Optimizing Early Growth of Laminaria hyperborea in Controlled Settings: A Pathway to Improved Restoration Efforts

Abstract

Laminaria hyperborea, a key species in marine forest ecosystems, is experiencing pressure at its southern distribution limit in northern Portugal due to climate change and human-induced stressors. The ongoing degradation of marine forests highlights the need for effective restoration strategies to protect biodiversity and maintain the essential services provided by these habitats. Cultivation of juvenile kelps in laboratory settings is a common approach shared across multiple strategies aimed at supporting reforestation efforts; however, the specific cultivation process for L. hyperborea remains largely underexplored. In this study, we tested two seeding densities to optimize the production of L. hyperborea recruits for reforestation initiatives. We assessed the effects of seeding density on juvenile development by measuring both area coverage and length. Our findings revealed that higher seeding density initially promoted greater area coverage (e.g., 8.69 ± 0.38 cm² vs. 3.35 ± 0.27 cm²) but reduced the length of individual recruits over time (e.g., 0.26 ± 0.0026 cm vs. 0.39 ± 0.003 cm at T3). This suggests that increased competition for resources at high densities limits individual growth. In contrast, lower densities produced larger, more robust individuals (e.g., 0.50 ± 0.004 cm vs. 0.262 ± 0.003 cm at T4), which may enhance post-transplantation survival in challenging environmental conditions. This suggests that utilizing lower seeding densities may improve individual growth while decreasing overall resource use and maintenance needs, promoting a more sustainable cultivation process and minimizing impacts on donor populations. However, further research is essential to refine the cultivation protocols and fully understand the variables influencing juvenile kelp development. Improving all phases of the restoration process, from laboratory cultivation to field deployment, will be critical for reducing costs, streamlining logistics, and ensuring the scalability of future kelp restoration efforts.

1. Introduction

Brown macroalgae, especially kelps and fucoids, are key species in marine ecosystems, particularly in cold-water environments where they dominate shallow rocky coasts, forming extensive marine forests [1,2]. Kelp forests provide a range of essential ecosystem services, such as enhancing biodiversity by offering complex, three-dimensional structures that serve as shelter, nursery grounds, and foraging areas for a variety of marine species [3,4]. These habitats are also highly productive, supporting coastal food webs and playing a significant role in nutrient cycling. Moreover, their potential for carbon sequestration contributes to climate change mitigation, while their dense canopies reduce coastal erosion by dampening wave energy [5]. These services make kelp forests not only essential for ecosystem health but also viable, nature-based solutions for coastal protection and resilience against climate change [6,7].

Kelp distribution patterns and dynamics have been extensively studied due to their ecological importance. Research on kelp forests in Southern Europe has shown that these habitats are now largely restricted to the Northwest Iberian Peninsula, with Portugal marking the southern distribution limit for several cold-adapted species, such as Laminaria hyperborea and Saccharina latissima [8,9,10]. In recent decades, anthropogenic stressors such as overfishing, pollution, and herbivore pressure have threatened the survival of these communities [3]. Besides these local stressors, climate change appears to be the predominant driver of kelp declines in the Iberian Peninsula. Rising seawater temperatures and the increasing frequency of extreme weather events, such as storms and heat waves, have been linked to significant population losses [9,11,12,13]. As environmental conditions in the region become less favorable, there is growing evidence that kelp populations are shifting toward more suitable, cooler habitats further north, resulting in local disappearances. This shift highlights the critical need for developing effective conservation strategies, particularly for populations at the edge of their distribution, such as those in the Iberian Peninsula, where these cold-water species are most vulnerable to environmental changes [8,9].

Laminaria hyperborea (Gunnerus) Foslie is a large kelp species, which dominates exposed rocky shores of the Northeast Atlantic, forming considerably extensive underwater forests [14,15,16]. Despite its ecological significance, many aspects of the biology and ecology of this species remain poorly understood, posing challenges for effective restoration efforts [15]. L. hyperborea is commercially harvested in several European countries, including Norway, UK, Scotland, Ireland, and Iceland, where sustainable harvesting practices such as rotational harvesting, size limits, and no-take zones have been implemented to account for the species’ long lifespan and ecological importance in both intertidal and subtidal ecosystems [17]. Although harvesting is rare in Portugal, L. hyperborea populations face other threats that jeopardize their stability. Climate change, particularly rising sea temperatures and the increased frequency of extreme weather events, combined with local stressors such as pollution, overgrazing, and coastal development, create synergistic pressures that further endanger kelp populations, underscoring the urgency for proactive and multifaceted restoration strategies [14,18].

