Halophila stipulacea descriptors in the native area (Red Sea): A baseline for future comparisons with native and non-native populations

Marine Environmental Research xxx (xxxx) xxx

Contents lists available at ScienceDirect

Marine Environmental Research journal homepage: http://www.elsevier.com/locate/marenvrev

Halophila stipulacea descriptors in the native area (Red Sea): A baseline for future comparisons with native and non-native populations Pedro Beca-Carretero a, e, f, *, Alice Rotini b, g, Astrid Mejia b, Luciana Migliore b, Salvatrice Vizzini c, d, Gidon Winters e a

Botany and Plant Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Department of Biology, Tor Vergata University, Via della Ricerca Scientifica snc, I-00133, Rome, Italy Department of Earth and Marine Sciences, University of Palermo, via Archirafi 18, 90123 Palermo, Italy d CoNISMa, Inter-University Consortium for Marine Sciences, Piazzale Flaminio 9, 00196 Roma, Italy e The Dead Sea-Arava Science Center, Tamar Regional Council, Neve Zohar, 86910, Israel f Department of Theoretical Ecology and Modelling, Leibniz Centre for Tropical Marine Research, Fahrenheitstrasse 6, 28359 Bremen, Germany g Institute for Environmental Protection and Research (ISPRA), Via Vitaliano Brancati 48, I-00144, Rome, Italy b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Seagrass Temporal changes Depth-adaptation Anthropogenic pressures Morphometric and population parameters Phenol content Nitrogen storage Stable isotopes

Halophila stipulacea is a small tropical seagrass species native to the Red Sea. Due to its invasive character, there is growing interest in understanding its ability to thrive in a broad range of ecological niches. We studied temporal (February 2014 and July 2014), depth (5, 9, 18 m) and spatial (NB and SB) related dynamics of H. stipulacea meadows in the northern Gulf of Aqaba. We evaluated changes in density, morphometry, biomass, and biochemical parameters alongside the reproductive effort. In both sites, maximal growth and vegetative performance occurred in the summer with a marked increase of 35% in shoot density and 18% in biomass; PAR reduction with season and depth induced a significant increase of 28% in leaf area. Sexual reproduction efforts were only observed in July, and the density of plants carrying male or female flowers decreased significantly with depth. The favorable growth responses of H. stipulacea plants observed in the N-enriched NB site suggests their capacity to acclimate to human-disturbed nearshore environments.

1. Introduction The Gulf of Aqaba (GoA, northern Red Sea) is a warm (21–27 � C; Winters et al., 2006), oligotrophic, tropical sea (Reiss and Hottinger, 1984). Although famous for its colourful coral reefs (Genin et al., 1995; Loya et al., 2004), the GoA also supports highly productive, but much less studied and internationally recognized, seagrass meadows domi­ nated by the small, dioecious, tropical seagrass Halophila stipulacea (Forsskal) Ascherson (Mejia et al., 2016; Winters et al., 2017; Nordlund et al., 2018). This seagrass species is native to the Red Sea, Persian Gulf, and Indian Ocean (Lipkin, 1975a). Within the GoA, H. stipulacea is by far the dominant, most widespread, and sometimes the only seagrass spe­ cies (Angel et al., 1995; Al-Rousan et al., 2011; El Shaffai, 2011), forming discontinuous meadows at depths ranging from 1 to 50 m ( Sharon et al., 2011; Winters et al., 2017). As ’ecosystem engineers’, seagrasses provide crucial ecological

services, including production and burial of organic carbon, coastal protection via sediment stabilization and the formation of an essential habitat for economically important fish and crustaceans (Orth et al., 2006; Waycott et al., 2009; Fourqurean et al., 2012). Also, seagrasses are capable to uptake and store nutrients from the seawater via roots and leaves, therefore, favoring nutrient cycling and acting as a sink of nu­ trients in N-enriched marine environments (e.g., Romero et al., 1994; Viana et al., 2019). In seagrasses, environmental conditions including temperature, irradiance and nutrient levels, have been identified as relevant factors controlling photosynthetic and respiration rates alongside morpholog­ ical changes, population dynamics and reproductive responses (e.g., Bulthuis, 1987; Erftemeijer and Herman, 1994; Stubler et al., 2017). H. stipulacea was reported to display maximal productivity during warmer periods, increasing leaf structures and growth rates and storing higher contents of energetic compounds such as carbohydrates and

Abbreviations: NB, North Beach; SB, South Beach; A/B, Above-ground/Below-ground; SST, Sea Surface Temperature; GoA, Gulf of Aqaba. * Corresponding author. Botany and Plant Science, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland. E-mail address: [email protected] (P. Beca-Carretero). https://doi.org/10.1016/j.marenvres.2019.104828 Received 27 June 2018; Received in revised form 2 September 2019; Accepted 24 October 2019 0141-1136/© 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Pedro Beca-Carretero, Marine Environmental Research, https://doi.org/10.1016/j.marenvres.2019.104828

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx

lipids (e.g., Wahbeh, 1984; Sharon et al., 2011; Cardini et al., 2017). Besides, H. stipulacea was shown to have the capacity to adapt to a wide range of irradiance levels and spectrums; this results in a high plasticity in photosynthetic structures allowing for a better capture of light (e.g., Lipkin, 1975b; Sharon et al., 2009). Variations in morphology, structure and biochemical compounds such as phenol content have been imple­ mented to assess the eco-physiological status of H. stipulacea in response to varying environmental conditions (i.e., Rotini et al., 2017; Beca-­ Carretero et al., 2019a). Following the opening of the Suez Canal in 1869, H. stipulacea soon became a putative Lessepsian migrant (Lipkin, 1975b) and has since established in many parts of the eastern Mediterranean Sea (Gambi et al., 2009; Sghaier et al., 2011). Surprisingly, in 2002 it was also re­ ported from the Caribbean Sea (Ruiz et al., 2017; Steiner and Willette, 2010), and merely a decade later it was found in most eastern Caribbean islands, as well as in the coast of the continental South American (Vera et al., 2014; Willette and Ambrose, 2009, 2014; Steiner and Willette, 2015; Smulders et al., 2017). The invasiveness of H. stipulacea has been attributed to its high adaptability to a wide range of environmental variables, such as salinity, temperature, light intensity, nutrient levels and anthropogenic pressures (Por, 1971; reviewed by Gambi et al., 2009; Sharon et al., 2009, 2011; Oscar et al., 2018). Indeed, in both native and non-native areas, H. stipulacea was observed to colonize and survive in human-disturbed habitats such as marinas, harbors or near to industrialized coastal areas (Kenworthy et al., 1993; Gambi et al., 2009;

Winters et al., 2017). Due to its invasive character, there is growing interest in under­ standing H. stipulacea’ ability to thrive in a broad range of ecological niches. However, studies providing quantitative data on H. stipulacea meadows growing in either native or invasive habitats are particularly scarce, and also these studies applied diverse methodological ap­ proaches (i.e., Wahbeh, 1988; Schwarz and Hellblom, 2002; Cardini et al., 2017). Lack of information regarding the year-round dynamics of both native and invasive populations of H. stipulacea limits our under­ standing of the current population dynamics of this seagrass species in both these habitats. Therefore, it is imperative to contribute to the growing dataset for this understudied tropical seagrass species. The present research was undertaken to learn the basic features concerning individual and population characteristics of H. stipulacea growing in its native habitat. In this study we hypothesized that (i) in comparison with winter season, the high temperatures and irradiance levels experienced during the summer period would induce significant morphological and struc­ tural responses alongside changes in biochemical composition in H. stipulacea plants in the region under study, and that (ii) increases in nutrient levels due to anthropogenic inputs in our nutrient impacted site might affect H. stipulacea’s vegetative development as the waters of GoA are considered to be very oligotrophic. Therefore, the objectives of this study were to (i) compare morphological, structural and biochemical descriptors of H. stipulacea in permanent transects in two contrasting

Fig. 1. Map of study area, northern Gulf of Aqaba (GoA), Red Sea, Israel, with the locations of the two study sites: North Beach (NB) and South Beach (SB). Halophila stipulacea distribution in the region is represented in green (adapted from Winters et al. (2017)). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article). 2

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx

meadows (impacted [NB] vs near-pristine [SB]) at 3 different depths (shallow [5 m], intermediate [9 m] and deep [18 m]) in Winter (February 2014) vs summer (July 2014), and (ii) examine the isotopic signature and nutrient content of the different H. stipulacea compart­ ments (leaves, rhizomes and roots), and correlate them with the nutrient levels in the seawater.

