Addressing reproductive stochasticity and grazing impacts in the restoration of a canopy-forming brown alga by implementing mitigation solutions

1. The worldwide decline of marine forests, due to human impacts and climate change, emphasizes the need to develop and implement effective and sustainable solutions to restore these endangered habitats and to re-establish the services they provide. 2. In this study, the ex situ restoration of Treptacantha barbata, a Mediterranean subtidal habitat-forming species of brown seaweed, was for the first time implemented in a marine protected area in the Adriatic Sea. Two restoration efforts were performed in 2019. The first one was started in winter, after a marine heatwave that triggered early fertility, the second one in spring, when the species usually reproduces. 3. This study aimed to evaluate: 1) the disruptive effects of a thermal anomaly on the reproductive biology and performance in culture of T. barbata; and 2) the impact of the grazing pressure on juveniles after the outplanting. 4. The first cultivation was more productive than the second one, in terms of zygote release and germling growth. To mitigate the low efficiency of the second culture and to avoid prolonged highly-demanding maintenance in the mesocosms, the cultivation period was extended outdoors using a structure suspended in the water column. 5. The modular frames conceived for outplanting T. barbata proved to be effective because of their easy operability and low cost. Controlling for herbivorous fish had significant positive effects on both juvenile survival and growth. 6. The outcomes highlighted that an unpredictable climatic event and fish grazing were major threats that impaired the restoration process of T. barbata. These stressors should be considered when developing plans to implement effective large-scale restoration of canopy-forming macroalgae

Cultivation of Gongolaria barbata (Fucales, Phaeophyceae) with a seaweed‐derived biostimulant in order to improve photophysiological fitness and promote fertility to advance the restoration of marine macroalgal forests

As a result of several anthropogenic factors, Cystoseira sensu lato forests have declined or become regionally extinct in many coastal regions of the Mediterranean. Given the low natural recovery of lost populations, research efforts have been encouraged to develop sustainable and efficient restoration of macroalgal forests on a large scale. By promoting growth and fertility of collected thallus branches under controlled laboratory conditions, the availability of seedlings for restoration could be ensured without jeopardizing natural populations. Here we investigated the effect of a commercial algal biostimulant (AlgatronCifo®) on the photophysiology, growth and fertility of Gongolaria barbata (Stackhouse) Kuntze (Fucales, Phaeophyceae). In a factorial laboratory experiment, two different temperatures (10 oC and 14 °C) and two culture media [i.e. seawater (SW) and Algatron (AT)] were tested. The photosynthetic performance of G. barbata doubled after three weeks of culture with AT, while it decreased by 25% when cultivated in SW. The highest photosynthetic performance and growth were achieved at 14oC with AT, where fertile receptacles also developed, followed by seedling settlements. The thalli cultured in AT had similar or better photosynthetic performance than the initial control thalli. AT-cultured thalli had a greater ability to quench energy via photochemical pathways (qP) than those from the SW, which on the contrary, had higher levels of non-photochemical responses (qN, NPQmax). This limited photosynthetic performance was probably linked to the higher P-limitation experienced under that treatment. The algal biostimulant enhanced the physiological performance and induced fertility of G. barbata, demonstrating its valorization potential and setting a new path for improved restoration applications

Where and how - new insight for brown algal forest restoration in the Adriatic

Several anthropogenic factors are responsible for the decline of Cystoseira sensu lato (hereafter Cystoseira) forests along Mediterranean coasts. Some Cystoseira species are already regionally extinct, and their decline has been widely recorded. Sustainable and efficient techniques for the restoration of Cystoseira are needed. In this context, the objectives of this study were i) to analyse the reproductive traits of three populations of Gongolaria barbata from three nearby donor sites in the northern Adriatic Sea, assessing the differences in their reproductive potential and reproductive success; and ii) to evaluate the outplanting success in terms of the effectiveness of G. barbata restoration, in relation to the different donor and receiving sites (Miramare MPA and in front of the Marine Biology Station Piran - MBSP) and implemented methods (ex situ and hybrid method combining a mesocosm cultivation and a suspended culture in the field). After 2 weeks of cultivation in mesocosms, half of the tiles with germlings were transported to the receiving sites and placed on suspended lantern nets (hybrid method), which were later (after 3 months) transferred to the seabed on concrete plates with protective cages. The remaining tiles were placed on the seabed on concrete plates with protective cages after a 4-week culture (ex situ method). At both sites, lantern nets and plates were randomly placed at 3 m depth. Thallus length was measured monthly in each treatment. Seedlings in suspended culture showed lower performance at the Miramare MPA, most likely due to the unfavourable environmental conditions. The satisfactory results obtained at MBSP demonstrate the efficiency of the hybrid method and confirm its potential to reduce the cost and time required for cultivation. Since unpredictable climatic events pose the greatest threat to restoration performance, these challenges must be considered when establishing new restoration practices. Moreover, herbivore regulation is extremely urgent and should be planned and implemented on a larger regional scale

