in meiofaunal variables between habitats, were related with changes in the sedimentary concentrations of phytopigments. The sediments of Polcevera and Bisagno canyons displayed indeed up to 3 times higher phytopigment contents than those found in the sediments of the western slope. Our results confirmed the important role of canyons in catching and channelling organic inputs and thus enhancing primary production (Ingels et al., 2011; Kiriakoulakis et al., 2011; Romano et al., 2013; Leduc et al., 2014; Amaro et al., 2016; Fernandez-Arcaya et al., 2017). The higher accumulation of organic matter and the presence of detritus patches can promote higher abundances and diversity of deep-sea benthic organisms compared to the adjacent open slope (Snelgrove and Smith, 2002; Ingels et al., 2009, 2011; Leduc et al., 2014; Amaro et al., 2016; Fernandez-Arcaya et al., 2017). In particular, meiobenthic fauna, capable of rapidly conveying the organic material produced in the surface layer into the food web, results favoured by the higher sedimentation rates and patches of detritus inside submarine canyons (Bianchelli et al., 2010). The variability of meiofaunal assemblages among canyons and slope was significantly influenced also by grain size. Polcevera and Bisagno canyons presented indeed

coarser sediments (i.e., higher contents of gravel) than those at the slope. Grain size has profound effects on meiobenthic assemblages. Greater diversity of benthic assemblages has been frequently reported in coarser sediments than in finer-grained ones, because of the range of microhabitats thought to be available in the former (Leduc et al., 2012). The increase of taxa richness with grain size has been recently reported also for other deep-sea canyons, located in the NW Mediterranean Sea (i.e., Blanes Canyon; Roman et al., 2016). The dominance of nematodes and harpacticoid copepods in deep-sea sediments (up to 98% of total meiofaunal abundance) can mask the changes in the relative importance of other taxa. We found that some taxa were ubiquitous, but Cladocera, Cumacea, Gastrotricha and Nemertina were exclusively found in the canyons. The rare taxonomic groups, showing preference for a specific habitat, were responsible for the high percentage of beta diversity found between canyons and slope (83%; Fig. 7b). In addition, when beta diversity was decomposed in its turnover and nestedness components, we found that variations in the composition of meiofaunal assemblages were mostly due to taxa loss than to replacement of taxa. This pattern is consistent with the higher spatial heterogeneity characterizing canyons compared to slope. Submarine canyons, indeed, are considered as the most heterogenic topographic systems, with great levels of within- and inter-canyon variability across a range of ecologically relevant processes, even at small spatial scale (Tyler et al., 2009; Ingels and Vanreusel, 2013). Finally, our results showed that the variability of meiofaunal assemblages can be driven also by the different geomorphological characteristics of canyons. Data reported by Harris and Whiteway (2011) in a study on worldwide occurrence and morphology of canyons indicate that the Polcevera and Bisagno canyons both belong to an active continental margin. Both canyons commence at ca. 340 m depth, but the canyon Polcevera is longer (46 km) and has a larger depth range (1700 m) and is more dendritic (20 limbs per 100,000 km2), than the canyon Bisagno. The results of the present study indicated that, the

high meiofaunal variability among the two canyons is also due to the different features characterizing each canyon. Besides grain size and trophic resources also geomorphological characteristics contributed to explain a large fraction of the variance of meiofaunal taxonomic

composition

between

Polcevera

and

Bisagno

canyons.

Peculiar

geomorphological features along with the higher food quality and quality allow canyons to become hotspots of benthic biodiversity in the deep sea, at least in terms of rare meiofaunal taxa (Danovaro et al., 2009; De Leo et al., 2010, 2014; Bianchelli et al., 2010; RamirezLlodra et al., 2010; Gambi and Danovaro, 2016; Roman et al., 2016).

Conclusions The present study provides the first data on the ecology of meiofaunal assemblages inhabiting the Ligurian canyons and adjacent open slope, indicating that the benthic biota is strongly influenced by the geomorphological characteristics of canyons (as termination depth). We conclude that the specific topographic settings, along with the interaction with grain size, food availability and high export of primary production to the seafloor, favor the presence of peculiar ecological conditions, which allow the establishment of hotspots of meiofaunal biodiversity in the Ligurian canyons.

