Establishment of intertidal seaweed beds of Sargassum thunbergii through habitat creation and germling seeding

Ecological Engineering 44 (2012) 10–17

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Establishment of intertidal seaweed beds of Sargassum thunbergii through habitat creation and germling seeding Yong Qiang Yu a , Quan Sheng Zhang a,∗ , Yong Zheng Tang a , Shu Bao Zhang a , Zhi Cheng Lu a , Shao Hua Chu a , Xue Xi Tang b a b

Ocean School, Yantai University, Yantai 264005, PR China Ecology Laboratory, Ocean University of China, Qingdao 266003, PR China

a r t i c l e

i n f o

Article history: Received 30 December 2011 Received in revised form 7 March 2012 Accepted 26 March 2012 Available online 2 May 2012 Keywords: Sargassum thunbergii Restoration Intertidal seaweed bed Habitat creation Artificial seeding

a b s t r a c t Many naturally occurring macroalgal beds on the coastal areas of China have been severely degraded by various anthropogenic perturbations. Few attempts have been made to develop restoration techniques for seaweed beds in intertidal ecosystems, owing to the complex and dynamic variations in physical conditions of the habitat. We developed a new Sargassum thunbergii restoration method involving creating intertidal habitat and seeding with artificially collected germlings. In June 2010, artificial rectangular pools constructed of a 4:5:1 rate of high-strength cement, sand and water on a rocky intertidal platform were seeded with S. thunbergii germlings released from fertile thalli during low tide. Artificial pools were covered with a double-layer shading net until the next tidal cycle to prevent germlings from being dislodged by water motion, resulting in a majority of young germlings successfully attaching to the pool bottom by rapid development of rhizoids. Two months after seeding, juvenile sporophytes attained a length of 15–20 mm. After one year following seeding, S. thunbergii in the restored bed reached a density of 118.5 ± 13.2 (mean ± SE) thalli m−2 , covered 32.7 ± 0.1% of the artificial substrate, and grew to an average length of 34.2 ± 1.6 cm with 7.3 ± 0.6 laterals per thallus. The proportion of fertile laterals of restored population was 73.6 ± 3.0%, indicating that these fertile thalli may serve as a source of new recruits to enhance the recovery of the algal population. Furthermore, restored S. thunbergii beds facilitated the presence of seven other species of macroalgae with species richness (R), diversity (H ) and evenness (J ) reaching 0.65 ± 0.04, 1.06 ± 0.09 and 0.67 ± 0.05, respectively. Therefore, the construction of artificial pools coupled with seeding germlings in natural habitat may be an effective approach for the restoration of S. thunbergii, and potentially other seaweeds in rocky intertidal habitats. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Macroalgal beds on rocky bottoms have been recognized to play an important role in ecological and biological function. They provide crucial habitats and spawning grounds for a wide variety of marine organisms and are an essential source of carbon for complex food webs in the rocky coastal ecosystem (Largo and Ohno, 1993; Terawaki et al., 2001; Oyamada et al., 2008). However, the rocky coastal ecosystems have been severely affected by various anthropogenically induced perturbations, such as land reclamation, nutrient pollution, over exploitation and climate change (Terawaki et al., 2003; Jones et al., 2010; Gallego-Fernández et al., 2011). Consequently, many naturally occurring macroalgal beds have been seriously degraded in a number of coastal areas of the