BASE KNOWLEDGE AND IMPLEMENTATIONS IN THE RESTORATION OF MEDITERRANEAN MARINE FORESTS

Brown macroalgae of the genus Cystoseira sensu lato (Fucales, Phaeophyta, henceforth Cystoseira) form dense canopy forests along the coasts of the Mediterranean Sea, sustaining biodiversity and biogeochemical cycles. In the last 4 decades, these habitats have undergone substantial and widespread regression, because of local natural and anthropogenic pressures and, more recently, global climatic stressors. Despite conservation policies have been adopted to passively counteract the deterioration of these habitats, the natural recovery is infrequent. For this reason, active restoration efforts are required and strongly recommended. In particular, restoration by ex situ recruitment enhancement is to be favoured. This is a sustainable and non-destructive methodology, which foresees the collection of fertile apices in a donor site, the reproduction and cultivation of propagules in mesocosm and the further outplanting of the cultivated germlings in a receiving site to be restored. In this thesis, the theme of restoration of Cystoseira forests by ex situ recruitment enhancement is addressed. The I part of the work provides base-knowledge on three Cystoseira species, useful to underpin further restoration efforts. In the first chapter, the early development of Ericaria barbatula (= C. barbatula) is described. This threatened species, particularly requiring in terms of water quality, could be a possible candidate for restoration efforts, thus the acquired knowledge could be exploited for the development of culturing protocols for ex situ recruitment enhancement. The second chapter represents the first description of the detrimental effects of a marine heatwave on the reproductive phenology of a macroalga, providing some considerations on the possible role of thermal anomalies and climate change in explaining the decline of Gongolaria barbata (= C. barbata) observed in the Gulf of Trieste (Northern Adriatic Sea) in the last thirty years, and of Cystoseira species in general. The third chapter focuses on the thermotolerance of early-life stages and adults of Ericaria giacconei (= C. hyblaea), a dotted endemism of the south-central Mediterranean Sea, discussing its conservation under ocean warming. Also the first description of the species’ embryology is provided. The II part of this work focuses on some of the first restoration efforts of Cystoseira species by ex situ recruitment enhancement in the Mediterranean Sea and the application of novel methods towards its upscaling. The first chapter reports the first restoration of G. barbata in the Adriatic Sea by ex situ recruitment enhancement, addressing the effects of the abovementioned marine heatwave and fish grazing on the restoration performance. Also in this case, the description of the species’ embryology is published for the first time. The second chapter investigates the effect of the donor population and the receiving site on the restoration performance and the feasibility of decreasing the restoration costs by reducing the cultivation time in mesocosm. This thesis contributes to the science behind the restoration of Cystoseira forests in the Mediterranean, which is still at the outset. The results obtained in the I part provide new knowledge on the biology of Cystoseira species from different parts of the Mediterranean Sea, useful to underpin future restoration efforts. In the II part, the implementation of restoration efforts shows how, despite biological and logistic challenges can be overcome, complex environmental dynamics and unpredictable climatic events represent the major threat to the success of restoration of Cystoseira s.l. forests, strongly limiting the possibilities of upscaling and the predictability of outcomes. In a context of ever increasing anomalous climatic events and environmental pressures on coastal habitats, the implementation of mitigation actions results as a crucial strategy to manage the stochasticity of restoration results.Brown macroalgae of the genus Cystoseira sensu lato (Fucales, Phaeophyta, henceforth Cystoseira) form dense canopy forests along the coasts of the Mediterranean Sea, sustaining biodiversity and biogeochemical cycles. In the last 4 decades, these habitats have undergone substantial and widespread regression, because of local natural and anthropogenic pressures and, more recently, global climatic stressors. Despite conservation policies have been adopted to passively counteract the deterioration of these habitats, the natural recovery is infrequent. For this reason, active restoration efforts are required and strongly recommended. In particular, restoration by ex situ recruitment enhancement is to be favoured. This is a sustainable and non-destructive methodology, which foresees the collection of fertile apices in a donor site, the reproduction and cultivation of propagules in mesocosm and the further outplanting of the cultivated germlings in a receiving site to be restored. In this thesis, the theme of restoration of Cystoseira forests by ex situ recruitment enhancement is addressed. The I part of the work provides base-knowledge on three Cystoseira species, useful to underpin further restoration efforts. In the first chapter, the early development of Ericaria barbatula (= C. barbatula) is described. This threatened species, particularly requiring in terms of water quality, could be a possible candidate for restoration efforts, thus the acquired knowledge could be exploited for the development of culturing protocols for ex situ recruitment enhancement. The second chapter represents the first description of the detrimental effects of a marine heatwave on the reproductive phenology of a macroalga, providing some considerations on the possible role of thermal anomalies and climate change in explaining the decline of Gongolaria barbata (= C. barbata) observed in the Gulf of Trieste (Northern Adriatic Sea) in the last thirty years, and of Cystoseira species in general. The third chapter focuses on the thermotolerance of early-life stages and adults of Ericaria giacconei (= C. hyblaea), a dotted endemism of the south-central Mediterranean Sea, discussing its conservation under ocean warming. Also the first description of the species’ embryology is provided. The II part of this work focuses on some of the first restoration efforts of Cystoseira species by ex situ recruitment enhancement in the Mediterranean Sea and the application of novel methods towards its upscaling. The first chapter reports the first restoration of G. barbata in the Adriatic Sea by ex situ recruitment enhancement, addressing the effects of the abovementioned marine heatwave and fish grazing on the restoration performance. Also in this case, the description of the species’ embryology is published for the first time. The second chapter investigates the effect of the donor population and the receiving site on the restoration performance and the feasibility of decreasing the restoration costs by reducing the cultivation time in mesocosm. This thesis contributes to the science behind the restoration of Cystoseira forests in the Mediterranean, which is still at the outset. The results obtained in the I part provide new knowledge on the biology of Cystoseira species from different parts of the Mediterranean Sea, useful to underpin future restoration efforts. In the II part, the implementation of restoration efforts shows how, despite biological and logistic challenges can be overcome, complex environmental dynamics and unpredictable climatic events represent the major threat to the success of restoration of Cystoseira s.l. forests, strongly limiting the possibilities of upscaling and the predictability of outcomes. In a context of ever increasing anomalous climatic events and environmental pressures on coastal habitats, the implementation of mitigation actions results as a crucial strategy to manage the stochasticity of restoration results