A total of 30 leaves from each sample quadrat (n ¼ 3) were used to measure leaf biometry. For this, leaves were scanned (Canon Lide 110 digital scanner) and measured using the ImageJ software (version 1.47; Abramoff et al., 2004) to calculate leaf area (mm2). Percent of plant fragments that were found to contain reproductive structures (male or female plant bearing segments) were counted in the three replicates.

2. Methods

2.3.2. Percentage of cover At each transect, plant % cover was estimated using photos taken at 12 quadrants (50 � 50 cm) randomly placed along the 50 m transect with an underwater camera (Canon S110, placed in a dedicated Ikelite housing) placed 80 cm above the quadrant. Photographs (taken under natural irradiance) were imported into Coral Point Count for Excel (CPCe; Kohler and Gill, 2006) where a uniform grid of 10 by 10 (¼ 100) points was overlaid onto the images Points intersecting with the occurrence of seagrasses were counted visually on the computer screen and used to calculate the percentage of seagrass cover (0–100%) (Win­ ters et al., 2017).

2.1. Study area H. stipulacea samples were collected from two different subtidal monospecific meadows, North Beach (NB) and South Beach (SB) on the western shores of the northern tip of the GoA, northern Red Sea, Israel (Fig. 1) in both winter (February) and summer (July) of 2014. The NB meadow (29.546150 N, 34.964819 E), is part of an extensive meadow in the area (343,032 m2; Winters et al., 2017; Mejia et al., 2016) and is characterized by a gentle bathymetric slope (2.5� ) of fine sediments with high turbidity and low hydrodynamic forces along a relatively anthropized coastal stretch (Mejia et al., 2016; Winters et al., 2017). The SB meadow (29.497664 N, 34.912737 E) is part of a smaller meadow in the area (61,900 m2), and is characterized by a steep slope (17.9� ), coarse-grained sand, high water clarity, higher hydrodynamic forces, widespread occurrence of corals and low anthropogenic pressures (Mejia et al., 2016; Winters et al., 2017; Rotini et al., 2017). Within each meadow, permanent 50 m-long transects were placed parallel to the shore at 5, 9 and 18 m depth by SCUBA-diving. Transects were fixed using three 1.5 m plastic poles that were left in the field throughout the sampling period. H. stipulacea plants were collected separately for density, morphometry, biomass and biochemical analyses (detailed below).

2.4. Seagrass biochemical descriptors 2.4.1. Total phenol content Since previous observations showed no significant variation in phenol contents in H. stipulacea growing from 4 to 18 m depth (Rotini et al., 2017), this present study focused the phenol measurements on one depth (9 m). At each season and site, shoots were collected at 9 m depth for phenol content analysis. Sampling was carried out at intervals of 5–10 m along the 50 m transect to prevent resampling of the same clonal type (n ¼ 20). Total phenol content was quantified in 200 mg (fresh weight ¼ FW) of leaf tissue (cleaned of epiphytes), powdered in liquid nitrogen. The extraction was conducted according to Migliore et al. (2007) and modified for Halophila spp. by Mejia et al. (2016). Total phenol content was expressed as mg/g of FW.

2.2. Environmental variables

2.4.2. Isotopic signature analysis and nutrient content Due to the costs associated with these measurements, for isotopic analysis, at each season and site, plants were collected only at 9 and 18 m depths (at 5-m deep, SB populations experience marked temporal fluctuations along the edges of the meadow). For these analyses, plants were divided into leaves, rhizomes, and roots and carefully cleaned, removing all the sand and epiphytic organisms. All samples were then oven-dried (60 � C, 72 h), ground to a fine powder using a Retsch micromill (MM200) and divided into two aliquots: one used for δ13C analysis and the other for C and N elemental content and δ15N analysis (Vizzini and Mazzola, 2004). Before δ13C analysis, samples were acidified with drop-by-drop 1N HCl to remove traces of carbonates and subsequently dried and powdered again. All samples were weighed in tin capsules (~2 mg for leaves, and ~3 mg for the other tissues) and analyzed for C and N elemental content and δ13C and δ15N through an Elemental Analyser-Isotope Ratio Mass Spectrometer system (Thermo Flash EA 1112 – IRMS Delta Plus XP). Isotopic data were reported in common delta (δ) units referred to VPDB and atmospheric nitrogen standards for carbon and nitrogen, respectively, following the formula: δX ¼ [(Rsample/Rstandard) 1] � 103, where X is the stable isotope mass (13 for C and 15 for N), and R is the corresponding 13C/12C or 15N/14N ratio. Analytical precision based on the standard deviation of replicates of internal standards (International Atomic Energy Agency IAEA-NO-3 for δ15N and IAEA-CH-6 for δ13C) was 0.2‰ for both δ13C and δ15N (Vizzini and Mazzola, 2006).

Long-term (2010–2015) salinity, turbidity (Secchi depth [m]), esti­ mated maximal underwater Photosynthetically Active Radiation (PAR), surface water temperature and surface water nutrients (NO3, NO2 and NH4) were obtained from Israel’s National Monitoring of the Gulf of Eilat (NMP; http://iui-eilat.ac.il/Research/NMPMeteoData.aspx; accessed 10/01/2017) from monitoring sites very close by (<400 m away) to the two studied meadows (NB and SB). For NB, environmental data was collected from the monitoring station called “North Beach”, and for SB the environmental data was collected from the monitoring station called “Taba”. To estimate maximal underwater PAR at the different depths and seasons, we used the Secchi depth (Sd) obtained for each site and season to calculate Kd, the attenuation coefficient of PAR according to the Duarte (1991) equation: Kd ¼ 1.7 Sd. We then applied the Lambert-Beer equation ID ¼ I0e-KD (van der Heide et al., 2007), with ID ¼ Light In­ tensity at depth (measured in Einstein); I0 ¼ Light Intensity at the Sur­ face; D ¼ Depth and Kd ¼ the attenuation coefficient of PAR. 2.3. Seagrass morphological and structural descriptors 2.3.1. Density, biomass, biometry, reproductive effort At each season, site and depth, entire H. stipulacea plant biomass (plant fragments with leaves, rhizomes, and roots) were collected from within a 25 � 25 cm quadrat (n ¼ 3) randomly placed along the 50 m transect. The collected samples were transported to the laboratory and carefully cleaned, removing all the sand and epiphytic organisms. Density measurements were conducted by counting the total number of shoots, leaves, and roots per quadrat (n ¼ 3) and then normalized per m2. Plant parts were then divided into above-ground (leaves) and belowground (roots and rhizomes) compartments, dried at 60 � C for 48 h and weighed, to estimate above- and below-ground dry biomass (g DW m 2) and above/below-ground biomass ratio (A/B) (Short and Duarte, 2001).

