Taking advantage of seagrass recovery potential to develop novel and effective meadow rehabilitation methods

Marine Pollution Bulletin 149 (2019) 110578

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Taking advantage of seagrass recovery potential to develop novel and effective meadow rehabilitation methods

T

Adriana Alagnaa,b, , Giovanni D'Annaa, Luigi Muscob, Tomás Vega Fernándezb, Martina Grestac, Natalia Pierozzic, Fabio Badalamentia,b,d ⁎

a

CNR-IAS, Institute for the study of Anthropic Impacts and Sustainability of the Marine Environment, Via G. da Verrazzano 17, 91014 Castellammare del Golfo, TP, Italy Stazione Zoologica Anton Dohrn, Integrative Marine Ecology Department, Villa Comunale, 80121 Naples, Italy c Saipem S.p.A., via Martiri di Cefalonia 67, 20097 San Donato Milanese, MI, Italy d School of Geosciences, Grant Institute, King's Buildings, University of Edinburgh, Edinburgh, United Kingdom b

ARTICLE INFO

ABSTRACT

Keywords: Restoration Conservation Habitat Substrate Ecological engineering Posidonia oceanica

Seagrasses are among the most threatened biomes worldwide. Until now, seagrass rehabilitation success has reached about 38% overall and more effective approaches to restoration are urgently needed. Here we report a novel method to rehabilitate Posidonia oceanica meadows based on observation of the species' natural recovery after disturbance. Posidonia oceanica rhizomes were transplanted on gabions filled with rocks of selected sizes in order to build a firm substrate with topographic complexity in the relevant scale range to propagules. Five techniques were tested, each involving a different anchoring device. The “slot” technique, which uses a wire-net pocket to retain the cuttings, was the most successful, with survival exceeding 85% after thirty months. Branching allowed final shoot survival to reach 422% of initial planting density. This study shows how an indepth knowledge of species life history processes provides a suitable foundation for developing effective restoration methods that benefit from species recovery ability.

1. Introduction In the last decades, seagrass systems have undergone global regression due to overexploitation of marine resources, coastal development, water quality degradation and climate change, with a decline rate increasing from 0.9% yr−1 before 1940 to 7% yr−1 since 1990 (Waycott et al., 2009; Orth et al., 2006). Meadow regression entails loss of associated high-value ecosystem services including nutrient cycling, protection against coastal erosion, carbon sequestration and support of diverse and productive biological communities whose cost was estimated at ~US$ 3.8 trillion year−1 (Costanza et al., 1997; Vassallo et al., 2013; Duarte et al., 2013). The growing awareness of the environmental, economic and social benefits provided by seagrass systems at a global scale has prompted policymakers to increase efforts for their conservation and to implement proper management strategies (the Ramsar Convention, the Great Barrier Reef Marine Park Act, the Bern Convention, the Barcelona Convention and SPA/BIO protocol, the EC

Habitat Directive (92/43/CEE)and the Marine Strategy Framework Directive (MSFD) (2008/56/EC) ). It is now evident that restoration and rehabilitation actions should be integrated within seagrass management strategies to allow or speed up recovery of degraded meadows from disturbance, with the final aim of re-establishing functional and self-sustaining habitats (Fonseca, 2011; Bell et al., 2008). The overall success of seagrass restoration reaches about 38% worldwide (Bayraktarov et al., 2016) and a recent review on restoration actions undertaken in Europe reveals that the success of transplantation is very low, with an average transplant survival of 15% (Cunha et al., 2012). Moreover, positive outcomes of restoration actions are assessed over periods shorter than 1 year, which is considered unsuitable for monitoring restoration success, especially for long-living seagrass species (Cunha et al., 2012). Re-establishing seagrass meadows is particularly challenging and costly (Bayraktarov et al., 2016). The success of restoration actions may be impaired by specific intolerance to transplantation, limited

⁎ Corresponding author at: CNR-IAS, Institute for the study of Anthropic Impacts and Sustainability of the Marine Environment, Via G. da Verrazzano 17, 91014 Castellammare del Golfo, TP, Italy. E-mail address: [email protected] (A. Alagna).

https://doi.org/10.1016/j.marpolbul.2019.110578 Received 7 March 2019; Received in revised form 6 September 2019; Accepted 7 September 2019 0025-326X/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Biological units used to describe transplant growth: a) an orthotropic rhizome (cutting) collected from the meadow in May 2012; b) a transplant collected in October 2014; c) orthotropic and plagiotropic rhizomes produced by branching of the initial cutting at the end of the experiment; d) leaf shoots developed by rhizome branching at the end of the experiment.

knowledge of the ecological requirements of target species and sitespecific constraints (Sanchez-Lizaso et al., 2009; McGlathery et al., 2012). Level of surf exposure, sediment mobility and long-term changes in habitat quality represent crucial features of restoration sites (Golden et al., 2010; Tanner et al., 2010). Traditional restoration techniques rely on the extraction of plugs, sods or bare-root adult plants from donor meadows, followed by transplantation at restoration sites. Besides uncertainty in outcome, transplantation interventions require high labour investment and related costs as well as the extraction of a large amount of biological material from the donor bed, resulting in the small scale (< 1 ha) of most restoration projects to date (Orth et al., 2006; Bayraktarov et al., 2016). Additional ecological and ethical concerns arise from the application of these methods at medium and large scales due to potential negative effects of the removal of large amounts of biological material on donor populations (Seddon, 2004). There is a critical need for more effective and sustainable approaches to seagrass meadow restoration, in particular for slowgrowing species such as Posidonia spp. and Thalassia spp., for which traditional techniques appear inadequate (Sanchez-Lizaso et al., 2009; Seddon, 2004). An emerging paradigm from seagrass restoration experiences across the world is the recommendation to give priority to the protection of existing beds and to the natural recovery potentials of species (Orth et al., 2006; Cunha et al., 2012; Elliott et al., 2007). Effective and sustainable approaches to seagrass rehabilitation should exploit knowledge of species' biological traits, and observations of life history processes, such as re-colonization and natural recovery after disturbance. This basic knowledge should be used to develop alternative restoration techniques that mimic