∗ Corresponding author. Tel.: +86 0535 6706011; fax: +86 0535 6706299. E-mail address: [email protected] (Q.S. Zhang). 0925-8574/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ecoleng.2012.03.016

world (Carney et al., 2005; Zhang and Sun, 2007; Yao et al., 2010). Given the essential function of macroalgal beds on rocky shores, much attention has been focused on effective methods to restore seaweed habitat on damaged rocky coastal ecosystems (Terawaki et al., 2003; Falace et al., 2006). Numerous attempts of construction of artificial macroalgal beds have been conducted in the last decade and several techniques have been developed (Stekoll and Deysher, 1996; HernándezCarmona et al., 2000; Terawaki et al., 2003; Correa et al., 2006; Yoshida et al., 2006; Yamamoto et al., 2010). Common methods for restoring macroalgal beds include supplying microscopic zoospores, transplanting juveniles and adults, deploying artificial reefs, and removing herbivorous fishes and animals (Largo and Ohno, 1993; Choi et al., 2002; Kang et al., 2008). These restoration studies demonstrated that seaweed populations could be reestablished successfully in the benthic or subtidal zones of many regions. However, restoration efforts on rocky intertidal shores have been largely unexplored (Carney et al., 2005; Susini et al., 2007; Whitaker

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Fig. 1. Location of the restoration and five reference sites around the Xiaoheishan Island, China.

et al., 2010). The marine intertidal zone is considered among the most stressful of all environments, because daily exposure at low tide results in daily fluctuation in a variety of physical factors such as temperature, humidity, salinity, wave exposure and light (Bell, 1993; Davison and Pearson, 1996; Wright et al., 2004; Chapman and Reed, 2006). Consequently, in comparison to subtidal habitats, restoration of the intertidal macroalgal beds may be more challenging in the stressful environment. Species of the genus Sargassum (Fucales) are a significant component of seaweed flora in the tropical and temperate coastal waters around the world (Phillips, 1995). They have a biogenic, habitat-forming role in shaping rocky intertidal community structure, making them especially good candidates for restoration. Sargassum thunbergii (Mert.) O. Kuntze, a brown canopy forming species of algae, distributes widely along the coasts of China, Japan and Korea (Umezaki, 1974; Koh et al., 1993; Zhang et al., 2009). This species occupies mainly rocky shores where it forms dense monospecific stands spanning low intertidal, including tide pools, to shallow subtidal regions (Zhao et al., 2008). In recent years, S. thunbergii is widely used in alginate production, extraction of bioactive products and biosorption of heavy metal ions (Padilha et al., 2005; Zhao et al., 2007; Chu et al., 2011). In addition, it is particularly used as a preferred food source for holothurian and abalone aquaculture. In recent years, natural populations have been largely harvested as desirable feed for the rapidly developing aquaculture of Stichopus japonicus in this region, which has resulted in the depletion of intertidal macroalgal beds (Zhao et al., 2007; Zhang et al., 2011). The decline of S. thunbergii from these extensive coastal areas highlights the urgent need to design effective strategies for restoration. To our knowledge, few attempts have been made to develop restoration techniques for intertidal S. thunbergii populations.

In this study, artificial cement pools were constructed on a rocky intertidal platform, and then inoculated with S. thunbergii germlings shortly released from fertile thalli at low tide. To evaluate the efficacy of this method, we measured abundance, density, and morphological characteristics of S. thunbergii, as well as macroalgal species diversity, in artificial seaweed beds after one year following seeding. 2. Materials and methods 2.1. Restoration area Our restoration was conducted on the island of Xiaoheishan (Changdao Archipelago), approximately 20 km off the northern side of Shandong Peninsula, China (Fig. 1). This island is 1.9 km long in a north-south direction and 1.2 km wide in an east-west direction, with an area of approximately 1.36 km2 . It is characterized by a warm temperate continental monsoon climate with distinctive seasons and rainy summers. The mean annual air temperature is 12 ◦ C and annual precipitation is 565 mm. The tides are regular semidiurnal, with spring tide amplitudes to 3 m. Annual ranges for sea surface temperature and salinity in this area were 1.8–23.3 ◦ C and 29–31 psu, respectively. S. thunbergii was the dominant species historically on the intertidal coast of this island but overexploitation has depleted the area of intertidal macroalgal beds. The target restoration site was established on the semi-exposed, west-facing coast of the island. An unvegetated intertidal rock platform with an area of ca. 120 m2 was selected for S. thunbergii bed restoration. This platform was composed of sandstone, and was backed by a 15–20 m high cliff. At low tide, the entire rock platform was completely exposed to air for approximately 8.5 h twice a day. S. thunbergii was absent within 100 m from the restoration area, and