In response to the urgent need for global action to reverse ecosystem degradation and mitigate the impending climate and biodiversity crises, the UN launched the Decade of Ecosystem Restoration, which encourages large-scale restoration projects to halt and reverse the degradation of marine ecosystems. To address the challenges posed by climate change and ensure the long-term success of restoration efforts, the recent literature on “futureproofing” restoration initiatives emphasizes the potential of using heat-tolerant kelp strains or selecting phenotypes with higher thermal resilience for out-planting. However, to effectively restore marine forests, the first critical step is to establish efficient nursery protocols that can support reforestation techniques, such as seeding on artificial substrates and out-planting of lab-reared juveniles [19,20]. These protocols are crucial not only for ensuring the successful growth of juvenile kelps but also for optimizing the entire restoration process and laying the groundwork for advanced approaches, reducing the time and expenses associated with cultivation [21,22].

This study contributes to the development of a cultivation protocol for L. hyperborea seedlings by testing how different seeding densities influence early developmental stages, with a focus on determining optimal conditions for spore germination to enhance the success of marine forest restoration initiatives. It is well established that culture density plays a critical role in juvenile competition for essential resources, such as light and nutrients, which, in turn, can affect growth rates and biomass yield [23,24]. By understanding these dynamics, we seek to refine cultivation methods that can improve both efficiency and the overall success of restoration efforts.

2. Materials and Methods

2.1. Fieldwork and Laboratory Procedure

Reproductive tissue was collected in February 2023 from random fertile individuals (n = 10) of Laminaria hyperborea from a population inhabiting shallow intertidal pools in Vila Chã (Vila do Conde, Portugal, 41°17042.800 N, 8°44012.100 W, Figure 1).

Spore release was induced in laboratory conditions following the protocol described in [25]. Reproductive tissue (sori) was isolated from the fertile individuals and cleaned to remove epiphytes and debris. The sori were then maintained in dark conditions at 5 °C for 2 h to induce spore release. The obtained spore culture was kept under red light (12 µmol m⁻² s⁻¹) to allow the germination into gametophytes and maintain the vegetative growth [26]. Before the seeding, the gametophyte culture was broken down into smaller fragments using a handheld electric blender, to stimulate reproduction and accelerate the cultivation phase.

To investigate the effect of seeding density on recruit development, two different gametophyte densities were tested: high (1.42 mg/mL) and low (0.71 mg/mL). The gametophyte solutions were evenly sprayed onto PVC spools (three replicates for each density), with 2 mm polypropylene rough-textured twine coiled around them. This type of structure has already been widely used in seaweed aquaculture, as referred in several previous works [25,27,28]. After spraying, the spools were incubated in a cultivation system formed by two 40 L acrylic tanks, with three spools per tank. Seawater temperature was regulated at 12 °C, within the optimal temperature range for L. hyperborea growth [29], using a cooler (Aqua Medic Titan 150). The initial light intensity was 30 µmol m⁻² s⁻¹, and after the first week, aeration was introduced, and light intensity was increased to 50 µmol m⁻² s⁻¹. Nutrient supplementation (Provasoli’s Enriched Seawater, PES) [30] was introduced after two weeks, along with germanium dioxide (GeO₂) to prevent diatom contamination [31]. Subsequently, the nutrient media (PES and GeO₂) were refreshed weekly.