natural processes (Bull et al., 2004; Irving et al., 2010; Wear et al., 2010; Badalamenti et al., 2011). Posidonia oceanica is one of the largest and most slow-growing seagrasses of the world (Duarte, 1991). Its meadows retain such structural complexity that full recovery of impacted beds is considered impossible on a human time-scale (Boudouresque et al., 2012). Traditionally, restoration attempts of P. oceanica beds occurs by transplanting rhizomes on sandy substrate or on dead matte, i.e. dead intertwined rhizomes and roots further compacted by trapped sediment. Artificial holders, such as metal stakes and grids, concrete frames inside which is inserted a wire net, bioengineering materials and partially biodegradable structures have been employed to anchor propagules to the bottom (Molenaar and Meinesz, 1995; Gobert et al., 2005; Cinelli et al., 2014, Sanchez-Lizaso et al., 2009; Carannante, 2011Carannante 2001; Calvo et al., 2014). Evidence suggests that plagiotropic cuttings are more successfully transplanted than orthotropic ones. Moreover, dead matte and sand colonized by Cymodocea nodosa appear to be more suitable recipient substrates than bare sand. However, restoration success appears highly variable, depending on the transplantation site and local environmental conditions (e.g. hydrodynamics, sediment instability) (Carannante, 2011; Molenaar and Meinesz, 1995). All these aspects make restoration of P. oceanica meadows a challenging task. Among seagrasses, P. oceanica is one of the few that are able to thrive on both hard and soft bottoms, showing high morphological and physiological plasticity (Hemminga and Duarte, 2000). In fact, root growth pattern and architectural traits may adapt to substrate topography to maximize anchorage and substrate exploration efficiency (Balestri et al., 2015). Meadows growing on exposed rocky outcrops exhibit lower rhizome growth rate, leaf length and shoot surface than

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Fig. 2. Map of the geographic location of the study area showing the two transplantation sites.

those growing on sand and matte. This evidence highlights a possible deficiency in nutrient availability on hard bottoms, due to lower sedimentation rate and higher hydrodynamic disturbance (Di Maida et al., 2013; Giovannetti et al., 2008). Between 1981 and 1993, during installation of the TransMediterranean Pipeline in southwestern Sicily (Italy), two dredging operations caused removal of P. oceanica meadow together with its primary substrate (i.e. calcareous and calcarenitic stones), impacting 80 ha overall. P. oceanica natural recovery, through vegetative fragments detached from adjacent meadow and settled on materials used to backfill the trench, was documented from 1993 to 2003 at Capo Feto (SW Sicily) (Badalamenti et al., 2011; Di Carlo et al., 2005). The establishment of vegetative fragments acting as recruitment units represents a colonization strategy documented for several seagrass species (Almela et al., 2008). In the Capo Feto area, substrate type was the major driver of P. oceanica natural re-colonization through vegetative fragments. In fact, substrate stability and topographical complexity at the spatial scale relevant to propagules were key requirements. Habitat features were determinant in initiating recovery, as vegetative fragments recruited only on the artificial mounds made of calcareous quarry rubble (length 19.2 ± 9.5 SD cm, width 15.9 ± 2.3 SD cm, height 10.6 ± 1.9 SD cm on average) deployed to fill and stabilize the pipeline trenches (Badalamenti et al., 2011). No recovery was observed on any of the other substrates present, which included dead matte, sand and large calcarenitic boulders (Badalamenti et al., 2011). In 2010

natural recovery on calcareous rubble mounds was persistent and is still ongoing. Re-colonized patches developed and have begun to merge into a small meadow (Badalamenti et al., 2010). Here, the lessons learned from the Capo Feto case-study were incorporated in designing a transplantation method that mimics the natural re-colonization process observed. An assemblage of rocks of selected sizes was used to build a firm substrate that provided vegetative propagules with suitable structural complexity (at the scale of a few centimetres). The rationale behind the use of rocks was that a boulder assemblage grants a stable base for propagule settlement, as it remains steady when subject to currents and waves. Crevices between the rocks trap sediments, offering propagules shelter from hydrodynamic disturbance and allowing rhizomes and roots to grow and anchor themselves. This ultimately increases the chance of propagule persistence and establishment. The aim of this study was to evaluate the effectiveness of a new transplantation method for P. oceanica inspired by its natural recovery process. Specifically we aimed to: (1) assess the effectiveness of transplantation on a rocky substrate provided with topographical complexity at scales relevant to propagule anchoring, (2) identify the best anchoring techniques from those tested, in terms of efficiency and reliability. The efficiency of anchoring techniques was evaluated from persistence, survival and growth performances of transplanted cuttings. These were expected to be higher when using a technique allowing better anchorage without hampering rhizome growth.