Acknowledgments This work has been funded by the Flagship Project RITMARE—The Italian Research for the Sea—coordinated by the Italian National Research Council and funded by the Italian Ministry of Education, University and Research within the National Research Program 2011–2013.

Competing Interests The authors have declared that no competing interests exist.

References Amaro, T., Huvenne, V.A.I., Allcock, A.L., Aslam, T., Davies, J.S., Danovero, R., De Stigter, H.C., Duineveld, G.C.A., Gambi, C., Gooday, A.J., Gunton, L.M., Hall, R., Howell, K.L., Ingels, J., Kiriakoulakis, K., Kershaw, C.E., Lavaleye, L.S.S., Robert, K., Stewart, H., Van Rooij, D., White, M. and Wilson, A.M., 2016. The Whittard Canyon: a case study of submarine canyon processes. Progress in Oceanography 146, pp. 38-57. Anderson, M.J., 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecology 26, 32–46. Anderson, M.J., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA+ for PRIMER: guide to software and statistical methods. PRIMER-E: Plymouth, UK. Anderson, M.J., Robinson, J., 2003. Generalised discriminant analysis based on distances. Aust. N. Z. J. Stat. 45, 301–318. Balsega, A., 2010. Partitioning the turnover and nestedness components of beta diversity. Global Ecol Biogeogr 19, 134-143. Balsega, A., 2013. Multiple site dissimilarity quantifi es compositional heterogeneity among several sites, while average pairwise dissimilarity may be misleading. Ecography 36, 124-128. Bianchelli, S., Gambi, C., Zeppilli, D., Danovaro, R., 2010. Metazoan meiofauna in deepsea canyons and adjacent open slopes: A large-scale comparison with focus on the rare taxa. Deep Sea Res. I 57, 420-433. Bray, J.R., Curtis, J.T., 1957. An ordination of the upland forest communities of southern Wisconsin Ecological Monographs 27, 325–49. Canals, M., Company, J.B., Martín, D., Sànchez-Vidal, A., Ramírez-Llodra, E., 2013. Integrated study of Mediterranean deep canyons: Novel results and future challenges. Prog. Oceanogr. 118, 1–27. Canals, M., Danovaro, R., Heussner, S., Lykousis, V., Puig, P., Trincardi, F., Calafat, A. M., Durrieude Madron, X., Palanques, A., Sanchez-Vidal, A., 2009. Cascades in Mediterranean submarine grand canyons. Oceanography 22(1), 26–43. Canals, M., Puig, P., de Madron, X.D., Heussner, S., Palanques, A., Fabres, J., 2006. Flushing submarine canyons. Nature 444, 354–357. Clarke, K.R., Gorley, R.N., 2006. PRIMER v6: User Manual/tutorial. PRIMER-E, Plymouth, UK, 192.