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other macroalgae, such as Ulva spp. and Gloiopeltis spp. were rare. Around the Xiaoheishan Island, five intertidal sites were selected (Fig. 1) where some patches of S. thunbergii were present in intertidal rock pools. The naturally growing thalli in the patches were used as reference plants. Previous investigations have shown that the reproduction of S. thunbergii in this area occurs during spring and early summer, from June through mid-August. During the sexual reproductive period, numerous receptacles are produced along the laterals of thalli. Eggs derive from receptacles and remain attached to the surface of the receptacles, where they are fertilized. After fertilization, young germlings are formed, shed naturally and are dispersed (Zhang et al., 2010; Chu et al., 2011). Parental thalli used as source of germlings were harvested from farmed population at Doungtou county, Zhejiang province (27◦ 54 N, 121◦ 09 E; Fig. 1), where is the principal cultivation ground of S. thunbergii in China.

their rhizoids on the bottom of vessel in the next 24 h following detachment from fertile thalli (Zhang et al., 2011). Therefore, artificial pools were immediately covered with a double-layer shading net made of polyethylene (mesh size 0.3 cm), and sandbags were then placed over the net to immobilize it firmly in place for 24 h after seeding, in order to reduce the loss of germlings due to wave motion. Supplement of NaNO3 and KH2 PO4 was added to facilitate growth once every three days at a concentration of 4 × 10−4 and 2 × 10−5 mol L−1 , respectively. In addition, artificial pools were sprayed one to two times a week with a water jet to remove the muddy sediments during tidal emersion. The entire cultivation periods lasted for 2 months until the seedlings attained a length of approximately 20 mm in artificial pools by late August.

2.2. Habitat creation

On 15–18 July 2011, one year following artificial seeding of pools, field surveys were conducted to measure abundance, density, size class, reproductive state and diversity of other macroalgae at the restored and reference sites. To facilitate artificial seaweed bed recovery, no destructive samples were taken at the restored site during this survey. In the present investigation, photographs were taken of each pool at low tide using a digital camera (Nikon D7000 at 16.2 megapixels). The abundance of S. thunbergii and other macroalgae was expressed as percentage cover quantified by tracing the total area within each photograph occupied by each species using image analysis software (Photoshop CS, Adobe System Inc.). Ten of the 50 artificial pools were selected for morphological measurements of S. thunbergii. Thallus densities were estimated by counting the number of individuals inside the selected pools. Six thalli were selected equidistantly along a diagonal direction from each pool, and thallus length, the number and length of primary laterals per thallus, and the presence of receptacles on each primary lateral were determined. Primary lateral length was measured from holdfast to apex of the longest of branch. Thallus length equals the length of the longest primary lateral. In addition, a total of 10 similarly sized natural rock pools were chosen at five reference sites (area between 0.5 and 1.25 m2 ; depth between 15 and 25 cm). In each pool, a 0.25 m2 quadrat (50 cm × 50 cm) was placed and photographed, and percentage cover of macroalgal species was estimated as described above. Within each quadrat, thallus density was determined, and the number and length of primary laterals per thallus and the length of 6 thalli of S. thunbergii selected equidistantly along a diagonal direction were also measured. The presence of receptacles was also recorded for each primary lateral in situ. Size frequency distributions of primary laterals were determined, based on pooled data for all selected thalli, distinguishing between fertile and sterile laterals. Twelve length hierarchies used in this study were in 5 cm intervals for the assessments of size structure in restored and reference populations of S. thunbergii. At the restored and reference sites, macroalgae present in each pool or quadrat were identified to species level in the field. Biodiversity indices, such as Margalef’s species richness index (R) and ShannonWeaver’s diversity index (H ), along with Pielou’s evenness index (J ) were calculated based on the proportional abundance of each species, as described by Díez et al. (2003).