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2. Materials and methods

aboveground biomass, rhizome biomass, root biomass and scale biomass where weighted (in g) after drying to constant weight at 60 °C (DW). Transplantation occurred on ad-hoc constructed gabions made of metallic grids (hereafter gabion mattresses) of 100x50x50 cm, filled with limestone rocks of two selected size ranges (70% “large size”: x [width] = 12.24 [mean] ± 0.60 [1 S.E.], y [height] = 8.92 ± 0.33, z [depth] = 5.63 ± 0.31; and 30% “small size”: x = 5.62 ± 0.33, y = 4.26 ± 0.23, z = 2.57 ± 0.22, in cm). The limestone rocks came from a quarry located in Zandobbio (western Lombardy, Italy). Such an arrangement of rocks reproduced the rubble mound described by Di Carlo et al. (2005), where the natural recovery of P. oceanica was reported in the area of Capo Feto. The gabion mattresses constituted the supporting structure (European Patent 2548435A1, describing the basic arrangement of the gabion mattresses) on which cuttings were secured according to different anchoring techniques. Five anchoring techniques were tested, namely cuttings inserted beneath rocks, hereafter “free cuttings” (FC); cuttings inserted beneath rocks and fastened with a cable tie to the gabion top, hereafter “cable tie” (CB); cuttings secured to a pebble with the aid of an elastic net and a cable tie and placed between rocks, hereafter “pebble” (PE); cuttings inserted inside a wire-net pocket, hereafter “slot” (SL, European Patent 2859789A1); cuttings inserted in a wire-net box filled with small pebbles, hereafter “box” (BO) (Fig. 3). Based on previous experiences, transplantation density was kept at 36 rhizomes m−2 (Carannante, 2011). Moreover, 4 batches of 18 cuttings were placed at each of the two sites close to the gabion mattresses on coarse sandy patches to compare P. oceanica rooting ability on sand to that on rocky substrate (“Substrate Control”, SCo). Each cutting was tied to a metal stake with a small rope and inserted beneath the substrate. Procedural controls were not performed as the structures used to anchor the cuttings to the rocks would not have held out to hydrodynamic drag forces if placed on the sandy substrate. Transplants were monitored over thirty months by non-destructive sampling. Technique FC was shown to be inefficient as, at the first check, most of the transplanted cuttings were not found in place (about 60%). For this reason, this technique was not considered further in the study. Similarly, most of the Substrate controls (SCo) set on sandy patches were not found in place after the first check (about 80%), and were no longer included in the study. At the end of the study period, subsamples of the transplants were extracted from the gabion mattresses for morphological and biomass analysis. The success of transplantation was evaluated by monitoring in situ persistence, survival of transplants and the number of rhizomes produced by branching of the transplanted cuttings over time (see Glossary). Transplantation was considered successful when survival was ≥50% (following Boudouresque et al., 2012) and highly successful when survival was ≥85% (following Bayraktarov et al., 2016) at the end of the experiment. Moreover, transplant growth performance was assessed by measuring the production of new rhizomes and new shoots and the increase in size and biomass of the transplants at the end of the experiment.

Glossary Transplant: An orthotropic rhizome (cutting) collected from the pristine meadow in May 2012 (Month0) and transplanted according to five different techniques. At Month0 the cuttings were all orthotropic, about 10 cm length, and bearing a single leaf shoot (Fig. 1). Rhizome: A recognizable rhizome, i.e. a lignified piece of stem, bearing one or more leaf shoots. A rhizome can correspond to an initial cutting collected from the pristine meadow or can be a new branch (Fig. 1). Shoot: A recognizable leaf bundle. As the transplant grows it tends to produce one or more new leaf shoots through branching. Each shoot develops new leaves and internodes over time, thus generating a new rhizome (Fig. 1). Experimental unit: A gabion mattress filled with rocks of selected size. Gabions differed according to the anchoring technique applied: “free cuttings” (FC), cuttings inserted beneath rocks; “cable tie” (CB), cuttings inserted beneath rocks and fastened with a cable tie to the gabion top; “pebble” (PE), cuttings secured to a pebble with the aid of an elastic net and a cable tie and placed between rocks; “slot” (SL), cuttings inserted inside a wire-net pocket; “box” (BO), cuttings inserted in a wire-net box filled with small pebbles; Gabions hosting FC, CB and PE did not have any specific anchoring structure, while gabions hosting SL were provided with three wire net pockets (slots), and those hosting BO had two boxes made of wire net filled with small calcareous pebbles (Fig. 3). Transplant persistence: Average number of transplanted cuttings, alive or dead, counted in each experimental unit. Persistence accounts for the ability of each transplantation technique to retain the transplants in place against hydrodynamic drag forces. Transplant survival: Average number of cuttings found alive in each experimental unit. Transplant survival takes into account the potential detrimental effect of the anchoring devices adopted in the different techniques on the transplanted cuttings, as differences between persistence and survival are due to transplant mortality.

2.1. Study site The study area is located on the SW coast of Sicily (Italy), near Capo Feto (Fig. 2), where one of the largest P. oceanica meadows of the Mediterranean Sea is present (Telesca et al., 2015). The meadow grows on matte and the primary substrate is a calcareous platform (Badalamenti et al., 2011). Two sites were selected at 12 m depth, close to the area where P. oceanica natural recovery on calcareous rubble occurred (Di Carlo et al., 2005). The two sites are about 400 m apart and the sea bottom is composed of coarse sand. 2.2. Transplantation method Posidonia oceanica orthotropic rhizomes, about 10–15 cm long, were collected from the meadow at two sites close to the western edge of the trench dug at Capo Feto (Badalamenti et al., 2006), at a depth of about 12 m. Only orthotropic rhizomes were collected, with an incision at the base, consistently keeping explant density lower than 2 cuttings m−2 (Boudouresque et al., 2012). We used orthotropic rhizomes instead of plagiotropic ones to minimize impact to the donor meadow. Rhizomes were trimmed at 10 cm (length measured from the rhizome trimming point to the leaf meristem) by cutting the base to ensure comparable biometry and biomass among individuals. Cuttings were stored underwater in nets placed on the sea bottom to minimize disturbance and transplanted within 2 days. A subsample (n = 10) of collected cuttings was brought to the laboratory, where rhizome and root length were measured using a rule and a calliper and total biomass,