Clarke, K.R., 1993. Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology 18, 117–43. Company, J.B., Puig, P., Sardà, F., Palanques, A., Latasa, M., Scharek, R., 2008. Climate influence on deep sea populations. PLoS One 3 (1), e1431. Corradi, N., Fierro, G., Piccazzo, M., Tucci, S., Zaccone, P., 1987. Répartition et transport de materiau en particules en suspension dans les canyons de Genes. Situation en hiver, Rev. Int. Océanogr. Méditerr. 85–86, pp. 117–121. Danovaro, R., 2010. Methods for the Study of Deep-sea Sediments, Their Functioning and Biodiversity. CRC Press Taylor & Francis Group. Danovaro, R., Bianchelli, S., Gambi, C., Mea, M., Zeppilli, D., 2009. Alpha-,beta-, gamma-, delta-and epsilon-diversity of deep-sea nematodes in canyons and open slopes of Northeast Atlantic and Mediterranean margins. Marine Ecology Progress Series 396, 197–209. Danovaro, R., Snelgrove, P.V., Tyler, P., 2014. Challenging the paradigms of deep-sea ecology. Trends in Ecology & Evolution 29, 465–475. Danovaro, R., Carugati, L., Corinaldesi, C., Gambi, C., Guilini, K., Pusceddu, A., Vanreusel, A., 2013. Multiple spatial scale analyses provide new clues on patterns and drivers of deep-sea nematode diversity. Deep Sea Research Part II: Topical Studies in Oceanography 92, 97-106. De Leo, F.C., Smith, C.R., Rowden, A.A., Bowden, D.A., Clark, M.R., 2010. Submarine canyons: hotspots of benthic biomass and productivity in the deep sea. Proceedings of the Royal Society B: Biological Sciences 277, 2783–2792. De Leo, F.C., Vetter, E.W., Smith, C.R., Rowden, A.A., Mc Granaghan, M., 2014. Spatial scale-dependent habitat heterogeneity influences submarine canyon macro-faunal abundance and diversity off the Main and Northwest Hawaiian Islands. Deep. Sea Res. Part II 104, 267–290. De Mol, L., Van Rooij, D., Pirlet, H., Greinert, J., Frank, N., Quemmerais, F., Henriet, J.- P., 2011. Cold-water coral habitats in the Penmarc’h and Guilvinec Canyons (Bay of Biscay): deep-water versus shallow-water settings. Marine Geology 282 (1– 2), 40–52. Fabiano, M., Danovaro, R., Fraschetti, S., 1995. A three-year time series of elemental and biochemical composition of organic matter in subtidal sandy sediments of the Ligurian Sea (north western Mediterranean). Continental Shelf Research 15, 1453–1469. Faith, D.P., Minchin, P.R., Belbin, L., 1987. Compositional dissimilarity as a robust measure of ecological distance. Vegetatio 69, 57–68.

Fernandez-Arcaya, U., Ramirez-Llodra, E., Aguzzi, J., Allcock, A.L., Davies, J.S., Dissanayake, A., Harris, P., Howell, K., Huvenne, V.A.I., Macmillan-Lawler, M., Martín, J., Menot, L., Nizinski, M., Puig, P., Rowden, A.A., Sanchez, F., Van den Beld, I.M.J., 2017. Ecological Role of Submarine Canyons and Need for Canyon Conservation: A Review. Front. Mar. Sci. 4, 5. Frache, R., Manfrinetti, P., Piccazzo, M., Tucci, S., 1986. Distribution and transport of particulate Fe and Cu in suspended matter of the Canyons of Genoa (northwestern Mediterranean), Mar. Poll. Bull. 17(3), pp. 123–127. Gambi, C., Danovaro, R., 2006. A multiple-scale analysis of metazoan meiofaunal distribution in the deep Mediterranean Sea. Deep-Sea Res. Part I Oceanogr. Res. Pap. 53, 1117–1134. Gambi, C., Danovaro, R., 2016. Biodiversity and life strategies of Deep-sea meiofauna and nematode assemblages in the Whittard Canyon (Celtic margin, NE Atlantic Ocean). Deep-Sea Res. I 108, 13–22. Gambi, C., Pusceddu, A., Benedetti-Cecchi, L., Danovaro, R., 2014. Species richness, species turnover, and functional diversity in nematodes of the deep Mediterranean Sea: searching for drivers at different spatial scales. Glob. Ecol. Bio-geogr. 23, 24–39. Garcia, R., Koho, K.A., De Stigter, H.C., Epping, E., Koning, E., Thomsen, L., 2007. Distribution of meiobenthos in the Nazare canyon and adjacent slope (western Iberian Margin) in relation to sedimentary composition. Mar. Ecol. Prog. Ser. 340, 207–220. Garcia, R., & Thomsen, L. (2008). Bioavailable organic matter in surface sediments of the Nazaré canyon and adjacent slope (Western Iberian Margin). Journal of Marine Systems, 74(1-2), 44-59. Giere, O., 2009. Meiobenthology: The Microscopic Motile Fauna of Aquatic Sediments. Springer-Verlag, Berlin, Heidelberg. Giere, O., 2009. Meiobenthology: The Microscopic Motile Fauna Of Aquatic Sediments. Springer, Berlin Gray, J.S., 2000. The measurement of marine species diversity, with an application to the benthic fauna of the Norwegian continental shelf. J. Exp. Mar. Biol. Ecol. 250, 23–49. Grémare, A., Medernach, L., Amouroux, J. M., Vétion, G., & Albert, P. (2002). Relationships between sedimentary organics and benthic meiofauna on the continental shelf and the upper slope of the Gulf of Lions (NW Mediterranean). Marine Ecology Progress Series, 234, 85-94.