2.5. Statistical analysis The effects of season, site, and depth (fixed and orthogonal factors) and their interactions were evaluated by performing 3-way ANOVAs and Tukey’s tests on morphological, structural, and biochemical plant de­ scriptors. Prior to conducting these analyses, the assumptions of 3

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx

normality were evaluated using the Kolmogorov–Smirnov test (Sokal and Rohlf, 1995) and homogeneity using Bartlett tests. To ensure these assumptions, when appropriate, we transformed the data into Ln. Pearson’s linear regressions were applied to assess potential correlations between (i) nitrogen content and N isotopic signatures of H. stipulacea leaves and (ii) seawater levels of nitrate (NO3), nitrite (NO2), and ammonium (NH4). Statistical analysis was performed using the software package IBM SPSS Inc., v.13. Data represented as mean � standard deviation. General linear models (GLMs) were used to test the effect of the environmental parameters, selected as independent variables (e.g., irradiance, temperature and seawater nutrients) on structural and biochemical descriptors (nutrient content and isotope signature), selected as dependent variables. We assumed a Gaussian distribution because we used negative values in the set of dependent variables (Vaz-Pinto et al., 2013). For performing the GLM, we used R-program (R Core Team, 2013).

p < 0.05), significantly influenced by light availability (Table S2) (GLM, p < 0.05). In addition to the seasonal effect, in both sites, leaf area increased with depth (ANOVA, p < 0.001), with significantly larger leaves in the NB compared with leaves from the SB in both seasons (ANOVA, p < 0.001). In both seasons, this increase in leaf area was particularly high at the SB site at 9 m (210 � 74.4 mm2) and 18 m (244.7 � 74.4 mm2), where winter leaf area was 2-fold higher than at 5 m (99.8 � 8.5 mm2) (Tukey’s, p < 0.01). Above- and below-ground biomasses significantly changed with season, site and depth (Table 2, Fig. 2 c-f), depending on temperature and nutrient content in the water column (Table S2) (GLM, p < 0.01). In both sites, the highest values of above- and below-ground biomasses were observed in summer (ANOVA, p < 0.05). Overall, average biomass values were larger in NB than in SB (ANOVA, p < 0.001); for instance, average values (5, 9, 18 m) of above-ground biomass was 132.5 � 52.2 g DW m 2 in NB vs 70.4 � 3 3.7 g DW m 2 in SB (Tukey’s, p < 0.05). Plants growing in the NB experienced a significantly higher increase in above-ground biomass (>30%) than SB (>5%) in the summer. In both seasons, at the NB site, biomass (above- and below-ground) was higher in the shallowest depth (5 m), while at SB it was higher in the deeper depths (9 and 18 m) (Fig. 2) (Tukey’s, p < 0.05).

3. Results 3.1. Environmental factors Secchi depth at the NB, ranging from 21.5 � 3.59 m in February to 22.7 � 3.5 m in July, were significantly lower compared with the SB values, that ranged from 25.4 � 2.9 m in February to 23.4 � 3.6 m in July (t-test, p < 0.05) (Table 1). Similarly, estimated maximal under­ water PAR was always significantly higher in the SB transects (February ¼ 730.5 and July ¼ 943.1) compared with their same-depth counterparts from the NB (February ¼ 660.7 and July ¼ 764.9) (t-test, p < 0.05). Temperatures ranged over seasons from 22.1 � C in February to 25.6 � C in July, with no differences between sites and depths (Beca-Carretero et al., 2019). Salinity remained stable throughout the year (range of 40.6–40.7 PSU), with no differences between sites (Table 1). Nutrient concentrations (NH4, NO2, NO3) in seawater significantly differed between seasons, with higher concentrations observed in February than in July; annual values of NH4 were marginally higher in the NB (104.0 nmol l 1) than SB (86.3 nmol l 1) (t-test, p ¼ 0.7); how­ ever there were no significant difference in both NO3 and NO2 (Table 1, Fig. S1).

3.3. Density, percentage of cover and reproductive effort Shoot density and percentage of cover changed according to seasons and depths (Fig. 3 a-h) (ANOVA, p < 0.05). Average values (across all depths) of shoot density (Fig. 3 a-b) increased from winter to summer in both sites (a 50.9% increase in the summer at the NB and 21.3% at the SB (ANOVA, p < 0.05). A similar pattern was observed for root densities (Fig. 3 e-f), which was significantly influenced by temperature (Table S2) (GLM, p < 0.001). In addition to the differences between seasons, differences in root densities were also found between sites (ANOVA, p < 0.001), with a higher average (across all depths) summer root density found in the SB (1,680 � 337 roots m 2) compared with an average (across all depths) summer root densities in the NB of 1,164 � 266 roots m 2 (Table 2, Fig. 3) (Tukey’s, p < 0.01). Depth had a different effect on the density parameters in both sites with the number of leaves, vegetative shoots, roots and reproductive shoots found to have a different trend at each site. In the NB, the density parameters decreased with depth from 5 to 18 m, while in the SB, the higher values of these density characteristics were found at 9 m. Overall, the percentage cover increased significantly from winter to summer in the NB (ANOVA, p < 0.001), while in SB, it remained stable over the seasons (Fig. 3 g-h). Moreover, in the SB, a significantly lower per­ centage cover was observed at 5 m than at 9 and 18 m (Tukey’s,

3.2. Biometry and biomass Leaf area increased significantly from summer to winter by 20.4% and 36.9% in NB and SB, respectively (Table 2, Fig. 2 a-b) (ANOVA,

Table 1 Data of the environmental variables measured in NB and SB in February and July 2014, and average data from 2010 to 2015 in February and July ((NMP; http://iui-ei lat.ac.il/Research/NMPMeteoData.aspx; accessed 10/01/2017). Data of turbidity (Secchi depth, m); estimated maximal PAR at 5, 9 and 18 m; surface water tem­ perature (� C); surface water content of NH4 (nmol l 1), NO2 (μmol l 1) and NO3 (μmol l 1). North Beach

Turbidity PAR Water temperature Nutrients

South Beach

Turbidity PAR Water temperature Nutrients

Feb-14

Jul-14

2010–2015 Feb average (�SE)

2010–2015 July average (�SE)

Secchi depth (m) Estimated maximal PAR at 5 m Estimated maximal PAR at 9 m Estimated maximal PAR at 18 m Surface water temperature. Surface water NH4 (nmol l 1) Surface water NO2 (μmol l 1) Surface water NO3 (μmol l 1)

21.5 975.8 657.2 349.1 22.0 107 0.13 0.85

18.0 1197.3 746.7 350.7 25.4 63 0.06 0.057

22.7 (�3.5) 1030.3 693.9 368.6 22.1 (�0.7) 158.5 (�32.3) 0.24 (�0.2) 0.92 (�0.1)

21.5 (�3.9) 1430.2 891.9 418.9 25.3 (�0.3) 105.3 (�55.7) 0.066 (�0.03) 0.55 (�0.3)

Secchi depth (m) Estimated maximal PAR at 5 m Estimated maximal PAR at 9 m Estimated maximal PAR at 18 m Surface water temperature. Surface water NH4 (nmol l 1) Surface water NO2 (μmol l 1) Surface water NO3 (μmol l 1)

25.0 1031.4 734.1 426.1 22.1 180 0.063 0.02

24.0 1347.4 945.5 536.5 25.8 103 0.03 0.06

25.4 (�2.9) 1152.8 776.4 412.4 22.2 (�0.5) 55.0 (�68.7) 0.21 (�0.2) 0.64 (�0.1)

23.38 (�3.6) 1555.2 969.8 455.6 25.7 (�0.3) 93.4 (�32.8) 0.030 (�0.03) 0.30 (�0.2)

4

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx

Table 2 Effect of season (February–July), site (NB - SB) and depth (5, 9 and 18 m) on structural variables of Halophila stipulacea. Phenol content were only measured on one depth (9 m). Reproductive shoots were only observed in one season (July). F-values of three-way ANOVA are shown along with significance levels (*p < 0.05; **p < 0.01, ***p < 0.001). Leaf area

Treatment Season (S) Site (Si) Depth (D) SxSi SxD SixD SxSixD

DF 1 1 2 1 2 2 2

DF 1 1 2 1 2 2 2

)

Above-ground

Below-ground

2

A/B ratio

Phenol content

2

(g DW m )

(g DW m )

F 1.3* 280.4*** 58.0*** 141.5 29.7 125.3*** 62

F 8.2* 425.0*** 6.8* 11.1* 8.0* 142.9*** 63.1***

F 10.6** 29.3*** 5.9** 8.8** 5.5** 24.0*** 17.1***

F 3.3 8.6* 1.8 5.7* 3.7* 0.2 0.2

Number of shoots (m2)

Number of leaves (m2)

Number of roots (m2)

Cover (%)