2.3. Experimental design and data analysis 2.3.1. Transplant persistence and survival In order to check for differences in transplant persistence and survival between techniques, a three-way ANOVA was performed with the following factors: Anchoring technique (Te) fixed, with four levels (CB, PE, SL, BO); Time (Ti) fixed, with three levels (Month0, Month6 and Month30) and Site (Si) random, with two levels (S1 and S2). Month0 was set at the moment of transplantation in May 2012, while Month6 and Month30 correspond to November 2012 and October 2014 respectively. There were two independent replicates for each combination of factor levels, each replicate consisting of a gabion mattress. There were 32 gabion mattresses overall, not 48, because measurements at Month0 4

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Fig. 3. Transplantation techniques and anchoring devices tested in the study. Higher panel: sketches of gabion matresses. (1) simple gabions without supporting structures, used in the free cuttings (FC), the cable tie (CB) and the pebble (PE) techniques; 2) gabions with three wire-net pockets inserted in the upper layer used in the slot (SL) technique; 3) gabions with two wire-net boxes inserted in the upper layer, used in the box (BO) technique. Lower panel: pictures showing details of transplantation techniques. A) free cuttings inserted beneath rocks (FC); B) cuttings inserted beneath rocks and fastened with a cable tie to the gabion top (CB); C) cuttings secured to a pebble with the aid of an elastic net and a cable tie and placed between rocks (PE); D) cuttings inserted inside the wire-net pocket (SL); E) cuttings inserted inside the wire-net box filled with small pebbles (BO).

were made on 20 randomly chosen gabions which were re-sampled at the next sample time.

number of new shoots produced by each transplant were analysed through a two-way ANOVA, with factor Anchoring technique (Te) fixed with four levels (CB, PE, SL, BO) and factor Site (Si) random and orthogonal to Te, with two levels (S1 and S2). There were 10 replicates for each combination of factors, each replicate consisting of a transplant extracted from the gabions at Month30.

2.3.2. Transplant branching Transplant branching, i.e. the number of rhizomes produced by branching of the transplanted cuttings, was analysed through a threeway ANOVA. The same experimental design used to analyse transplant persistence and survival was applied, except for the level of factor Time corresponding to Month6 that was substituted by Month14 (July 2013). This was because six months after transplantation it was not possible to distinguish in situ new rhizomes produced by branching using nondestructive sampling.

2.3.4. Overall rhizome and shoot percent survival In order to estimate the overall rhizome and shoot density variations at the end of the experiment the mean number of new rhizomes and new shoots produced by transplants within each technique at Month30 (measured by destructive sampling) was multiplied by the mean percentage of survived transplants at Month30. This gave the overall rhizome and shoot percent survival at Month30.

2.3.3. Transplant growth The growth of transplants was evaluated by comparing morphological and biomass variables of the transplants at the end of the experiment with those measured on cuttings gathered at two sites before transplantation in May 2012 (Month0). The following response variables were measured: total rhizome length, as the sum of the length of all the rhizomes produced by a transplant; total root length, as the sum of the length of all the (first order) roots produced by a transplant; total biomass; aboveground biomass; rhizome biomass; root biomass; and scale biomass. Morphological and biomass data were analysed through a two-way ANOVA, with factor Transplant growth (Gro) fixed, with five levels (CB, PE, SL, BO, Month0), and factor Site (Si) random and orthogonal to Gro, with two levels (S1 and S2). There were 10 replicates for each combination of factors, each replicate consisting of a transplant or, in the case of Month0, of a cutting collected from the meadow at the beginning of the experiment. The rate at which transplants changed their growth mode from orthotropic to plagiotropic was scored as P.ratio, defined as the ratio between the number of plagiotropic rhizomes and that of orthotropic ones in each transplant. P.ratio, the number of new rhizomes, and the

2.3.5. Early recovery of structural traits In order to evaluate early recovery of P. oceanica structural traits at the end of the experiment, aboveground morphological and biomass variables of transplants were compared with those of orthotropic rhizomes collected from the nearby mature meadow (Control, Co) in the same period (October 2014). The sum of the leaf surface and biomass of all the shoots produced by each transplant (hereafter termed Total leaf surface and Total leaf biomass respectively) and the leaf surface and biomass of the largest shoot within a transplant (hereafter termed Maximum leaf surface and Maximum shoot biomass respectively) were analysed via two-way ANOVA with factor Technique (Te) fixed, with five levels (CB, PE, SL, BO, Co), and factor Si, random and orthogonal to Te, with two levels (S1 and S2). There were 10 replicates for each combination of factors, each replicate consisting of a transplant for CB, PE, SL, BO, and of an orthotropic rhizome for Co. Prior to running all the analyses, the assumption of homogeneity of variances was checked via Cochran's C tests for all the response variables. Levels of significant effects were subsequently compared via 5

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(Table 1). No significant differences were found between techniques CB, PE and BO during the same period (Table 1).

Table 1 Results of ANOVA on the effects of transplantation technique (Te) on P. oceanica transplant survival and branching over time (Ti). CB = cable tie; PE = pebble; SL = slot; BO = box; Month0 = May 2012; Month6 = November 2012; Month14 = July 2013; Month30 = October 2014. SNK = StudentNewman-Keuls test; NS = not significant. Numbers in bold indicate significant effects.

3.2. Transplant branching Fourteen months after transplantation it was possible to observe in situ active branching of transplants with production of new rhizomes in all the techniques (Fig. 4). The number of rhizomes produced by transplant branching from Month14 to Month30 ranged from 22.5 ± 3.66 m−2 rhizomes in PE to 57 ± 2.81 m−2 in SL, although differences were only significant in SL, where the number of rhizomes increased to 106.5 ± 12.45 m−2 at Month30 (Fig. 4, Table 1). At the end of the experiment transplant branching was higher in SL than in the remaining techniques, which did not differ among themselves (Table 1).