Hajdu, L.J., 1981. Graphical comparison of resemblance measures in phytosociology. Vegetatio 48, 47–59. Harris, P.T., Whiteway, T., 2011. Global distribution of large submarine canyons: geomorphic differences between active and passive continental margins. Mar. Geol. 285, 69–86. Hicks, G.R., 1983. The ecology of marine meiobenthic harpacticoid copepods. Oceanogr, Mar. Biol. Anu. Rev., 21, 67-175. Ingels, J., Kiriakoulakis, K., Wolff, G.A., Vanreusel, A., 2009. Nematode diversity and its relation to quantity and quality of sedimentary organic matter in the Nazaré Canyon, Western Iberian Margin. Deep-Sea Res. I: Oceanogr. Res. Pap. 56, 1521–1539. Ingels, J., & Vanreusel, A. (2013). The importance of different spatial scales in determining structural and

functional

characteristics

of

deep-sea

infauna

communities.

Biogeosciences, 10(7), 4547-4563. Ingels, J., Tchesunov, A.V., Vanreusel, A., 2011. Meiofauna in the Gollum Channels and the Whittard Canyon, Celtic Margin—How Local Environmental Conditions Shape Nematode Structure and Function. PLoS ONE 6 (5), e20094. Ingels, J., Vanreusel, A., Romano, C., Coenjaerts, J., Flexas, M. M., Zúñiga, D., Martin, D., 2013. Spatial and temporal infaunal dynamics of the Blanes submarine canyon-slope system (NW Mediterranean); changes in nematode standing stocks, feeding types and gender-life stage ratios. Prog. Oceanogr. 118, 159-174. Koho, K.A., Garcia, R., de Stigter, H.C., Epping, E., Koning, E., Kouwenhoven, T.J., van der Zwaan, G.J., 2008. Sedimentary labile organic carbon and pore water redox control on species distribution of benthic foraminifera: a case study from LisbonSetubal Canyon (sourthern Portugal). Prog. Oceanogr. 79, 55–82. Kiriakoulakis, K., Blackbird, S., Ingels, J., Vanreusel, A., & Wolff, G. A. (2011). Organic geochemistry of submarine canyons: the Portuguese Margin. Deep Sea Research Part II: Topical Studies in Oceanography, 58(23-24), 2477-2488. Leduc, D., Rowden, A. A., Nodder, S. D., Berkenbusch, K., Probert, P. K., & Hadfield, M. G. (2014). Unusually high food availability in Kaikoura Canyon linked to distinct deep-sea nematode community. Deep Sea Research Part II: Topical Studies in Oceanography, 104, 310-318. Leduc, D., Rowden, A. A., Probert, P. K., Pilditch, C. A., Nodder, S. D., Vanreusel, A., et al. (2012). Further evidence for the effect of particle-size diversity on deep-sea benthic biodiversity, Deep-Sea Res. Pt. I, 63, 164–169