F 5.9* 0.5 6.1* 0.7 0.9 14.6*** 2

F 0.4 3.9* 3.5* 1.6 1.3 5.3* 0.2

F 66.0*** 5.0* 12.6*** 2 3.0* 18.4*** 11.3***

F 71.2*** 62.1*** 34.1*** 44.5*** 58.5*** 2.3� 17.4***

(mm Treatment Season (S) Site (Si) Depth (D) SxSi SxD SixD SxSixD

2

(mg g DF 1 1 1 -

1

FW 1)

F 11.7*** 85.7*** 3.3 Reproductive shoots (m2)

DF 1 1 1 -

7.5* 3.3� 0.05 -

p < 0.001). Reproductive shoots were only observed in summer, with a higher percentage of plants with male or female reproductive shoots across all depths found in the SB (602 � 497 no. of rep. shoots; 22.1% � 20.0) than in the NB (82 � 4 no. of rep. shoots; 3.8% � 4.7). Besides, in both NB and SB, there was a significantly higher percentage of reproductive plants in shallow and intermediate depths than at 18 m (Tukey’s, p < 0.01).

remained more stable between seasons (Fig. 5). Winter and summer concentration of NO3 (μmol l 1), NO2 (μmol l 1) and NH4 (μmol l 1) in the water column (averages from values measured during February and July months of 2010–2015) were significantly correlated (p < 0.05–0.001, R2 ¼ 0.4–0.8, n ¼ 8) with δ15N. Also, NO3 (μmol l 1) and NO2 (μmol l 1) concentration were correlated (p < 0.1–0.001, R2 ¼ 0.3–0.8, n ¼ 8).

3.4. Total phenol content

4. Discussion

Total phenol contents in plants differed significantly across seasons and sites (Table 2, Fig. 4) (ANOVA, p < 0.001). In both sites, total phenol concentrations at 9 m were higher in winter than in summer (NB: 27.7% and SB: 46.3%). The average annual values (average of summer and winter total phenol contents values) were higher at the SB (2.67 � 0.79 mg g 1 FW) than at NB (1.78 � 0.6 mg g 1 FW) (Fig. 4).

4.1. Temporal, spatial and depth-related variability in plant descriptors H. stipulacea is a perennial species in GoA, although our results show strong summer-winter dynamics in plant descriptors, changing with the environmental variations. Larger leaves observed in February, were replaced by smaller ones in July, alongside a significant increase in above-ground biomass, density, and percentage cover, confirm July (Temperature ~26–27 � C and PAR ¼ 854.0) as the most productive season in the GoA during the study period. Previous studies on H. stipulacea in the GoA also showed temporal patterns with growth peaks attained in warmer seasons (Wahbeh, 1988; Cardini et al., 2017). Morphological and population structure responses to varying climatic conditions represents a common adaptative mechanism of seagrass to adjust photosynthetic activity and optimize metabolic costs (Touchette and Burkholder, 2000; Alcoverro et al., 2001; Collier et al., 2011). In the Arabian Gulf, where H. stipulacea is also considered a native species, summer seawater temperatures reach 28–31 � C in local meadows (Naser, 2014; Campbell et al., 2015); in Cyprus, a colonized Mediter­ ranean area, H. stipulacea showed maximum photosynthetic rates and clonal growth at 30 � C (Georgiou et al., 2016). This adaptability to high temperatures suggests the capability of the species to thrive under future ocean warming in the area under study by the end of this century (~31–22 � C, IPCC, 2014). Indeed, this thermal tolerance suggests a potential future expansion under climate change scenarios in the Med­ iterranean Sea. The capacity of H. stiuplacea to cope with differences in environ­ mental conditions is clearly shown in this study. The leaf surface area, more extensive in plants from the NB than the SB in both seasons, is probably due to the environmental differences between the two sites, as suggested by Mejia et al., (2016) and Winters et al., (2017); for instance, the lower Kd and the attenuation coefficient of PAR, at the same depths in the NB compared to the SB (Table 1). These differences are associated

3.5. Stable nitrogen isotopes and nutrient content Overall, when pooling values of δ15N values from different seasons, sites and depths, values of δ15N differed among the different seagrass tissues, and were higher (i.e., more enriched) in leaves (1.4 � 0.7‰) than in roots (0.90 � 0.3‰) and rhizomes (0.51 � 0.3‰) (Fig. 5). In both sites, leaves had significantly higher δ15N values in February than in July, and values from NB plants across the different depths were higher in both seasons than in plants from the SB over the studied period (Table 3) (ANOVA, p < 0.01). Similarly, N% was overall higher (more enriched) in leaves (1.5 � 0.2%) than in roots (0.6 � 0.2%) and rhi­ zomes (0.32 � 0.1‰) (Fig. 5). Also, in both δ15N and N%, there was a significant interaction between season and site, indicating that in SB and NB, there was a different seasonal variability (ANOVA, p < 0.01). N% in leaves from the SB displayed significant differences temporally with larger N% values observed in February than in July (ANOVA, p < 0.001); however, there were no marked differences in N% in leaves from the NB over the seasons (Table 3). Particularly, in the NB, N% contents in leaves were stable between winter and summer, while in leaves from the SB, levels of N fluctuated significantly with lower levels observed in summer and higher levels in the winter. In contrast to δ15N and N%, carbon content was more constant among seagrass tissues as it averaged across depths from 25.4 � 4.9% in leaves to 27.1 � 5.5% in roots and 23.7 � 1.1% in rhizomes. The C:N ratios decreased markedly in leaves in comparison with roots and especially rhizomes, where ratios 5

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx

Fig. 2. Leaf area (mm2; n ¼ 30; A-B), above-ground (C-D) and below-ground (E-F) biomass (g DW m 2; n ¼ 3), and above/below-ground biomass ratio (n ¼ 3; G-H) of Halophila stipulacea at each season (February and July 2014), site (north beach and south beach) and depth (5, 9 and 18 m). Data represented as means � standard deviations. Significant differences among seasons based on post-hoc Tukey’s test are indicated with asterisks (*p < 0.05; **p < 0.01, ***p < 0.001).

with higher nutrient concentrations, which lead to higher water turbidity in the NB than in the SB. Also, biomass (AG and BG) and density were always higher in NB than in SB. A separate discussion deserves the remarkable differences in biomass/number of shoots that was found at the shallow depth (5 m) at the SB site, probably related to the type of rubble sediment found at the 5 m depth of the SB, in contrast with the sandy sediment and the higher nutrient availability in the NB (Mejia et al., 2016; Rotini et al., 2017). The 5 m at the SB site represents the shallow edge of the meadow, which experience marked seasonal variability regarding biomass and percentage cover, hence, far from the ideal environmental conditions for H. stiuplacea plants. The ability of Halophila to adjust leaf morphology and their photo­ synthetic apparatus clearly indicates its efficacy to adapt to light-limited conditions; prior studies demonstrated that depletions in PAR with depth induced an increase in both leaf area, chlorophyll a-b and omega-3

polyunsaturated fatty acids (Rotini et al., 2017; Beca-Carretero et al., 2019). This adaptation is a well-known described mechanism of sea­ grasses to cope with low irradiance levels (e.g., Krause-Jensen et al., 2000; Sharon et al., 2009; Beca-Carretero et al., 2019; Schubert et al., 2015: Beca-Carretero et al., 2019b); H. stipulacea was described as one of the deepest adapted seagrasses worldwide (Duarte, 1991). It was observed at maximum depths of more than 50 m in GoA, native area (Sharon et al., 2011; Winters et al., 2017) and 45 in the Mediterranean, newly colonized area (Cyprus and Greece, Lipkin, 1975a). The decrease of shoot density and below-ground biomass with depth is a common feature of seagrasses to adjust their productivity and metabolism ac­ cording to environmental resources such as irradiance (e.g., Alcoverro et al., 1995, 2001; Krause-Jensen et al., 2000). The reproductive activity of H. stipulacea is restricted to the summer period; reproductive shoots were only present in this season as reported 6