A Source of variation

Time (Ti) Technique (Te) Site (Si) TiXTe TiXSi TeXSi TiXTeXSi Residual Total

df

2 3 1 6 2 3 6 24 47

Transplant survival

Transplant branching

F

P

F

P

152.47 12.09 0.17 12.05 0.17 0.3 0.11

0.0065 0.035 0.681 0.004 0.8421 0.8282 0.9941

13.84 396.99 1.45 6.32 2.36 0.05 1.05

0.0674 0.0002 0.2399 0.0205 0.1163 0.985 0.4199

3.3. Transplant growth Overall transplant growth mode switched from orthotropic to plagiotropic with an average 0.88 ± 0.03 ratio. P.ratio was lower in SL (0.76 ± 0.08) than in the other techniques, although differences were not statistically significant (F3,72 = 8.24; P = .058). After thirty months, 97.5% of transplants branched, producing at least 1 new shoot and 77.5% produced new rhizomes. The number of rhizomes produced by branching in each transplant ranged between 2.90 ± 0.38 in BO and 2.40 ± 0.36 in SL, with no significant differences between transplantation techniques (F3,72 = 4.46; P = .126). The number of shoots produced by branching of initial cuttings ranged between 4.60 ± 0.44 in PE and 6.00 ± 0.68 in BO, which showed the highest number of new shoots (F3,72 = 12.45; P = .034), while the other techniques did not differ from each other (Fig. 5). Total rhizome length significantly increased from Month0 to Month30 (F4,90 = 7.92; P = .035), with a mean elongation rate of 36.57 ± 2.78 mm yr−1 (Fig. 5). Similarly, total root length increased from 19.83 ± 8.39 mm at Month0 to 531.7 ± 38.11 mm at Month30 (F4,90 = 148.71; P < .0001) (Fig. 5). Total transplant biomass significantly increased from 5.49 ± 0.34 gDW at Month0 to 10.29 ± 0.42 gDW at Month30, without any difference between techniques (F4,90 = 15.82; P = .010) (Fig. 6). Biomass growth was ascribed to the increment of belowground biomass that significantly raised from 3.80 ± 0.30 gDW at Month0 to 9.18 ± 0.37 gDW at Month30 (F4,90 = 22.44; P = .005), while no differences in aboveground biomass was detected across time (F4,90 = 4.21; P = .096). Both rhizomes and roots contributed to the increase in belowground biomass across time, growing respectively from 1.04 ± 0.12 gDW and 0.03 ± 0.02 gDW at Month0 to 4.40 ± 0.20 gDW and 1.17 ± 0.08 gDW at Month30 (F4,90 = 26.72; P = .004 and F4,90 = 12.91; P = .015 respectively) (Fig. 6). As a result, the relative contribution of transplant organs to total biomass changed sharply during the study. At Month0 leaf biomass represented 31% of total biomass, while rhizome and roots constituted only 19% and 1% respectively (Fig. 7). At Month30, the relative contribution of rhizomes and roots to total transplant biomass increased to 43% and 11% respectively, while that of leaf biomass decreased to 11% (Fig. 7). Accordingly, the ratio between aboveground and belowground biomass decreased significantly from 0.47 ± 0.03 at Month0 to 0.12 ± 0.01 at Month30 (F4,90 = 59.78; P < .001) (Fig. 6). Biomass trend did not vary significantly between transplant techniques.

B SNK test Variable

Effect

Interpretation

Transplant survival

TiXTe

Transplant branching

TiXTe

a) Ti (Te) CB Month0 > Month6 > Month30 PE Month0 > Month6 > Month30 SL Month0 > Month6 = Month30 BO Month0 > Month6 = Month30 b) Te (Ti) T0 NS T1 SL > CB = PE = BO T2 SL > CB = PE = BO a) Ti (Te) CB NS PE NS SL Month0 = Month14 < Month30 BO NS b) Te (Ti) T0 NS T1 NS T2 CB = PE = BO < SL

Student-Newman-Keuls (SNK) multiple test, with the nominal alphalevel set at 0.05. 3. Results 3.1. Transplant persistence and survival A significant interaction between factors Time and Anchoring Technique was detected for transplant persistence (F6,24 = 8.44; P = .010). Transplant persistence decreased significantly across time in CB (55.56 ± 7.17%, mean ± 1 SE at Month30), PE (58.33 ± 9.76%) and BO (66.67 ± 5.07%), but not in SL (93.06 ± 5.73). This anchoring technique showed higher persistence than the other techniques, which in turn did not differ from each other. The ANOVA revealed a significant interaction between Time and Anchoring technique also for transplant survival (Table 1). Survival decreased in CB and PE at Month6 lowering further at Month30, reaching values of 54.17 ± 6.16% and 59.72 ± 12.5% respectively (Fig. 4, Table 1). In SL and BO transplant survival decreased during the first six months, and then remained stable from Month6 to Month30, with values of 88.89 ± 3.93% in SL and 65.28 ± 4.74% in BO by Month30 (Fig. 4, Table 1). Transplant survival was significantly higher in SL than in the other techniques at both Month6 and Month30

3.4. Overall rhizome and shoot percent survival At the end of the experiment, overall rhizome survival ranged from 146.32% in technique PE to 213.33% in SL, with intermediate values of 151.67% and of 189.31% in CB and BO, respectively. Overall shoot survival at Month30 showed values of 274.72% in PE and 278.96% in CB respectively, and reached 391.67% in BO and 422.22% in SL. 6

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Fig. 4. Mean % and standard error of P. oceanica transplant survival and branching over the time phases (Month0, Month6 for survival and Month14 for branching, Month30) of the experiment according to the transplantation technique. In the upper panel light grey and dark grey dotted lines indicate the significant threshold for transplant success. In the lower panel the dotted line indicates the initial planting density (36 cutting m−2). CB = cable tie; PE = pebble; SL = slot; BO = box; Month0 = May 2012; Month6 = November 2012; Month14 = July 2013; Month30 = October 2014.