Reproductive phenology, zygote embryology and germling development of the threatened Carpodesmia barbatula (= Cystoseira barbatula) (Fucales, Phaeophyta) towards its possible restoration

Carpodesmia barbatula (=\ua0Cystoseira barbatula) (Fucales, Phaeophyta) is a species endemic to the Southern Mediterranean Sea, which grows in clean waters on exposed sublittoral rocky shores. Together with other species of the genus Cystoseira sensu lato, it is an ecosystem engineer that forms dense canopy forests, sustaining biodiversity, productivity and nutrient cycling in temperate rocky reefs. Human impacts and climate stressors are seriously hampering these ecosystems and actual conservation efforts appear insufficient for their preservation. Restoration actions could be a solution for the loss of these habitats, but they require the deep knowledge of the target species. In the present study, we describe egg release, zygote development and germling growth of C. barbatula, poorly known, despite its importance in the upper sublittoral seaweed flora. The aim is to improve the knowledge on the early life stages of this rare and threatened species, towards its possible future restoration

Turning biomass into functional composite materials: rice husk for fully renewable immobilized biocatalysts

Rice husk is an underexploited, low density and highly robust composite material, massively available from rice processing. Here we report two new procedures for the formulation of immobilized lipases applicable in fats and oils transformations. The enzymes were covalently anchored on aldehyde groups introduced on rice husk by laccase-catalysed oxidation of the cellulose component. The method avoids the use of toxic glutaraldehyde while allows for the application and recycling of the biocatalysts in aqueous media. The second method used a fluidized bed granulator for the coating of the particles of rice husk (200\u2013400 m) in the presence of water-soluble binders. The formulations are mechanically stable and suitable for applications in different hydrophobic media. Both methods allow for the recovery and reuse of the rice husk at the end of the life cycle of the biocatalysts

Climatic anomalies may create a long-lasting ecological phase shift by altering the reproduction of a foundation species

The resilience of ecological communities is often defined by one or a few species that have disproportionately important roles influencing many other species in the community. This is true for some areas of the Mediterranean Sea that are characterized by large brown fucoid algae in the genus Cystoseira that form dense underwater forests structurally similar to the giant kelps of the Pacific. While shorter than the giant kelp, Cystoseira form dense underwater stands, contributing to the three\u2010dimensional complexity of the seascape (Fig. 1). These canopy\u2010forming seaweeds play a crucial role in primary production and nutrient cycling of temperate coastal ecosystems from the Mediterranean Sea to the Atlantic Ocean (Mineur et al., 2015) and act as \u2018ecosystem engineers', providing food, nursery, and shelter for a rich associated biota. Our study highlighted potential disruptive effects of winter hot spells on reproductive timing, recruitment, and adult survival that could severely affect the persistence of Cystoseira populations. Because extreme climate episodes are increasing in intensity and frequency, implementing coordinated initiatives connecting centers for climate alerts and algologists may shed light on how these phenomena impact population dynamics of Cystoseira species, and help current attempts to restore algal forests