Artificial cement pools were established in the target restoration site on 20 May 2010, about one month before seeding with S. thunbergii germlings in order to leach out the potential toxicity of cement. Based on the actual topography, a total of 50 rectangular pools were constructed of a 4:5:1 ratio of high-strength cement, sand and water, on the surface of the rock platform with the long axis perpendicular to the wave crests during daytime low tide. The length, width and height of each pool were 2.0, 1.0 and 0.1 m, respectively. As the tide was receding, all artificial pools contained seawater as well as intertidal rock pools. Seawater temperature in the pools rose as high as 38–40 ◦ C caused by direct solar radiation during low tide at noon. This situation generally lasted for less than 3 h due to tidal immersion, and seawater temperature was then returned to ambient level. 2.3. Collection of germlings On 19 June, a total of 50 kg parental S. thunbergii plants with well-developed receptacles were collected from farmed population at Doungtou county. Following cleaning of surface epiphytes and sediments, plants were placed in plastic foam boxes with ice and transported by airplane to the experimental hatchery site within 10 h, which lies adjacent to the restoration location. They were submerged into an aerated 8 m3 concrete tank (4 m × 2 m × 1 m) filled with sand-filtered seawater for the release of gametes. The tank was kept at 22 ◦ C, 10 ␮mol photons m−2 s−1 and a 12L:12D (light:dark cycle) photoperiod, which has been proven to be favorable for massive discharge of S. thunbergii gametes (Chu et al., 2011). After removal of thalli from the tank, germlings settled on the bottom of tank were collected by siphonage at 24-h intervals, and the duration of release was maintained for 3 days. To remove fouling, dross and epiphytes, collected germlings were repeatedly washed with filtered seawater using a 75-␮m nylon sieve, followed by filtration using a 180-␮m nylon sieve. The number of germlings was determined by dilution method and a total of ten million germlings were obtained. 2.4. Seeding of germlings The collected germling suspension was then diluted with seawater to produce an inoculum for the field. This inoculum was divided equally and subsequently sprinkled over the seawater surface of each artificial pool as soon as the tide had receded. Germlings were seeded onto the rock bottom of pools at a density of 5–10 individuals cm−2 during the experimental period. Our previous work indicated that almost all germlings attached firmly with

2.5. Data acquisition

2.6. Statistical analysis All data are reported as mean values ± standard error (SE). Statistical analyses were conducted using SPSS version 13.0 for windows. The collected data for reference sites were pooled for each variable in the aim to compare with artificial macroalgal beds. An independent t-test was used to test for differences in the

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Fig. 2. Observation on early growth of juvenile sporophyte of Sargassum thunbergii in restored bed. (A) Young sporophytes with one erect leaflet at 7–10 days after seeding; (B) branch differentiated from young sporophytes by 17–18 days after seeding; (C) young sporophytes with four to five branches after 4 weeks; (D) tiller buds formed on young sporophytes by 38–40 days after seeding; (E) young sporophytes with tiller buds 8–10 mm in length at 48 days after seeding; (F) young sporophytes with a length of 20 mm after 2 months. The inset in each image shows a partial enlarged view of young sporophytes at different growing stages. (G) Healthy stands of S. thunbergii were established after one year of restoration.

thallus densities, morphological variables (thallus length, number and length of primary laterals, and percentage of fertile laterals), biodiversity indices and species abundance between the restored and reference sites. Size frequency distribution of primary laterals

between restored and reference populations were compared with the Kolmogorov–Smirnov test. Prior to t-test analysis, the data were tested for normality and homogeneity of variance and were transformed when necessary (Sokal and Rohlf, 1995). A non-parametric