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx

Fig. 3. Vegetative shoot (A-B), leaf (C-D) and root (E-F) densities (n ¼ 3; per m 2) and percentage cover (n ¼ 12; G-H) of Halophila stipulacea at each season (February and July 2014), site (north beach and south beach) and depth (5, 9 and 18 m). Data represented as means � standard deviations. Significant differences among seasons based on post-hoc Tukey’s test are indicated with asterisks (*p < 0.05; **p < 0.01, ***p < 0.001).

in previous studies in the same region, indicating that in this species, reproductive effort is actively controlled by increases in temperature and light (Malm, 2006; Diaz-Almela et al., 2007; Nguyen et al., 2018), as was also observed in other tropical species (Durako and Moffler, 1987; �n et al., 2003). Differences in irradiance levels are known to affect Rollo reproductive effort, seed germination, and settlement processes of sea­ grass species (Diaz-Almela et al., 2007). Reproductive effort was different in the two meadows, with lower incidences of reproductive shoots in plants from the NB compared with their counterparts from the SB. According to the observations of Cabaco and Santos (2012), which stated that seagrass sexual reproductive effort increased in unfavorable environments, these differences may suggest that the SB might not represent the ideal environment for H. stipulacea plants, as hypothesized by Mejia et al. (2016). In the Mediterranean Sea, the recent documen­ tation of both female and male H. stipulacea flowers alongside the finding of mature seed capsules (Gerakaris and Tsiamis, 2015; Nguyen et al., 2018), changed the previous suggestions of a dominance of clonal propagation in the basin (Procaccini et al., 1999; Gambi et al., 2009).

Fig. 4. Mean total phenol content in leaves of Halophila stipulacea at each season (February and July 2014), site (north beach and south beach) at 9 m depth Data represented as mean � standard deviation. Significant differences among seasons based on post-hoc Tukey’s test are indicated with asterisks (*p < 0.05; **p < 0.01, ***p < 0.001).

7

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx

Fig. 5. Stable nitrogen isotope ratio (δ15N), nitrogen content (N), stable carbon isotope (δ13C), carbon content (C) and carbon to nitrogen ratio (C:N) in the different plant compartments (rhizomes, roots and leaves) (n ¼ 3) of Halophila stipulacea at each season (February and July 2014), site (north beach and south beach) and depth (9 and 18 m). Data are represented as means � standard deviations. Significant differences among seasons based on post-hoc Tukey’s test are indicated with asterisks (*p < 0.05; **p < 0.01, ***p < 0.001).

8

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx

Table 3 δ15N, nitrogen and carbon content (%) and C:N ratio in different plant compartments (rhizome, root and leaf) of Halophila stipulacea according to season (Februar­ y–July), location (north beach -south beach) and depth (5, 9 and 18 m). F-values of three-way ANOVA are shown along with significance levels (*p < 0.05; **p < 0.01, ***p < 0.001). Rhizome 15

Treatment Season (S) Site (Si) Depth (D) SxSi SxD SixD SxSixD

DF 1 1 1 1 1 1 1

Root

δ N (‰)

N (%)

C (%)

C:N

F 2.67 2.83 10.81 * 0.14 0.09 10.21 ** 0.88

F 0.1 10.4* 7.0* 2.1 1.6 0.2 0.7

F 0.3 2.9 0.1 1.2 0.5 0.5 0.1

F 0.09 13.36 ** 7.20 ** 1.62 1.83 0.12 0.76

Leaf

15

DF 1 1 1 1 1 1 1

δ N (‰)

N (%)

C (%)

C:N

F 6.3* 2.0 6.5* 13.6** 0.9 22.3** 18.3**

F 0.7 0.0 3.2* 1.1 1.3 0.8 1.8

F 1.7 0.2 1.2 0.2 1.5 0.4 0.4

F 0.0 0.1 2.3 2.4 0.3 2.6 4.7*

Furthermore, Nguyen et al. (2018) also showed higher plasticity in the sex ratio of invasive H. stipulacea plants, compared with native pop­ ulations in the GoA, further supporting the high sexual plasticity of H. stipulacea. The total phenol contents in marine angiosperms is considered a reliable descriptor of the seagrass eco-physiological status in response to different environmental conditions, including grazing pressure; the in­ crease of total phenol content being a stress-related response (e.g., Rotini et al., 2013; Silva et al., 2013; Rotini et al., 2017; Ceccherelli et al., 2018). In both SB and NB, higher phenol content was observed in winter, when the relatively low temperatures and irradiances represent less suitable environmental conditions for H. stipulacea in the GoA (Wahebe, 1988). Furthermore, higher phenol concentrations were found in plants from the SB, compared with plants from the NB, suggesting again that NB conditions are more suitable for H. stipulacea than SB, as suggested by Mejia et al. (2016).

DF 1 1 1 1 1 1 1

δ15N (‰)

N (%)

C (%)

C:N

F 25.6*** 3.7* 9.0** 5.1* 1.0 0.1 10.0**

F 58.1*** 0.9 7.1* 20.7** 0.7 3.1 7.0*

F 21.0*** 17.4*** 6.9* 4.0 8.2* 2.8 3.7

F 3.8 6.9* 0.0 3.1 2.8 6.9* 0.2

leaves (Fig. 6). Our results reinforce the suggestion of Cardini et al. (2017) that H. stipulacea lives in N limited conditions in its native tropical habitat. A slight input of anthropogenic origin nutrients (δ15N) (Winters et al., 2017) may favor H. stipulacea growth and vegetative performance, as was observed in the NB. Previous studies have demonstrated a better assimilation of NH4 than NO3 in several seagrass species (e.g., Thallassia testudinum or Phyllospadix torrey), which was explained by the higher affinity of these seagrasses to reduced N forms (NH4) with lower meta­ bolic costs, as compared with the metabolic costs associated with the uptake of NO3 (e.g., McRoy, 1974; Lee and Dunton, 1999b). In contrast to N, C content remained quite stable over the study sites, depths and seasons, as was previously reported (Wahbeh, 1988; Schwarz and Hellblom, 2002) and may be explained by the lower variability of car­ bon compounds in oligotrophic marine systems (Atkinson and Smith, 1984; Duarte, 1992). Noticeable, these results suggest the capability of H. stipulacea to acquire and store anthropogenic nutrients from the water column, therefore increasing water quality for neighboring ecosystems such as coral communities (Lamb et al., 2017), enhancing the impor­ tance of preserving of local seagrass meadows. Finally, with the rapid rate of climate change in the Mediterranean Sea (warming at 2 x global average global mean coastal SST trend (IPCC, 2014) alongside it becoming saltier (e.g., Bianchi and Morri, 2000; Ozer et al., 2016), it has been predicted that the ongoing tropicalization of the Mediterranean Sea might cause it to become less favorable for native seagrasses (e.g., the slow-growing and endemic climax Posidonia oce­ anica; Jord� a et al., 2012), and more welcoming for fast-growing and warmer-tolerant seagrass species, such as the invasive species H. stipulacea. Recent studies from the Mediterranean have already re­ ported diebacks and regressions of P. oceanica populations under anomalous high temperatures conditions reached in recent summers � and Duarte, (28.83 and 28.54 � C in 2003 and 2006, respectively) (Marba � et al., 2012) which can make available new habitats for 2010; Jorda other seagrass species (e.g., Cymodocea nodosa or H. stipulacea) or green algae, as Caulerpa spp. (De Vill� ele and Verlaque, 1995; Peirano et al., 2005). The plasticity of morphological, structural, biochemical and reproductive descriptors observed in this study and in previous ones confirms the ability of H. stipulacea to thrive under different environ­ mental conditions (Por, 1971; Gambi et al., 2009; Sharon et al., 2009, 2011; Oscar et al., 2018), and may represents an advantage in the spreading, competition with other species, and colonisation of new habitats. It is urgent to set up H. stipulacea monitoring campaigns, in both native and invasive sites to further comprehend its behavior, document changes in its distribution, and potentially predict its inva­ siveness capacity in the coming future. Overall, understanding how seagrass communities respond to global change alongside the impacts of invasive species is particularly relevant since shifts in the native seagrass community have an implicit impact on the associated ecosystem func­ tionality affecting all the tropic levels.