Levin, L.A., Sibuet, M., 2012. Understanding Continental Margin Biodiversity: a New Imperative. Annu. Revis. Mar. Sci. 4, 79–112. Lorenzen, C.J., Jeffrey, S.W., 1980. Determination of chlorophyll and phaeopigments spectrophotometric equations. Limnol. Oceanogr. 12, 343e346 McArdle, B.H., Anderson, M.J., 2001. Fitting multivariate models to community data: a comment on distance-based redundancy analysis. Ecology 82, 290–297. Mc Ardle, B.H., Anderson, M.J., 2004. Variance heterogeneity, transformations and models of species abundance: a cautionary tale. Canadian Journal of Fisheries and Aquatic Sciences 61, 1294–1302. Odum, E.P., 1950. Bird populations of the Highlands (North Carolina) Plateau in relation to plant succession and avian invasion. Ecology 31, 587–605. Orsi Relini, L., Relini, G., 1998. Long term observations of Aristeus antennatus: Sizestructures of the fished stock and growth parameters, with some remarks about the" recruitment". Cahiers Options Mediterraneennes (CIHEAM). Pape, E., Bezerra, T., Jones, D.O., Vanreusel, A., 2013a. Unravelling the environmental drivers of deep-sea nematode biodiversity and its relation with carbon mineralisation along longitudinal primary productivity gradient. Biogeosciences 10 (5), 3127–3143. Pape, E., Manini, E., Bezerra, T., Vanreusel, A., Jones, D.O., 2013b. Benthic-Pelagic Coupling: effects on Nematode Communities along Southern European Continental Margins. PLoS One 8 (4), 1–14. Pasqual, C., Sanchez-Vidal, A., Zúñiga, D., Calafat, A., Canals, M., Durrieu de Madron, X., Pere, P., Heussner, S., Palanques Monteys, A., Delsaut, N., 2010. Flux and composition of settling particles across the continental margin of the Gulf of Lion: the role of dense shelf water cascading. Biogeosciences 7, 217–231. Pierce, J.W., Tucci, S., Fierro, G., 1981. Assessing variation in suspensates, Ligurian Sea (northwestern Mediterranean), Geo-Marine Lett. 1, pp. 149–154 Puig, P., Palanques, A., Martín, J., 2014. Contemporary sediment-transport processes in Submarine Canyons. Annual Review of Marine Science 6, 53–77. Pusceddu, A., Dell’Anno, A., Fabiano, M., Danovaro, R., 2009. Quantity and bioavailability of sediment organic matter as signatures of benthic trophic status. Mar. Ecol. Prog. Ser. 375, 41e52. Pusceddu, A., Mea, M., Canals, M., Heussner, S., Durrieude Madron, X., Sanchez-Vidal, A., Bianchelli, S., Corinaldesi, C., Dell'Anno, A., Thomsen, L., Danovaro, R., 2013.

Major consequences of an intense dense self water cascading event on deep-sea trophic conditions and meiofauna biodiversity. Biogeosciences10(4), 2659–2670. Ramalho, S. P., Adão, H., Kiriakoulakis, K., Wolff, G. A., Vanreusel, A., Ingels, J., 2014. Temporal and spatial variation in the Nazaré Canyon (Western Iberian margin): Interannual and canyon heterogeneity effects on meiofauna biomass and diversity. Deep Sea Research Part I: Oceanographic Research Papers 83, 102-114. Ramírez-Llodra, E., Company, J.B., Sarda, F., Rotllant, G., 2010. Megabenthic diversity patterns and community sttructure of the Blanes submarine canyon and adjacent slope in the Northwestern Mediterranean: a human overprint? Mar. Ecol. 31, 167–182. Román, S., Vanreusel, A., Romano, C., Ingels, J., Puig, P., Martin, D., 2016. High spatiotemporal variability in meiofaunal assemblages in Blanes Canyon (NW Mediterranean) subject to anthropogenic and natural disturbances. Deep Sea Research Part I: Oceanographic Research Papers 117, 70-83. Romano, C., Coenjaerts, J., Flexas, M. M., Zúñiga, D., Vanreusel, A., Martin, D., 2013. Spatial and temporal variability of meiobenthic density in the Blanes submarine canyon (NW Mediterranean). Prog. Oceanogr. 118, 144-158. Skliris, N., Denidi, S., 2006. Plankton dynamics controlled by hydrodynamic processes near a submarine canyon off NW corsican coast: a numerical modelling study. Continental Shelf Research 26, 1336–1358. Smith, J.K.L., Druffel, E., 1998. Longtime-series monitoring of an abyssal site in the NE Pacific an introduction. Deep-Sea Research Part II: Topical Studies in Oceanography 45 (4–5), 573–586. Snelgrove, P.V.R. and Smith, C.R. (2002). A riot of species in an environmental calm: the paradox of the species-rich deep-sea floor. Oceanogr. Mar. Biol. Ann. Rev. 40: 311342 In: Oceanography and Marine Biology: An Annual Review. Aberdeen University Press/Allen & Unwin: London. ISSN 0078-3218 Tesi, T., Puig, P., Palanques, A., Goñi, M.A., 2010. Lateral advection of organic matter in cascading-dominated submarine canyons. Prog. Oceanogr. 84(3–4), 185–203. Thistle, D., Sedlacek, L., Carman, K. R., & Barry, J. P. (2017). Influence of habitat heterogeneity on the community structure of deep-sea harpacticoid communities from a canyon and an escarpment site on the continental rise off California. Deep Sea Research Part I: Oceanographic Research Papers, 123, 56-61.