3.5. Early recovery of structural traits

natural recovery process. Transplanting P. oceanica vegetative fragments into rock-filled gabion mattresses using the wire-net pocket, here termed “slot”, proved to be highly successful, with transplant persistence and survival exceeding 85% at the end of the experiment. Securing cuttings with cable ties, loading them with pebbles with the aid of an elastic net, and placing them into a wire-net box filled with small rocks was also successful, with transplant persistence and survival of over 50% after thirty months. It should be noted that transplantation sites were exposed to strong south-easterly winds and, although fishing is banned in the study area, gabions were clearly subject to disturbance from fishing gears. Mechanical impacts, together with strong hydrodynamic disturbance from winter storms, were presumably responsible for the loss of a number of transplants from the gabion mattresses, particularly those used for the “free cuttings” technique. Lacking additional anchoring devices, this last technique was the most prone to transplant displacement among the five tested. When designing a novel transplantation methodology, the chances of transplant survival need to be maximized in order to reduce labour and cost efforts. The anchoring devices introduced here optimized transplantation efficiency and reduced labour effort, and thus costs. Transplant survival aided by the slot device was, on average, higher than that of previous long-term transplantations performed with P.

Thirty months after transplantation the total leaf surface of transplants ranged from 202.79 ± 24.87 cm2 in technique SL to 247.30 ± 28.13 cm2 in BO, while it was 269.49 ± 12.30 cm2 in controls. Total leaf biomass of transplants was on average 1.21 ± 0.05 gDW and 1.55 ± 0.10 gDW in controls. No differences in the total leaf surface and biomass between transplantation techniques, nor between transplants and controls, were detected (respectively F4,90 = 2.23; P = .228 and F4,90 = 6.46; P = .059). The maximum leaf surface ranged from 65.68 ± 5.28 cm2 in BO to 86.34 ± 6.39 cm2 in PE, and was significantly lower than that of controls (269.49 ± 12.30 cm2) (F4,90 = 53.4; P < .001) (Fig. 8). Similarly, the maximum shoot biomass ranged between 0.36 ± 0.02 gDW in CB and 0.44 ± 0.4 gDW in PE and was in any case significantly lower than that in controls (1.55 ± 0.10 gDW) (F4,90 = 175.41; P < .0001) (Fig. 8). No differences in leaf surface and biomass of the largest shoot were detected between transplantation techniques. 4. Discussion This study presents the first attempt to transplant rhizomes of P. oceanica on a rocky substrate. Results demonstrate the effectiveness of a new transplantation method inspired by observations of the seagrass 7

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Fig. 5. Boxplot showing the effect of growth (Gro) and of transplantation technique (Te) on transplant morphological variables (total rhizome length, total root length, P-rate, number of rhizomes and shoots produced by each transplant). Results are shown only for variables for which a significant effect of factor Gro or Te was detected. CB = cable tie; PE = pebble; SL = slot; BO = box; Month0 = May 2012; Median = horizontal line; 25th and 75th percentiles = vertical boxes; 10th and 90th percentiles = whiskers. n = 20 for each technique. Letters above boxes indicate significant differences between treatment levels.

oceanica. Previous studies using plastic, metal grids or single metal pegs to anchor vegetative fragments to dead matte or sandy substrates reported survival ranging from 20 to 73% (Meinesz et al., 1993), 0 to 84% (Molenaar and Meinesz, 1995), 70 to 66% (Piazzi et al., 1998), and 31% (Calvo et al., 2014). Similar experiences with natural materials such as woven bamboo grids and sisal fibres reported less successful results, with overall survival of 16% after three years (Vangeluwe, 2007). Survival values from this study for the slot device are comparable with the best values obtained in medium- (1600 m2) and large-scale (10.000 m2) restoration actions using concrete frames deployed on a sandy substrate. In the latter, transplant survival approached 100% in some cases, although with high variability across depths and years (Carannante, 2011). In the present study, comparison of transplant persistence and survival over time revealed that all the techniques tested were able to retain a high number of propagules, but a marginal percentage of them died. Mortality ranged from 20% among transplants fixed with cable ties, to 18% among those placed into the slots, and 12% in grid “boxes” at Month6 (data not shown). This difference suggests that the structure of the anchoring devices could be improved. Transplant branching was visible for all the techniques during in situ inspections one year after transplantation. At Month6 branching was not sufficient to compensate for the loss of propagules, except for

slots. After thirty months from the beginning of the experiment, branching allowed initial planting density to be reached and even outperformed in every experimental condition, with a three-fold increase in transplants fixed with slots. After thirty months overall rhizome and shoot percent survival exceeded 200% and 400% respectively when using the slot technique, and were higher than the best values obtained in previous studies for P. oceanica (Carannante, 2011). Such findings highlight the good health of the transplants growing on rocks and the strong re-colonization potential of vegetative fragments once they are well-established. During the experiment, transplants changed their growth mode from orthotropic to plagiotropic, as observed in previous studies (Molenaar and Meinesz, 1995; Piazzi et al., 1998; Vangeluwe, 2007). Change in growth form allows propagules to begin colonization and spread over bare areas. The rate at which seagrasses occupy space largely depends on the horizontal rhizome elongation rate and branching rate (Marbà and Duarte, 1998). In P. oceanica mature meadows the elongation rate of plagiotropic rhizomes ranges from 1 to 6 cm yr−1, with a mean branching rate of 0.51% of internodes developing branches (Marbà and Duarte, 1998; Duarte, 1991). In previous studies transplanted cuttings showed rhizome elongation rates that varied highly according to the morphological type used for transplantation, ranging from 3.44 cm yr−1 to 1.36 cm yr−1 for plagiotropic 8