4.2. Effect of anthropogenic nutrient inputs We observed significantly larger contents of N in leaves than in rhizomes or roots, as was previously reported for other Halophila spp. (i. e, Lee and Dunton, 1999a; Evrad et al., 2005). Prior studies have demonstrated that seagrasses are capable of changing from leaf-dominated to root-dominated nutrient uptake depending on different factors, such as water or sediment nutrient content (Carignan and Kalff, 1980). In our measurements, levels of N in both leaves and roots/rhizome were always lower than 1.8% indicating a marked N limitation in the oligotrophic GoA environments, as previously sug­ gested (Powell et al., 1989; Duarte, 1992; Schwarz and Hellblom, 2002; Cardini et al., 2017). GoA is characterized by deep winter mixing processes (with a maximum mixing depth of up to 500–900 m) observed until early spring which entails increases in nutrient levels in the upper seawater layers (Genin et al., 1995; Carlson et al., 2014). In both the NB and SB sites, the higher concentrations of N observed in both the water column and in H. stipulacea plants in winter may be associated with the winter mixing processes in the GoA, which significantly increases the nutrient levels in the upper layer of the seawater (Genin et al., 1995; Labiosa et al., 2003). Interestingly, we found higher levels of NH4 in seawater in the NB than in the SB alongside a higher annual signal of δ15N in plants living in the anthropized site (NB) compared with the more pristine site (SB). These results indicate a potential anthropogenic input of N in the seawater, which is reflected in the plant content. Indeed, this is further supported by the finding that plants from the NB maintained similar annual levels of N content during the study period (February–July), while plants from the SB experienced a marked decrease from February to July. Therefore, these results may suggest that the NB site may experience a constant anthropogenic nutrient contribution (Mejia et al., 2016; Winters et al., 2017); these assumptions are supported by the most of positive corre­ lations between nutrient concentrations in the seawater column (NO3, NO2, and NH4) and the signal of δ15N and the N content in the seagrass 9

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx

Fig. 6. Correlations between nitrogen contents (N%) (A–C) and δ15N (‰) (D–F) of Halophila stipulacea leaves with the nitrate (NO3) (μmol/l), nitrite contents (NO2) (μmol/l) and ammonium (NH4) (nmol/l) in surficial seawater (2 m depth) in the two seasons (taken from averages for February and July from NMP data for the years 2010–2015) and sites (NB and SB). Black lines represent the linear regression line (n ¼ 8).

5. Conclusions

Appendix A. Supplementary data

Our findings indicate the high capacity of H. stipulacea to thrive in varied environmental conditions, including N-enriched seawater con­ ditions, suggesting a highly morphological, structural, and biochemical plasticity. The multidisciplinary approach described/applied here showed its great potential and usefulness; thus, a similar methodology is recommended for future assessments of H. stipulacea meadows. The present study includes the most detailed quantitative information on H. stipulacea providing a general framework and background for future monitoring efforts to measure impacts of local and global dynamics as well as a baseline for comparison with other native and non-native populations.

Supplementary data to this article can be found online at https://doi. org/10.1016/j.marenvres.2019.104828. References Abramoff, M.D., Magalhaes, P.J., Ram, S.J., 2004. Image processing with image. J. Biophotonics. Int. 11, 36–42. Al-Rousan, S., Al-Horani, F., Eid, E., Khalaf, M., 2011. Assessment of seagrass communities along the Jordanian coast of the Gulf of Aqaba, Red Sea. Mar. Biol. Res. 7, 93–99. Alcoverro, T., Duarte, C.M., Romero, J., 1995. Annual growth dynamics of Posidonia oceanica: contribution of large-scale versus local factors to seasonality. Mar. Ecol. Prog. Ser. 120, 203–210. Alcoverro, T., Manzanera, M., Romero, J., 2001. Annual metabolic carbon balance of the seagrass Posidonia oceanica: the importance of carbohydrate reserves. Mar. Ecol. Prog. Ser. 211, 105–116. Angel, D.L., Eden, N., Susel, L., 1995. The influence of environmental variables on Halophila stipulacea growth. In: Rosenthal, H., Moav, B., Gordin, H. (Eds.), Improving the Knowledge Base in Modern Aquaculture, vol. 25. Eur. Aquac. Soc. Spec. Publ, pp. 103–128. Atkinson, M.J., Smith, S.V., 1984. C:N:P ratios of benthic marine plants. Limnol. Oceanogr. 28, 568–574. Beca-Carretero, P., Guih�eneuf, F., Winters, G., Stengel, D.B., 2019. Depth-induced adjustment of fatty acid and pigment composition suggests high biochemical plasticity in the tropical seagrass Halophila stipulacea. Mar. Ecol. Prog. Ser. 608, 105–177. Beca-Carretero, P., Stanschewski, C.L., Julia-Miralles, M., Sanchez-Gallego, A., Stengel, D.B., 2019. Structure, morphometry, and productivity of pristine Zostera marina meadows in western Ireland. Aquat. Bot. 155, 5–17. Bianchi, C.N., Morri, C., 2000. Marine biodiversity of the Mediterranean Sea: situation, problems, and prospects for future research. Mar. Pollut. Bull. 40, 367–376. Bulthuis, D.A., 1987. Effects of temperature on photosynthesis and growth of seagrasses. Aquat. Bot. 27, 27–40. Cabaco, S., Santos, R., 2012. Seagrass reproductive effort as an ecological indicator of disturbance. Ecol. Indicat. 23, 116–122. Campbell, J.E., Lacey, E.A., Decker, R.A., Crooks, S., Fourqurean, J.W., 2015. Carbon storage in seagrass beds of Abu Dhabi, United Arab Emirates. Estuar. Coasts 38, 242–251.

Acknowledgments We are highly thankful to the Associate Editor and the anonymous reviewer for their valuable suggestions and constructive criticisms during the review process. This work was supported by the Israeli Ministry of Environmental Protection (grant number121-4-1), the Israeli Nature Park Authority (INPA) and the Dead Sea-Arava Science Center (ADSSC). AYM and AR were recipients of a Short-term Scientific Mission grant from the COST Action scientific program (ES0906) on “Seagrass productivity: from genes to ecosystem management” to travel to Israel and conduct sampling activities (COST-STSM-ES0906-06445 and ES0906-15001). AYM was funded by a PhD grant (International PhD program) from the University of Tor Vergata (XXVII Cycle). AR was funded by a Postdoctoral grant from the University of Tor Vergata/ RegioneLazio Research Program “Certificazione di prodotti agro­ �: dal DNA barcoding al naso elettronico – Area alimentaridi qualita tematicaDTB: Bioscienze & Biotec Verdi”.