Tyler, P., Amaro, T., ArzolA, R., Cunha, M. R., de Stigter, H., Gooday, A., ... & Masson, D. (2009). Europe's grand canyon Nazare submarine canyon. Oceanography, 22(1), 4657. Tselepides, A., Lampadariou, N., 2004. Deep-sea meiofaunal community structure in the Eastern Mediterranean: are trenches benthic hotspots?. Deep Sea Research Part I: Oceanographic Research Papers, 51(6), 833-847. Tudela, S., Sarda, F., Maynou, F., Demestre, M., 2003. Influence of submarine canyons on the distribution of the deep-water shrimp, Aristeus antennatus (Risso, 1816) in the NW Mediterranean. Crustaceana 76, 217–225. Vincx, M., Bett, B.J., Dinet, A., Ferrero, T., Gooday, A.J., Lambshead, P.J.D., Pfannkuche, O., Soltwedel, T., Vanreusel, A., 1994. Meiobenthos of the north-east Atlantic. Adv. Mar. Biol. 30, 1–88. Würtz, M., 2012. Mediterranean Submarine Canyons: Ecology and Governance. Gland; Málaga: IUCN. Zeppilli, D., Pusceddu, A., Trincardi, F., Danovaro, R, 2016. Seafloor heterogeneity influences the biodiversity–ecosystem functioning relationships in the deep sea. Scientific Reports 6, 26352.

Figures’ captions Figure 1. Study area and sampling stations along Polcevera and Bisagno canyons and the western open slope. Figure 2. Concentration of biopolymeric C (A) and percentage of algal fraction of biopolymeric C (BPC) (B) in the sediments of the investigated stations. Reported are average values ± standard deviation (P = Polcevera canyon; B = Bisagno canyon). Figure 3. Biochemical composition of organic matter in the sediments of the investigated stations (P = Polcevera canyon; B = Bisagno canyon). Figure 4. Meiofaunal abundance (A) and richness of higher taxa (B) in the sediments of the investigated stations. Reported are average values ± standard deviation (P = Polcevera canyon; B = Bisagno canyon). Figure 5. Meiofaunal taxonomic composition of all (A) and rare taxa (B). Reported are number of higher taxa identified at each sampling station (P = Polcevera canyon; B = Bisagno canyon). Figure 6. Principal component analysis (PCA) ordination based on meiofaunal taxonomic composition of all taxa among slope and the two canyons. Figure 7. Principal component analysis (PCA) ordination based on meiofaunal composition of rare taxa among slope and the two canyons (A). Number of rare taxa found along the open slope and in the two canyons. Reported is also the beta diversity (% of dissimilarity calculated on the taxonomic composition of rare meiofaunal taxa by using SIMPER analyses) between open slope and canyons (B).

253 m 225 m

222 m 496 m

1001 m

507 m 1055 m

1620 m

1516 m 1945 m

2004 m

Fig. 1

a) Biopolymeric C (mgC g-1)

3,50 3,00

2,50 2,00 1,50

1,00 0,50 0,00 slope

P

B

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Algal fraction of BPC (%)

60 50 40

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1946-2004

100% 80% 60% 40% 20% 0% slope

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Depth (m) Protein

Fig. 3

Carbohydrate

P

Lipid

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1946-2004

a)

1000 900

Ind. 10 cm -2

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n higher taxa

10 8

6 4

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B

slope

B

496-507

slope

944-1055

Depth Depth(m) (m)

Fig. 4

P

P

1515-1600

B

1946-2004

a)

100% 95% 90% 85% 80% 75% slope

P

B

225-252

slope

slope

496-507 Nematoda

b)

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Fig. 5

Ostracoda Gastrotricha Acarina

Kinorhyncha Cumacea Nemertina

Oligochaeta Amphiopoda Amphipoda Cladocera

Fig. 6

a)

b)