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Fig. 6. Boxplot showing the effect of transplant growth (Gro) on P. oceanica transplant biomass variables (total, aboveground, belowground, rhizome, root, scale, aboveground/belowground biomass). Results are shown only for variables for which a significant effect of factor Gro was detected. CB = cable tie; PE = pebble; SL = slot; BO = box; Month0 = May 2012; Median = horizontal line; 25th and 75th percentiles = vertical boxes; 10th and 90th percentiles = whiskers. n = 20 for each technique. Letters above boxes indicate significant differences between treatment levels.

was one order of magnitude higher than those of initial orthotropic cuttings in previous studies, and was slightly higher than those of plagiotropic ones (Piazzi et al., 1998; Meinesz et al., 1992; Balestri et al., 2011). Moreover, the percentage of branching transplants and the number of new rhizomes produced by each transplant were substantially higher than those reported for (initial) orthotropic cuttings in previous transplantation experiments performed on sand and dead matte (Piazzi et al., 1998; Vangeluwe, 2007). These results suggest that transplant growth over the rocky mattresses was not limited by nutrient availability, as may be expected for P. oceanica growing on rocky compared to sandy substrate (Di Maida et al., 2013; Romero et al., 2006). The nutrient content of sediment deposited among rocks inside the gabions thus appeared sufficient to meet plant demand. Previous studies reported variability in root architecture of P. oceanica seedlings growing on different substrates, with a superficial, dense “herringbone” branching pattern on rock and a vertical, loose and more dichotomous growth pattern on sand (Balestri et al., 2015). In this study, roots of vegetative fragments transplanted on rocks were densely packed and developed according to the substrate topography, around and downward between rocks. These observations confirm the high morphological plasticity of the P. oceanica root growth pattern, which allows the plant to maximize anchoring efficiency. The effective development and growth of transplants was confirmed

Fig. 7. Percent contribution of root, rhizome, scale and leaf biomass to the total transplant biomass measured at the beginning of the experiment, in May 2012 (Month0) and on transplants collected in October 2014 (Month30).

cuttings and from 0.88 cm yr−1 to 0.16 cm yr−1 for (initial) orthotropic cuttings (Piazzi et al., 1998; Meinesz et al., 1992; Balestri et al., 2011). In this study the mean horizontal rhizome elongation rate of transplants 9

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Fig. 8. Boxplot showing the effect of transplantation technique (Te) on early recovery of P. oceanica structural traits: total leaf surface, maximum leaf surface, total leaf biomass and maximum shoot biomass. Results are shown only for variables for which a significant effect of factor Te was detected. CB = cable tie; PE = pebble; SL = slot; BO = box; Median = horizontal line; 25th and 75th percentiles = vertical boxes; 10th and 90th percentiles = whiskers. n = 20 for each technique. Letters above boxes indicate significant differences between treatment levels.

by the global increase in biomass, which nearly doubled after thirty months. P. oceanica biomass allocation varied during the study, with a decrease in aboveground percent biomass and a strong increase in rhizome and root percent biomass over time. As observed in other transplantation studies (Gobert et al., 2005) and in vegetative fragments spontaneously colonizing rocky bottoms (Di Carlo et al., 2007), vegetative propagules invest large amounts of energy in developing belowground organs. Roots play two functions of primary importance to propagules, namely to anchor the plant to the substrate, and to contribute to nutrient uptake from interstitial pore waters. Conversely, rhizome development helps to anchor the plant to the bottom and allows colonization of bare new areas. On the other hand, recovery of the aboveground structural traits of the plant appears to be a very slow process. After thirty months the overall leaf standing crop of one transplant (composed of several shoots) did not differ from that of a single orthotropic shoot of the mature meadow, showing that the entire transplant recovered the initial leaf surface and aboveground biomass, but the leaf surface and biomass of a single shoot were still lower than in natural meadows. The transplantation techniques tested in this study proved to be either successful or highly successful, with the wire-net pockets (slots) emerging as the best anchoring technique thus seen. Other experiments at small and medium spatial scales are needed to assess survival and growth variability across sites and to further develop this technique. Future upgrading could include an increase in gabion mattress planting surface, and a reduction in the height of the mattress (e.g. to 25–35 cm) in order to limit hydrodynamic disturbance and fishing gear exposure. Although statistically not significant, the lower P.ratio and number of shoots produced by each transplant where slots were used suggest that slots may constrain transplant development. The design of slots should therefore be improved by taking care to leave transplant meristems outside the grid to allow the transplant to grow among nearby rocks. Moreover, the transplantation techniques made use of zinc-coated wirenets to make the slots and boxes, and of plastic ties to secure the rhizomes to the slots, with the potential risk of metal and plastic contamination of the environment. Efforts should be made to develop and test anchoring structures using more biocompatible materials. Posidonia oceanica meadows thrive on hard and soft bottoms (Hemminga and Duarte, 2000). However when it comes to spontaneous colonization or restoration of bare substrates, it should be acknowledged that P. oceanica early life history phases display specific habitat requirements. In particular, substrate features needed for propagule