10

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx

Cardini, U., van Hoytema, N., Bednarz, V.N., Al-Rshaidat, M.M.D., Wild, C., 2017. N 2 fixation and primary productivity in a red sea Halophila stipulacea meadow exposed to seasonality. Limnol. Oceanogr. 63, 786–798. Carignan, R., Kalff, J., 1980. Phosphorous sources for aquatic weeds: water or sediment? Science 207, 987–989. Carlson, D.F., Fredj, E., Gildor, H., 2014. The annual cycle of vertical mixing and restratification in the northern Gulf of Eilat/Aqaba (Red Sea) based on high temporal and vertical resolution observations. Deep-Sea Res. 84, 1–17. Ceccherelli, G., Oliva, S., Pinna, S., Piazzi, L., Procaccini, G., Marin-Guirao, L., Dattolo, E., Gallia, R., La Manna, G., Gennaro, P., Costa, M.M., Barrote, I., Silva, J., Bulleri, F., 2018. Seagrass collapse due to synergistic stressors is not anticipated by phenological changes. Oecol 186, 1137–115. Collier, C.J., Uthicke, S., Waycott, M., 2011. Thermal tolerance of two seagrass species at contrasting light levels: implications for future distribution in the Great Barrier Reef. Limnol. Oceanogr. 56, 2200–2210. De Vill� ele, X., Verlaque, M., 1995. Changes and degradation in a Posidonia oceanica bed invaded by the introduced tropical alga Caulerpa taxifolia in the north western Mediterranean. Bot. Mar. 38, 79–88. Diaz-Almela, E., Marba, N., Duarte, C.M., 2007. Consequences of Mediterranean warming events in seagrass (Posidonia oceanica) flowering records. Glob. Chang. Biol. 13, 224–235. Duarte, C.M., 1991. Seagrass depth limits. Aquat. Bot. 40, 363–377. Duarte, C.M., 1992. Nutrient concentration of aquatic plants: patterns across species. Limnol. Oceanogr. 37, 882–889. Durako, M.J., Moffler, M.D., 1987. Factors affecting the reproductive ecology of Thalassia testudinum (Hydrocharitaceae). Aquat. Bot. 27, 79–95. El Shaffai, A., 2011. A Field Guide to Seagrasses of the Red Sea, first ed. Total Foundation, Gland, Switzerland. IUCN and Courbevoie, France. Erftemeijer, P.L., Herman, P.M., 1994. Seasonal changes in environmental variables, biomass, production and nutrient contents in two contrasting tropical intertidal seagrass beds in South Sulawesi, Indonesia. Oecologia 99, 45–59. Evrad, V., Kiswara, W., Bouma, T.J., Middelburg, J.J., 2005. Nutrient dynamics of seagrass ecosystems: 15N evidence for the importance of particulate organic matter and root systems. Mar. Ecol. Prog. Ser. 295, 49–55. Fourqurean, J.W., Duarte, C.M., Kennedy, H., Marba, N., Holmer, M., Mateo, M.A., Apostolaki, E.T., Kendrick, Ga, Krause-Jensen, D., McGlathery, K.J., Serrano, O., 2012. Seagrass ecosystems as a globally significant carbon stock. Nat. Geosci. 5, 505–509. Gambi, M.C., Barbieri, F., Bianchi, C.N., 2009. New record of the alien seagrass Halophila stipulacea (Hydrocharitaceae) in the western Mediterranean: a further clue to changing Mediterranean Sea biogeography. Mar. Biodivers. Rec. 2, 84. Genin, A., Gal, G., Haury, L., 1995. Copepod carcasses in the ocean. II. Near coral reefs. Mar. Ecol. Prog. Ser. 123, 65–71. Georgiou, D., Alexandre, A., Luis, J., Santos, R., 2016. Temperature is not a limiting factor for the expansion of Halophila stipulacea throughout the Mediterranean Sea. Mar. Ecol. Prog. Ser. 544, 159–167. Gerakaris, V., Tsiamis, K., 2015. Sexual reproduction of the Lessepsian seagrass Halophila stipulacea in the Mediterranean Sea. Bot. Mar. 58, 51–53. IPCC (Intergovernmental Panel on Climate Change), 2014. Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. Jord� a, G., Marb� a, N., Duarte, C.M., 2012. Mediterranean seagrass vulnerable to regional climate warming. Nat. Clim. Chang. 2, 821–824. Kenworthy, W.J., Durako, M.J., Fatemy, S.M.R., Valavi, H., Thayer, G.W., 1993. Ecology of seagrasses in northeastern Saudi Arabia one year after the Gulf War oil spill. Marine pollution bulletin 27, 213–222. Kohler, K.E., Gill, S.M., 2006. Coral point count with excel extensions (CPCe): a visual basic program for the determination of coral and substrate coverage using random point count methodology. Comput. Geosci. 32, 1259–1269. Krause-Jensen, D., Middelboe, A.L., Sand-Jensen, K., Christensen, P.B., 2000. Eelgrass, Zostera marina, growth along depth gradients. Oikos 91, 233–244. Labiosa, R.G., Arrigo, K.R., Genin, A., Monismith, S.G., van Dijken, G., 2003. The interplay between upwelling and deep convective mixing in determining the seasonal phytoplankton dynamics in the Gulf of Aqaba: evidence from SeaWiFS and MODIS. Limnol. Oceanogr. 48, 2355–2368. Lamb, J.B., Van De Water, J.A., Bourne, D.G., Altier, C., Hein, M.Y., Fiorenza, E.A., Harvell, C.D., 2017. Seagrass ecosystems reduce exposure to bacterial pathogens of humans, fishes, and invertebrates. Science 35, 731–733. Lee, K.-S., Dunton, K.H., 1999. Inorganic nitrogen acquisition in the seagrass Thalassia testudinum: development of a whole-plant nitrogen budget. Limnol. Oceanogr. 44, 1204–1215. Lee, K.-S., Dunton, K.H., 1999. Influence of sediment nitrogen availability on carbon and nitrogen dynamics in the seagrass Thalassia testudinum. Mar. Biol. 134, 217–226. Lipkin, Y., 1975. Halophila stipulacea, a review of a successful immigration. Aquat. Bot. 1, 203–215. Lipkin, Y., 1975. Halophila stipulacea in Cyprus and Rhodes, 1967–1970. Aquat. Bot. 1, 309–320. Loya, Y., Lubinevsky, H., Rosenfeld, M., Kramarsky-Winter, E., 2004. Nutrient enrichment caused by in situ fish farms at Eilat, Red Sea is detrimental to coral reproduction. Mar. Pollut. Bull. 49, 344–353. Malm, T., 2006. Reproduction and recruitment of the seagrass Halophila stipulacea. Aquat. Bot. 85, 345–349. Marb� a, N., Duarte, C.M., 2010. Mediterranean warming triggers seagrass (Posidonia oceanica) shoot mortality. Glob. Chang. Biol. 16, 2366–2375. McRoy, C.P., 1974. Seagrass productivity: carbon uptake experiments in eelgrass, Zostera marina. Aquac. Environ. Interact. 4, 131–137.