2.2. Data Analysis

Once the sporophytes became visible, approximately one month after seeding, their growth (i.e., total length) and coverage were monitored weekly for one month. This monitoring began in week 1 (T1), when the cultures were approximately five weeks old. Weekly photographic sampling was conducted for four weeks (T1–T4), allowing for the assessment of growth and coverage at each time point. The time points correspond to specific weeks post-seeding: T1 is at 5 weeks, T2 at 6 weeks, T3 at 7 weeks, and T4 at 8 weeks. At each time point, the spools were removed from the tanks and positioned at a fixed distance (30 cm) from the camera (Sony Alpha 6400, Sony, Tokyo, Japan, 16–50 mm lens) next to a scale for reference. This setup was consistently maintained for each monitoring. Eight distinct frames were captured by rotating the spool, with each frame covering a field of view of 80 × 60 mm. After the photographic sampling, three 21 cm² sections of each frame were randomly selected and used to calculate the area covered by the juveniles. For length measurements, 10 juveniles were randomly selected from each section. All photographic replicates were analyzed using the ImageJ software, version 1.54g (U. S. National Institutes of Health, Bethesda, ML, USA). All the measurements were expressed as average ± standard error.

A linear mixed-effects model (LMM) was used to assess the effect of seeding density and time on the area covered by recruits. The model was constructed to test the influence of seeding density, time, and their interaction, where seeding density and time were treated as fixed effects, and spool was included as a random effect to account for variation between spools. The response variable was the area covered by recruits. The same model was also employed to evaluate the impact of seeding density and time on recruit length.

The LMM was performed using R (version 3.3.0; R Core Team 2016) with the lme4 package [32] to fit the model. Model assumptions, including normality and homoscedasticity, were checked through residual analysis. Tukey’s HSD post hoc test was applied for pairwise comparisons to identify significant differences between treatments, where applicable. Model significance was determined using p-values, with a significance threshold set at p < 0.05.

3. Results

The LLM carried out highlighted a significant effect of the interaction between the seeding density and time on the juvenile length (p < 0.001; Figure 2). The results of Tukey’s pairwise comparisons showed that higher length values were initially observed in the high-density recruits compared to the low-density ones (0.23 ± 0.0027 cm vs. 0.193 ± 0.0022 cm; p < 0.0001). While on the second monitoring time, the length of the individuals showed no significant differences between the two seeding treatments, on the last two dates, the individual length resulted higher at low seeding density (T3: 0.39 ± 0.003 cm vs. 0.26 ± 0.0026). On the last monitoring time, the average length of the high-density individuals reached almost double the values of the low-density individuals (0.50 ± 0.004 cm vs. 0.262 ± 0.003 cm) (Figure 2).

The LLM carried out on the coverage area of recruits showed significant differences in the interaction between the seeding density and week (p < 0.001; Figure 3). Lower coverage was observed in the low-density treatment compared to the higher-density one at all monitoring times excluding the third week (T1: 8.69 ± 0.38 cm² vs. 3.35 ± 0.27 cm², p < 0.0001; T2: 5.35 ± 0.23 cm² vs. 2.81 ± 0.14 cm², p < 0.0001; T4: 14.42 ± 0.2 cm² vs. 13.08 ± 0.56 cm², p < 0.01). Moreover, between the third and fourth weeks, the results show a significant increase in recruit coverage for both density treatments, with values nearly tripling (HD: 5.17 ± 0.13 cm² vs. 14.42 ± 0.2 cm²; LD: 4.15 ± 0.13 cm² vs. 13.08 ± 0.56 cm²) (Figure 3 and Figure 4).

4. Discussion

This study aimed to optimize protocols for cultivating Laminaria hyperborea juveniles by assessing the effects of seeding density on early growth stages. The results demonstrated that seeding density significantly influenced both the coverage area and individual juvenile length, providing valuable insights into improving marine forest restoration techniques.

The significant effect of seeding density on both recruit length and coverage area confirms that density plays a critical role in the development of L. hyperborea juveniles. At lower seeding densities, juveniles exhibited greater individual lengths, likely due to reduced competition for essential resources such as light and nutrients. This reduced competition allows for more robust growth, as each individual has access to a larger share of available resources [33,34]. This aligns with previous studies that have shown that high-density cultures often lead to stunted growth due to these resource constraints [23,24].