establishment are more restrictive than those required for adult rhizome in well-established patches, which are able to progress on sand corridors (Gobert et al., 2016). Vegetative and sexual propagules, which lack a well-developed anchoring system, are subject to wave and current drag forces that can pull plantlets away. Recent studies show that settlement and recruitment of P. oceanica vegetative fragments, as well as seedlings, take place preferentially on firm substrates displaying a level of topographical complexity that matches the size of the propagules (tens of centimetres for vegetative fragments, a few centimetres for seedlings) (Badalamenti et al., 2011; Alagna et al., 2013; Alagna et al., 2015; Piazzi et al., 1999). On the contrary, propagule establishment does not occur on unconsolidated substrates characterized by sediment instability (Badalamenti et al., 2011; Alagna et al., 2013; Alagna et al., 2015; Piazzi et al., 1999). Moreover, recent observations have documented early life history traits in P. oceanica seedlings which represent features that are adaptive to rocky substrates, favouring their establishment on hard bottoms (Badalamenti et al., 2015; Borovec and Vohník, 2018). These findings lead us to re-evaluate P. oceanica substrate requirements, at least during the early colonization of bare substrates. In some cases, restoring the characteristics of the habitat where the species spontaneously settles and grows is preferable to increasingly sophisticated techniques of seagrass replanting, especially when largescale interventions are needed. This approach is helpful to develop innovative restoration methods that take advantage of the natural recovery potential of the species, like the one tested here. Our approach entails 1) restoring the original features of the habitat where the seagrass originally established itself by mimicking the primary rocky substrate or 2) introducing a small layer of rocks where the meadow primary substrate was made of sand to facilitate seagrass colonization and recovery, thus modifying the habitat. In areas experiencing sediment deposition, low profile rock layers could be infilled by sediment in a few years, thus reproducing a soft bottom environment. Further studies are needed to understand how ecosystem functions and services provided by P. oceanica meadow established on hard bottoms differ from those of meadow established on soft ones and in turn, from meadows growing on artificially-restored hard substrates. The applicability of the method proposed here is related to those cases in which physical impact had caused removal of the meadow together with its original substrate, entailing the loss of above- and belowground plant organs. The method can potentially be applied to other seagrass species able to colonize hard substrates, such as species 10

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of the genera Phyllospadix, Thalassodendron, and Amphibolis (Cooper and McRoy, 1988; Bandeira, 2002; Koch et al., 2006). The same method can also be used when stabilization of incoherent substrate is required to allow seagrasses to initiate recovery. Coastal development involves marina construction and expansion, deployment of cables and pipelines for communication and gas, oil electricity, and water transportation. These interventions may cause burial or removal of seagrass beds. In some areas, decades of urban and industrial wastewater discharge has led to the replacement of the original rocky bottoms with fine sand and muddy substrates, with consequent loss of high-value biological communities. The introduction of boulders and engineered rocky artificial reefs has already been implemented in coastal areas for sea ranching and for habitat restoration and enhancement, giving hard-bottom communities the chance to recover, with positive effects on overall biodiversity (Firth et al., 2014; Liversage and Chapman, 2018; Støttrup et al., 2017; Miller and Barimo, 2001). Moreover, protection, restoration and creation of vegetated coastal habitats, such as seagrasses, are now being included in ecoengineering approaches to mitigate climate change, since they provide protection from coastal erosion and represent key ocean carbon sinks (Duarte et al., 2013, Bilkovic and Mitchell, 2013). In this scenario, revegetated rocky mattresses can be used as “habitat enhancement units” for seagrass rehabilitation in wider rocky and sandy bottoms requiring general environmental restoration.

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Authors' contributions Conceived and designed the experiments: AA GD LM TVF MG FB. Performed the experiments: AA GD LM TVF NP FB. Analysed the data: AA FB. Led the writing: AA. Contributed critically to the drafts: GD LM TVF FB. All authors gave final approval for publication. Acknowledgement This work was funded by Saipem S.p.A. Support to AA was provided by a research fellowship from IAS – CNR of Castellammare del Golfo (Tp), project “Sperimentazione su rizomi di Posidonia oceanica, contratto N. 634522/2011”. We are grateful to Carlo Magliola, along with all the Saipem project team, for their contribution to the development of the project, and to Giuseppe Di Stefano, Marilena Coppola, Barbara Mikac, Davide Agnetta, Nicola Galasso and Rosaria Prestia for co-operation during field work and laboratory analyses. Permissions and authorizations The authorization by the competent authority to carry out the trial was acquired with the delivery report No 16530 issued by the Regional Council of the Territory and Environment of the Sicilian Region (Italy) on March 15, 2012. References Alagna, A., Vega Fernández, T., Terlizzi, A., Badalamenti, F., 2013. Influence of microhabitat on seedling survival and growth of the Mediterranean seagrass Posidonia oceanica (L.) Delile. Estuar. Coast. Shelf Sci. 119, 119–125. Alagna, A., Vega Fernández, T., D’Anna, D., Magliola, C., Mazzola, S., Badalamenti, F., 2015. Assessing Posidonia oceanica seedling substrate preference: an experimental determination of seedling anchorage success in rocky vs. sandy substrates. PLoS One 10, e0125321. Almela, E.D., Marbà, N., Álvarez, E., Santiago, R., Martínez, R., Duarte, C.M., 2008. Patch dynamics of the Mediterranean seagrass Posidonia oceanica: implications for recolonisation process. Aquat. Bot. 89 (4), 397–403. Badalamenti, F., Di Carlo, G., D’Anna, G., Gristina, M., Toccaceli, M., 2006. Effects of dredging activities on population dynamics of Posidonia oceanica (L.) Delile in the Mediterranean Sea: the case study of capo Feto (SW Sicily, Italy). Hydrobiologia 555, 253–261. Badalamenti, F., Alagna, A., D’Anna, G., Di Stefano, G., Vega Fernández, T., 2010. Ipotesi teoriche per la sperimentazione di metodologie di facilitazione del reclutamento di germogli di Posidonia oceanica finalizzate al ripristino delle praterie. Rapporto Tecnico, contratto n. 506019 del 19/04/2010. Consulenza scientifica nel campo del

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