12

N. rare taxa

10 8

Beta diversity 83%

6

4 2 0

Open slope

Fig. 7

Canyon

Table 1. Location, depth of sampling stations, grain size, concentrations of phytopigments and of biopolymeric C, prokaryotic abundance, meiofaunal abundance and taxa richness. Data are reported as average value. SD=standard deviation. Habitat

Slope

Polcevera canyon

Bisagno Canyon

Latitude

Longitude

Depth

N

E

44° 18'47.1036"

Total Phytopigments

Biopolymeric Carbon

Prokaryotic abundance

Gravel

Sand

Silt

Clay

m

%

%

%

%

µg g

SD

mg g

SD

Cells DW

08° 40'13.6722"

225

0

2

61

37

15.1

0.6

1.8

0.1

44° 16'13.7496"

08° 40'17.7864"

507

0

1

55

44

6.6

2.3

1.2

44° 11'22.4316"

08°40'31.0470"

1055

0

1

57

42

5.5

1.4

44° 03'24.2676"

08° 39'09.2184"

1516

0

1

51

48

3.8

43° 55'01.9546"

08° 38'10.0365"

2004

0

4

52

44

44° 21'52.3032"

08° 49'19.5666"

253

0

1

58

44° 18'43.1392"

08° 49'52.6998"

1001

0

6

44° 10'35.0634"

08° 45'42.5256"'

1620

0

44° 21'07.5126"

08° 54'43.9164"

222

44° 20'07.0032"

08° 54'38.0784"

44° 00'42.46682"

08° 49'06.0750"

Meiofaunal abundance

−1

SD

n ind -2 10cm

SD

2.19E+08

7.70E+07

650.7

222.5

9

0.2

1.78E+08

3.27E+07

109.8

66.5

5

1.7

0.2

2.83E+08

4.66E+07

356.1

123.0

6

1.5

1.5

0.1

2.32E+08

7.17E+07

160.7

44.4

3

2.4

0.5

1.3

0.3

2.15E+08

6.85E+07

162.7

15.9

3

41

16.0

1.0

2.5

0.3

1.99E+08

2.68E+07

642.9

132.7

11

53

41

11.0

3.5

1.4

0.2

2.37E+08

5.71E+06

149.0

29.2

8

1

50

49

5.9

2.2

1.4

0.3

2.53E+08

2.59E+07

138.2

36.0

6

0

5

55

40

10.3

0.3

2.7

0.4

4.97E+08

8.63E+07

161.4

12.1

6

496

2

6

52

40

24.4

1.6

2.1

0.1

3.55E+08

8.80E+06

551.7

172.9

11

1945

0

2

53

45

5.8

0.7

1.2

0.4

3.40E+08

4.31E+07

67.9

10.3

4

-1

-1

g

Meiofaunal taxa richness

Table 2. Output of the PERMANOVA analysis carried out to test for differences in quantity and quality of sedimentary organic matter and prokaryotic abundance among habitats (Ha) and depths (De) (DF = degrees of freedom; MS = mean square; Pseudo-F = F statistic; P(MC) = probability level); *** P<0.001; ** P<0.01; * P<0.05; ns: not significant. Variable

Source

df

MS

Pseudo-F

P(MC)