Although higher densities were associated with greater total coverage in the early stages, this pattern likely reflects the initial presence of more recruits, rather than an increase in individual growth or resource availability. However, it is important to note that our results only capture the first four weeks of development. Over longer periods, this dynamic may shift as competition for resources intensifies, potentially leading to self-thinning among recruits, i.e., a decreased survival resulting from intraspecific competition in dense macroalgae stands [35,36]. This pattern has been observed in previous studies with other kelp species; for example, in an experiment using green gravel with the kelp Ecklonia radiata, high sporophyte density initially covered the substrate but eventually thinned to just 1–4 juvenile sporophytes per stone after 12 months of cultivation [36]. These results present an important trade-off: lower seeding densities favor the development of larger, more robust individuals, while higher seeding densities may accelerate early substrate coverage.

While lower seeding densities are likely more suitable for producing recruits with a greater capacity to survive harsh environmental conditions, higher densities could promote competitive selection, where only the most resilient individuals survive. This mechanism might enhance the kelp’s long-term resilience once out-planted, though such effects would require validation through extended field trials. In restoration projects, where survivability post-transplantation is critical, cultivating larger individuals at lower seeding densities may be more beneficial. Larger recruits have a greater capacity to survive in harsh environmental conditions, potentially improving the success of restoration efforts.

Tailoring cultivation protocols by adjusting variables such as seeding density, nutrient supply, and light exposure to match specific environmental factors (like hydrodynamic conditions, nutrient availability, competition, and grazing) can significantly enhance restoration outcomes. However, since our experiments were conducted in controlled laboratory settings, further studies are needed to assess the effects of additional stressors, such as wave activity and grazing pressure, commonly found in natural habitats. While higher seeding densities promote rapid early-stage coverage, lower seeding densities ensure sufficient resource availability and space, allowing each recruit to thrive and increasing survivability post-transplantation. Moreover, lower seeding densities reduce production costs by minimizing the need for extensive fieldwork to collect reproductive tissue, lowering the demand for reagents and materials required to maintain cultivation systems and cultures, and decreasing labor associated with their upkeep. By reducing the scale of these resource-intensive activities, lower densities make large-scale restoration efforts more economically feasible while maintaining effectiveness.

Selection of an appropriate seeding twine is crucial for successful kelp cultivation. Previous research on the effect of seeding twine on the hatchery of the kelp E. radiata found small but significant differences in hatchery performance among three seeding twine types (e.g., nylon, polyester, and kuralon), depending on their surface complexity and structural form [37]. In our study, we used a polypropylene twine that was pre-treated to roughen its surface and increase the available attachment area for juvenile kelps. This surface modification resulted in excellent hatchery performance, demonstrating the importance of surface texture in promoting successful kelp attachment and growth.

Incorporating biodegradable fibers as an alternative to the synthetic fibers used in our study could also offer significant environmental benefits for marine restoration projects. Unlike synthetic materials, which may persist in the environment and contribute to marine pollution, biodegradable fibers would naturally decompose over time, reducing the ecological footprint of restoration efforts. Selecting the right type of biodegradable fiber is crucial to improve the likelihood of restoration actions: the fiber should be durable enough to support juvenile growth during the critical early stages but also capable of decomposing naturally without harming the environment. Additionally, the choice of fiber should be matched with the species being cultivated, as different species of macroalgae may have varying attachment needs and growth rates. Previous studies have explored various biodegradable twines, such as cotton, jute, and sisal, for their potential in kelp cultivation. Cotton twine has shown promising results with comparable bioadhesion strength and biomass yield to synthetic materials like polyvinyl alcohol (PVA), though further research is needed to optimize its physical structure. In contrast, jute and sisal have shown toxic effects on meiospores, though they were successful for sporophyte seeding, albeit with weak bioadhesion [38]. As biodegradable materials become more advanced and accessible, they could play a key role in scaling up restoration initiatives while maintaining ecological integrity [39].

Progressing with sustainable and tailored cultivation protocols for kelp restoration, improving laboratory processes alongside the use of environmentally friendly materials, and the integration of complementary conservation techniques are essential for enhancing the scalability and long-term success of marine reforestation efforts. By adopting a multifaceted approach that considers both biological (e.g., growth requirements and survival rates of the target species) and environmental factors, future restoration projects will be better equipped to restore degraded marine ecosystems, playing a significant role in achieving global conservation objectives.