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Fig. 3. Sargassum thunbergii. Thallus density (A), thallus length (hatched bars) and mean length of primary laterals per thallus (black bars) (B), mean number of primary laterals per thallus (C) and mean proportion of fertile primary laterals per thallus (D) in restored and reference populations during July 2011. Vertical error bars indicate the standard error of the mean (n = 10). *Significant (P < 0.05) difference between restored and reference populations. NS = non-significant.

test (Mann–Whitney’s U-test) was performed on those data that did not meet the assumptions required for parametric statistics. Statistical significance was set at the 0.05 level for all tests. 3. Results 3.1. Early growth of young sporophytes S. thunbergii germlings developed rhizoids rapidly for attaching to the substratum and became macroscopic in the artificial pools at 24 h post-seeding. Young sporophytes appeared in the form of one erect leaflet during the first week (Fig. 2A). Branchlets were differentiated and young sporophytes reached approximately 2 mm in length at 17–18 days after seeding (Fig. 2B). In the following two weeks, the number and length of branchlet increased (Fig. 2C and D), and young sporophytes had 6–8 branchlets, with a length of 8 mm, and also initiated to form tiller buds by 38–40 days after seeding (Fig. 2D). One week later, a majority of tiller buds grew up to a length of 10 mm (Fig. 2E). After two months, young sporophytes present in artificial pools attained a length of approximately 20 mm (Fig. 2F). In July 2011, one year after seeding, healthy stands of fully developed S. thunbergii were established at the restoration site (Fig. 2G).

thallus length was found between the restored and reference populations (34.2 ± 1.6 cm vs. 40.7 ± 3.5 cm) (t-test, t = −1.820, P = 0.086; Fig. 3B). In contrast, the mean length of primary laterals per thallus was significantly shorter in the restored population (12.0 ± 0.9 cm) than in the reference population (16.6 ± 1.4 cm) (t-test, t = −2.992, P = 0.008; Fig. 3B). The mean number of laterals per thallus from the restored population was significantly lower (7.3 ± 0.6) than that recorded in the reference population (12.8 ± 1.2; t-test, t = −4.431, P < 0.001; Fig. 3C). Furthermore, there was no significant difference in the proportion of fertile laterals per thallus between the restored (73.62 ± 2.97%) and reference populations (69.67 ± 6.44%; t-test, t = 0.610, P = 0.550; Fig. 3D). The size frequency distributions of primary laterals, pooled for all thalli, were similar between the restored and reference populations (Kolmogorov–Smirnov test, Z = 0.816, P = 0.518), with many small laterals and few large ones (Fig. 4). The primary laterals in the 5–10 cm size class predominated in both populations, with a relative frequency of over 20%. In the reference population, in total 10% of the laterals grew to 40–60 cm in July 2011, whereas all laterals from the restored population were less than 40 cm long. Furthermore, fertile laterals were represented in all size classes except in the smallest class of 0–5 cm, and all laterals >15 cm became reproductive in the restored and reference populations during the survey (Fig. 4, white bars).

3.2. Thallus density and morphology 3.3. Diversity and abundance of macroalgae After one year of restoration, the density of S. thunbergii was 118.5 ± 13.2 thalli m−2 at the restored site, which was significantly higher than 63.8 ± 15.6 thalli m−2 observed at the reference sites (t-test, t = 2.449, P = 0.028; Fig. 3A). No significant difference in the

A total of 23 macroalgal species were identified during the study, including 3 Chlorophyta, 5 Phaeophyta and 15 Rhodophyta (Table 1). The number of species present in the restored and

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Table 1 Abundance of macroalgal species (mean ± SE) and values of total abundance (A), Margalef’s species richness index (R), Shannon–Weaver’s diversity index (H ) and Pielou’s evenness index (J ), measured in restored and reference beds during July 2011. Species Rhodophyta Gelidium amansii Heterosiphonia japonica Ahnfeltiopsis flabelliformis Gloiopeltis furcata Pterocladiella capillacea Chondrus ocellatus Gracilaria verrucosa Corallina officinalis Dumontia simplex Grateloupia filicina Ceramium kondoi Grateloupia turuturu Chondrophycus intermedia Gracilaria lemaneiformis Caulacanthus okamurai