Mejia, A.Y., Rotini, A., Lacasella, F., Bookman, R., Thaller, M.C., Shem-Tov, R., Winters, G., Migliore, L., 2016. Assessing the ecological status of seagrasses using morphology, biochemical descriptors and microbial community analyses. A study in Halophila stipulacea (Forsk) Aschers meadows in the northern Red Sea. Ecol. Indicat. 60, 1150–1163. Migliore, L., Rotini, A., Randazzo, D., Albanese, N.N., Giallongo, A., 2007. Phenols content and 2-D electrophoresis protein pattern: a promising tool to monitor Posidonia meadows health state. BMC Ecol. 7, 6. Naser, H.A., 2014. Marine ecosystem diversity in the Arabian Gulf: threats and conservation. In: Biodiversity-The Dynamic Balance of the Planet. IntechOpen. Nguyen, H.M., Kleitou, P., Kletou, D., Sapir, Y., Winters, G., 2018. Differences in flowering sex ratios between native and invasive populations of the seagrass Halophila stipulacea. Bot. Mar. 61, 337–342. Nordlund, L.M., Jackson, E.L., Nakaoka, M., Samper-Villareal, J., Beca-Carretero, P., Creed, J.C., 2018. Seagrass ecosystem services – what’s next? Mar. Pollut. Bull. 134, 145–151. Orth, R.J., Carruthers, T.J.B., Dennison, W.C., Duarte, C.M., Fourqurean, J.W., et al., 2006. A global crisis for seagrass ecosystems. Bioscience 56, 987–996. Oscar, M.A., Barak, S., Winters, G., 2018. The tropical invasive seagrass, Halophila stipulacea has a superior ability to tolerate dynamic changes in salinity levels compared to its freshwater relative, Vallisneria americana. Front. Plant Sci. 9, 950. Ozer, T., Gertman, I., Kress, N., Silverman, J., Herut, B., 2016. Interannual thermohaline (1979–2014) and nutrient (2002–2014) dynamics in the Levantine surface and intermediate water masses, SE Mediterranean Sea. Glob. Planet. Chang. Peirano, A., Damasso, V., Montefalcone, M., Morri, C., Bianchi, C.N., 2005. Effects of climate, invasive species and anthropogenic impacts on the growth of the seagrass Posidonia oceanica (L.) Delile in Liguria (NW Mediterranean Sea). Mar. Pollut. Bull. 50, 817–822. Por, F.D., 1971. One hundred years of Suez Canal-a century of Lessepsian migration: retrospect and viewpoints. Syst. Zool. 20, 138–159. Powell, G.V.N., Kenworthy, W.J., Fourqurean, J.W., 1989. Experimental evidence for nutrient limitations of seagrass growth in a tropical estuary with respect to circulation. Bull. Mar. Sci. 44, 324–340. Procaccini, G., Acunto, S., Fam� a, P., Maltagliati, F., 1999. Structural, morphological and genetic variability in Halophila stipulacea (Hydrocharitaceae) populations in the western Mediterranean. Mar. Biol. 135, 181–189. R Core Team, 2013. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/. Reiss, Z., Hottinger, L., 1984. The Gulf of Aqaba: Ecological Micropaleontology. Springer, Berlin. Roll� on, R.N., van Steveninck, E.D.D.R., van Vierssen, W., 2003. Spatio-temporal variation in sexual reproduction of the tropical seagrass Enhalus acoroides (Lf) Royle in Cape Bolinao, NW Philippines. Aquat. Bot. 76, 339–354. Romero, J., P� erez, M., Mateo, M.A., Sala, E., 1994. The belowground organs of the Mediterranean seagrass Posidonia oceanica as a biogeochemical sink. Aquat. Bot. 47, 13–19. Rotini, A., Belmonte, A., Barrote, I., Micheli, C., Peirano, A., Santos, R., Silva, J., Migliore, L., 2013. Effectiveness and consistency of a suite of descriptors to assess the ecological status of seagrass meadows (Posidonia oceanica L Delile). Estuar. Coast Shelf Sci. 130, 252–259. Rotini, A., Mejia, A.Y., Costa, R., Migliore, L., Winters, G., 2017. Ecophysiological plasticity and bacteriome shift in the seagrass Halophila stipulacea along a depth gradient in the northern Red Sea. Front. Plant Sci. 7, 2015. Ruiz, H., Ballantine, D.L., Sabater, J., 2017. Continued spread of the seagrass Halophila stipulacea in the Caribbean: documentation in Puerto Rico and the British Virgin Islands. Gulf Caribb. Res. 28, 5–7. Schubert, N., Colombo-Pallota, M.F., Enríquez, S., 2015. Leaf and canopy scale characterization of the photoprotective response to high-light stress of the seagrass Thalassia testudinum. Limnol. Oceanogr. 60, 286–302. Schwarz, A.M., Hellblom, F., 2002. The photosynthetic light response of Halophila stipulacea growing along a depth gradient in the Gulf of Aqaba, the Red Sea. Aquat. Bot. 74, 263–272. Sghaier, Y.R., Zakhama-Sraieb, R., Benamer, I., Charfi-Cheikhrouha, F., 2011. Occurrence of the seagrass Halophila stipulacea (Hydrocharitaceae) in the southern Mediterranean Sea. Bot. Mar. 54, 575–582. Sharon, Y., Silva, J., Santos, R., Runcie, J., Chernihovsky, M., Beer, S., 2009. Photosynthetic responses of Halophila stipulacea to a light gradient: II – plastic acclimations following transplantations. Aquat. Biol. 7, 153–157. Sharon, Y., Dishon, G., Beer, S., 2011. The effect of UV radiation on chloroplast clumping and photosynthesis in the seagrass Halophila stipulacea grown under high-PAR conditions. J. Mar. Biol. 2011, 483–428. Short, F.T., Duarte, C.M., 2001. Methods for the measurement of seagrass growth and production. In: Coles, R.G. (Ed.), Global Seagrass Research Methods. Elsevier, Amsterdam, pp. 155–182. Silva, J., Barrote, I., Costa, M., Albano, S., Santos, R., 2013. Physiological responses of Zostera marina and Cymodocea nodosa to light-limitation stress. PLoS One 8 e81058. Smulders, F.O.H., Vonk, J.A., Engel, M.S., Christianen, M.J.A., 2017. Expansion and fragment settlement of the non-native seagrass Halophila stipulacea in a Caribbean bay. Mar. Biol. Res. 13, 967–974. Sokal, R.R., Rohlf, F.J., 1995. Biometry. W. H. Freeman, New York. Steiner, S.C.C., Willette, D.A., 2010. Distribution and size of benthic marine habitats in Dominica, Lesser Antilles. Rev. Biol. Trop. 58, 589–602. Steiner, S.C.C., Willette, D.A., 2015. The expansion of Halophila stipulacea (Hydrocharitaceae, Angiospermae) is changing the seagrass landscape in the Commonwealth of Dominica, Lesser Antilles. Caribb. Nat. 22, 1–19.

11

P. Beca-Carretero et al.

Marine Environmental Research xxx (xxxx) xxx Wahbeh, M.I., 1984. The growth and production of the leaves of the seagrass Halophila stipulacea (Forsk.) Aschers. from Aqaba, Jordan. Aquat. Bot. 20, 33–41. Wahbeh, M.I., 1988. Seasonal distribution and variation in the nutritional quality of different fractions of two seagrass species from Aqaba (Red Sea), Jordan. Aquatic botany 32 (4), 383–392. Waycott, M., Duarte, C.M., Carruthers, T.J.B., Orth, R.J., Dennison, W.C., 2009. Accelerating loss of seagrasses across the globe threatens coastal ecosystems. PNAS 106, 12377–12381. Willette, D.A., Ambrose, R.F., 2009. The distribution and expansion of the invasive seagrass Halophila stipulacea in Dominica, West Indies, with a preliminary report from St. Lucia. Aquat. Bot. 91, 137–142. Willette, D.A., Chalifour, J., Debrot, A.O.D., Engel, M.S., Miller, J., Oxenford, H.A., Short, F.T., Steiner, S.C.C., V� edie, F., 2014. Continued expansion of the transAtlantic invasive marine angiosperm Halophila stipulacea in the Caribbean. Aquat. Bot. 112, 98–102. Winters, G., Loya, Y., Beer, S., 2006. In situ measured seasonal variations in Fv/Fm of two common Red Sea corals. Coral Reefs 25, 593–598. Winters, G., Edelist, D., Shem-Tov, R., Beer, S., Rilov, G., 2017. A low-cost field-survey method for mapping seagrasses and their potential threats: an example from the northern Gulf of Aqaba, Red Sea. Aquat. Conserv. Mar. Freshw. Ecosyst. 27, 324–339.

Stubler, A.D., Jackson, L.J., Furman, B.T., Peterson, B.J., 2017. Seed production patterns in Zostera marina: effects of patch size and landscape configuration. Estuar. Coasts 40, 564–572. Touchette, B.W., Burkholder, J.M., 2000. Overview of the physiological ecology of carbon metabolism in seagrasses. JEMBE 250, 169–205. van der Heide, T., van Nes, E.H., Geerling, G.W., Smolders, A.J.P., Bouma, T.J., van Katwijk, M.M., 2007. Positive feedbacks in seagrass ecosystems: implications for success in conservation and restoration. Ecosystems 10, 1311–1322. Vaz-Pinto, F., Olabarria, C., Arenas, F., 2013. Role of top-down and bottom-up forces on the invasibility of intertidal macroalgal assemblages. Journal of sea research 76, 178–186. Vera, B., Collado-Vides, L., Moreno, C., van Tussenbroek, B.I., 2014. Halophila stipulacea (Hydrocharitaceae): a recent introduction to the continental waters of Venezuela. Caribb. J. Sci. 48, 66–70. Viana, I.G., Saavedra-Hortúa, D.A., Mtolera, M., Teichberg, M., 2019. Different strategies of nitrogen acquisition in two tropical seagrasses under nitrogen enrichment. New Phytol. 223, 1217–1229. Vizzini, S., Mazzola, A., 2004. Stable isotope evidence for the environmental impact of a land-based fish farm in the western Mediterranean. Mar. Pollut. Bull. 49, 61–70. Vizzini, S., Mazzola, A., 2006. The effects of anthropogenic organic matter inputs on stable carbon and nitrogen isotopes in organisms from different trophic levels in a southern Mediterranean coastal area. Sci. Total Environ. 368, 723–731.

12