Biopolymeric C

Ha

2

1,836

8,527

**

De

4

4,013

18,643

***

Ha x De

4

1,505

6,991

**

Residual

22

0,215

Ha

2

2,289

41,096

***

De

4

3,616

64,903

***

Ha x De

4

2,415

43,351

***

Residual

22

0,056

Ha

2

3,082

11,834

**

De

4

3,121

11,983

***

Ha x De

4

1,348

5,176

**

Residual

22

0,260

Ha

2

2,678

11,855

**

De

4

3,934

17,415

***

Ha x De

4

0,725

3,209

*

Residual

22

0,226

Ha

2

1,962

9,163

**

De

4

2,786

13,016

***

Ha x De

4

2,920

13,639

***

Residual

22

0,214

Ha

2

7,270

5,814

**

De

4

18,813

15,047

***

Ha x De

4

8,764

7,009

**

Residual

22

1,250

Ha

2

10,945

40,056

***

De

4

1,079

3,950

**

Ha x De

4

0,619

2,264

ns

Residual

22

0,273

Total

32

Phytopigments

Protein to biopolymeric C %

Protein to carbohydrate ratio

Algal fraction %

Biochemical composition

Prokaryotic abundance

Table 3. Output of the PERMANOVA analysis carried out to test for differences in meiofaunal abundance, richness of taxa and composition as all and rare taxa among habitats (Ha) and depths (De) (DF = degrees of freedom; MS = mean square; Pseudo-F = F statistic; P(MC) = probability level); *** P<0.001; ** P<0.01; * P<0.05. ns: not significant. Variable

Source

df

MS

Pseudo-F

P(MC)

Abundance

Ha

2

20413,00

0,618

ns

De

4

172790,00

5,228

**

Ha x De

4

200530,00

6,068

***

Residual

22

33049,00

Ha

2

7,51

2,455

ns

De

4

17,29

5,648

**

Ha x De

4

9,59

3,134

*

Residual

22

3,06

Ha

2

649,86

1,005

ns

De

4

2924,90

4,522

***

Ha x De

4

3489,50

5,395

***

Residual

22

646,84

Ha

2

6867,90

2,863

**

De

4

6415,90

2,675

***

Ha x De

4

3400,80

1,418

ns

Residual

22

2398,70

Total

32

Richness of higher taxa

Composition as all taxa

Composition as rare taxa

Table 4. Summary of the conditional tests of the DISTLM (distance-based multivariate analysis for a linear model using forward selection) analyses performed to test interactions between environmental and trophic conditions on meiofaunal variables. F, F statistic; P, probability level; Var (%) is the percentage of the variance explained by that explanatory variable; Cum (%) is the cumulative percentage of variance explained by the explanatory variables. Reported are those variables that display a P level: *** P<0.001; ** P<0.01; * P<0.05. Explained variance (%) 2

Dependent variable

Explanatory variable

R

P

Proportion

Cumulative

a) Meiofaunal abundance

Total phytopigments Silt

0,365 0,488

*** **

37 12

37 49

PRT:CHO

0,550

*

6

55

Algal fraction %

0,611

*

6

61

Total phytopigments

0,462

***

46

46

Gravel

0,550

*

9

55

Total phytopigments Algal fraction %

0,265 0,357

** *

27 9

27 35

Protein to biopolymeric C %

0,443

**

9

44

Silt

0,505

*

6

50

Total phytopigments

0,153

***

15

15

Protein to biopolymeric C %

0,427

*

6

21

b) Richness of meiofaunal taxa

c) Composition as all taxa

d) Composition as rare taxa

Table 5. Summary of the conditional tests of the DISTLM (distance-based multivariate analysis for a linear model using forward selection) analyses performed to test interactions between environmental, trophic conditions and canyons geomorphological characteristics on meiofaunal variables, between Polcevera and Bisagno canyons. F, F statistic; P, probability level; Var (%) is the percentage of the variance explained by that explanatory variable; Cum (%) is the cumulative percentage of variance explained by the explanatory variables. Reported are those variables that display a P level: *** P<0.001; ** P<0.01; * P<0.05. Explained variance (%) Dependent variable

2

Explanatory variable

R

P

Proportion

Cumulative

Total phytopigments

0.486

**

49

49

Sand

0,621

*

14

63

b) Richness of meiofaunal taxa

Total phytopigments

0.324

**

32

32

c) Composition as all taxa

Total phytopigments Sand Depth

0.459 0,583 0,679

*** ** **

46 12 10

46 58 68

Depth

0,149

**

15

15

Polcevera vs Bisagno Canyons a) Meiofaunal abundance

c) Composition as rare taxa

Protein to biopolymeric C % Maximum depth

0,268 0,386

** **

12 12

27 39

Highlights  Organic matter and biodiversity were investigated in Ligurian canyons and slope  The richness of meiofaunal taxa was higher in canyons than in the adjacent slope  Canyons were depocenter of primary organic matter  Polcevera and Bisagno canyons and slope showed different taxa  Geomorphology, food availability and grain size influenced benthic biota of canyons