Restored bed 0.00 0.43(0.02) 0.02(0.02) 0.26(0.01) 0.00 0.00 0.00 0.69(0.03) 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Reference bed 1.70(0.05) 2.45(0.08) 1.50(0.07) 0.14(0.02) 1.07(0.03) 1.08(0.01) 0.90(0.03) 1.45(0.06) 0.96(0.04) 1.06(0.04) 1.00(0.01) 1.13(0.04) 1.00(0.02) 1.27(0.02) 0.98(0.03)

reference beds was 8 and 23, respectively. All the macroalgae recorded in the restored bed were also found in the reference bed. Except for the dominant species, S. thunbergii, the green alga Ulva pertusa was the most abundant seaweed at study sites, especially in the restored bed. After one year of restoration, Margalef’s species richness index (R), Shannon-Weaver’s diversity index (H ) and Pielou’s evenness index (J ) for macroalgal species in the restored bed was significantly lower than that of reference bed (Mann–Whitney’s U-test, U = 0, P < 0.001 for R; t-test, t = −10.632, P < 0.001 for H ; U = 12, P = 0.02 for J ; Table 1). The total abundance of macroalgae was not significantly different between the restored and natural beds at the time of investigation in July 2011 (t-test, t = 1.436, P = 0.175). A greater percentage cover value was recorded in the restored bed. This dense coverage was mainly dominated by S. thunbergii, U. pertusa,

Fig. 4. Sargassum thunbergii. Size frequency distribution of primary laterals in restored (A) and reference (B) populations during July 2011. Hatched and white bars respectively indicate sterile and fertile laterals. Total number (n) of laterals was indicated, based on pooled data for 60 thalli.

Species

Restored bed

Reference bed

Chlorophyta Enteromorpha intestinalis Ulva pertusa Codium fragile

2.07(0.08) 17.51(0.03) 0.00

2.34(0.02) 10.39(0.07) 0.55(0.01)

Phaeophyta Ectocarpus confervoides Sargassum thunbergii Scytosiphon lomentaria Colpomenia sinuosa Hizikia fusiformis

5.12(0.07) 32.66(0.09) 0.00 0.00 0.00

1.13(0.06) 12.03(0.11) 0.81(0.01) 1.18(0.03) 2.07(0.02)

A R H J

58.75(0.08) 0.65(0.04) 1.06(0.09) 0.67(0.05)

48.21(0.12) 2.81(0.16) 2.37(0.08) 0.90(0.01)

and Heterosiphonia japonica. In comparison to the restored bed, S. thunbergii and U. pertusa showed lower percentage covers in the reference bed, and the difference was significant for S. thunbergii (ttest, t = 3.091, P = 0.009), not for U. pertusa (t = 1.653, P = 0.119). The majority of red algae were observed in the reference bed; however, this category contributed to a low level of coverage at the restored site (Table 1). 4. Discussion Reestablishing or enhancing depleted biogenic species are common strategies to restore abundances and ecosystem functions in damaged rocky coastal habitats (Correa et al., 2006; Rodríguez-Salinas et al., 2010; Whitaker et al., 2010). Although numerous examples of degraded rocky intertidal habitats are found in many parts of the world, restoration efforts in these ecosystems are rare, particularly for species restoration purposes. As discussed by Chu et al. (2011), a major challenge in constructing artificial seaweed beds in the intertidal zone of rocky habitats is attributable to large variations of physical conditions. Stekoll and Deysher (1996) observed that Fucus gardneri germlings, which were inoculated onto mats and rock surfaces for restoration of damaged Fucus populations following the Exxon Valdez oil spill, were especially susceptible to mortality from high temperature and desiccation stress. Moreover, the rocky intertidal zone is often accompanied by increased frequency and intensity of extreme weather events, such as windstorms, hurricanes, typhoons and heavy rainfall, which could lead to potential threats to success of seaweed bed restoration in this area (Tomanek and Helmuth, 2002; Ruiz-Jaen and Mitchell Aide, 2005). For example, Vásquez and Tala (1995) used nontoxic epoxy cement to glue Lessonia nigrescens plants to rocky intertidal substrata; however, 100% of the plants disappeared due to heavy surge resulting from winter storms in only two weeks. The appropriate restoration technique will depend on the seaweed species and conditions at the target sites (e.g., water velocities, substrate types, and expected grazers) (Choi et al., 2003; Park and Lee, 2010). For reconstruction of artificial seaweed beds, species of Sargassum are the main components in consideration for restoration due to their large biomass, wide distribution and relative tolerance to stressful habitat conditions (Choi et al., 2008; Olabarria et al., 2009). Around the Xiaoheishan Island coast, S. thunbergii is generally restricted to natural rock pools in the upper rocky intertidal zone, often being the dominant alga during summer. Rock pools afford shelter from desiccation stress and wave impact during low tide, and are favorable habitats for the growth of seaweed

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and other coastal vegetation (Baer and Stengel, 2010). Our previous works have shown that S. thunbergii germlings exhibit strong tolerance to thermal and osmotic stresses; however, desiccation is the predominant factor affecting survival and growth of germlings (Chu et al., 2011). Based on these results, construction of artificial pools in natural habitat to minimize desiccation may be an effective approach for S. thunbergii restoration using germlings. Nevertheless, this measure may cause negative effects, such as fluctuating salinity in the pools resulted from heavy rain in summer in this area, which is likely a damage to seeded germlings. Hence, another study was conducted to investigate the influence of salinity fluctuation on the survival and growth of S. thunbergii germlings. We found that germlings of S. thunbergii displayed a high tolerance to salinity stress, reflecting the feasibility of restoration of intertidal seaweed beds by seeding germlings in artificial pools (Chu et al., 2012). In the present study, healthy stands of fully developed S. thunbergii were established at the target site after one year following seeding. S. thunbergii in the restored bed reached a density of 118.5 thalli m−2 , covered 32.7% of the artificial substrate. Furthermore, seven other species of macroalgae were also found in the restoration area and biodiversity of algae was increased (Table 1). Additionally, 73.6% of primary laterals became fertile in July during one year after seeding. In this way we can achieve an expected idea of the construction of seaweed beds in the intertidal zone, where artificial S. thunbergii beds were established locally in order to fan out from “point” to “area” and enlarge population size, via vegetative regeneration of thalli to maintain population abundance and sexual reproduction to expand spatially. Another key point of restoration techniques for intertidal seaweed beds by the dispersal of microscopic lifestages is to reduce the dislodgement caused by wave action (Stekoll and Deysher, 1996; Carney et al., 2005). In this study, artificial pools built on the rock platform were seeded with S. thunbergii germlings, and subsequently the rhizoids developed rapidly and attached onto the pool bottom within following 24 h. After seeding, all pools were immediately covered with double-layer shading nets, which provided refuges for young germlings with the same role as natural canopy cover, thus avoiding germlings to be dislodged by water movement during high tide period (Yoshida et al., 2006; Whitaker et al., 2010). The technique for seeding artificial habitat with Sargassum germlings has proven to be inexpensive and not labor intensive. In this study, a total of 18 person hours were required for pools construction, germlings collection, transportation and seeding, and the restoration cost was approximately 320 US dollars, including materials, fuel, and hourly rates for labors and boat drivers. In addition, grazing pressure is considered to be a significant factor limiting the success of artificial seaweed beds as they consume or damage thalli (Largo and Ohno, 1993; Terawaki et al., 2003). Choi et al. (2003) attempted to transplant juveniles of S. horneri using artificial structures, but the majority of the juveniles were lost due to grazing by herbivores (sea urchins and snails) within a few months of transplantation. Carney et al. (2005) found that stipe breakage caused by grazing gastropod Lacuna vincta posed the largest limiting factor on survival of juvenile transplants of Nereocystis luetkeana. In the present study, the major grazer Chlorostoma rustica occurred at the restoration site with a density of approximately 7 individuals per 0.25 m2 during our experimental period (data not published). Although they did not obviously affect the establishment of S. thunbergii population, effective methods to exclude herbivores should be taken into account in future experimental studies. Comparing the morphological and physiological characteristics of restored plants and naturally growing seaweed individuals can lead to the development of criteria for defining successful seaweed restoration (Hernández-Carmona et al., 2000; Lee and Park, 2008).

In this study, thallus size, number and length of primary laterals per thallus of the restored population were lower than those of natural population at the time of investigation. S. thunbergii population at the restoration site appeared much more dense compared with reference sites, and individuals could respond to crowding by reduced branching as well as other seaweeds (Baer and Stengel, 2010). The increased duration of air exposure might be regarded another important cause of a reduction in growth performance of S. thunbergii thalli in the restored bed, as already reported by Zhang et al. (2010). The established individuals at the restoration site were exposed to air more than 3 h compared with the reference sites during a single low-tide period (personal observation), and thus subjected to more severe thermal and desiccation stress, especially in hot summer. Therefore, individuals in this habitat would need to spend more energy to mitigate physical stress, which could lead to a corresponding decrease of energy allocated to growth (Davison and Pearson, 1996). Regardless of their diminished growth performance, our results showed that the proportion of fertile laterals of the restored population was comparable to, or even higher than, that of naturally growing population. It suggested that these fertile thalli might serve as a source of new recruits to enhance the recovery of the S. thunbergii population in this study area. Diversity of species is one of the most widely used parameters for measuring restoration success (Choi et al., 2002; Ruiz-Jaen and Mitchell Aide, 2005; Oyamada et al., 2008). It has been suggested that restored seaweed beds should have similar diversity and community structure in comparison with reference sites (RuizJaen and Mitchell Aide, 2005). In the present study, the macroalgal species diversity in the artificially restored bed was significantly lower than that in the neighboring natural bed after one year of restoration. This might be because reference sites had a higher habitat complexity compared with the study area, which was a bare rock platform without any species before experimental restoration. Thus, it is possible that a recovery time is needed for the restored bed to reach the level of diversity observed in natural seaweed bed (Kang et al., 2008). Nevertheless, our results showed that the total abundance of macroalgae was higher in the restored bed compared with reference bed. And S. thunbergii abundance at the restoration site was significantly greater than that of reference bed, indicating that S. thunbergii, a depleted important canopy-forming seaweed species, can be established on the rocky platform using this method. Moreover, other algae were also found in the restored bed, such as Enteromorpha intestinalis, Ulva pertusa, Ectocarpus confervoides, Heterosiphonia japonica, Ahnfeltiopsis flabelliformis, Gloiopeltis furcata, and Corallina officinalis, even though these species contributed to a low level of coverage at the restoration site. This suggests that creation of artificial habitat is likely to have beneficial effects on macroalgal assemblages in the rocky intertidal zone. Since the intertidal zone of rocky shores is particularly harsh with very high solar heating and desiccation stress, restoration techniques previously developed for benthic or subtidal habitats could not applicable to intertidal areas. In this study, the construction of artificial pools in natural habitat to mitigate desiccation stress at low tide may be an effective and feasible strategy for S. thunbergii bed restoration using its germlings. The present technique of constructing artificial habitat coupled with seeding germlings was a successful demonstration for the restoration of intertidal seaweed beds.

Acknowledgement This work was financially supported by the National Natural Science Foundation of China (no. 31070376).

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