Elena Balestri

Sexual reproductive success in Posidonia oceanica

Abstract To identify the factors that might contribute to limiting the sexual reproduction of Posidonia oceanica we examined: (1) flowering and fruiting phenology; (2) variability in flowering frequency, seed production and reproductive success (i.e. the proportion of flowers setting mature fruits per unit area) over a 2-year period; and (3) losses of potential seed production to fruit abortion and/or pre-dispersal seed predation. The flowering frequency of P. oceanica varied among years, ranging between 7.9 and 19.8%. Despite the large number of flowers and ovules, few of these produced mature fruits owing to abortion. Moreover, about 84% of developing inflorescences were damaged by herbivores. Fruit production varied among years, ranging from 4.7 to 13.5 fruits per m2, but reproductive success remained constant (2.0–2.4%). Exclusion of herbivores showed that abortion alone was reponsible for the loss of about 87% of the reproductive potential. Since reduced fertilization did not completely explain the observed frequency of abortion, it was hypothesized that post-fertilization factors could affect seed formation. The higher reproductive success of protected plants (11.2%) compared to herbivore-exposed plants (3.1%) indicated that predation significantly reduced the number of seeds available for establishment. We concluded that pre-dispersal seed losses to abortion and predation may seriously reduce the reproductive success of the species.

Global analysis of seagrass restoration: the importance of large-scale planting

In coastal and estuarine systems, foundation species like seagrasses, mangroves, saltmarshes or corals provide important ecosystem services. Seagrasses are globally declining and their reintroduction has been shown to restore ecosystem functions. However, seagrass restoration is often challenging, given the dynamic and stressful environment that seagrasses often grow in. From our world-wide meta-analysis of seagrass restoration trials (1786 trials), we describe general features and best practice for seagrass restoration. We confirm that removal of threats is important prior to replanting. Reduced water quality (mainly eutrophication), and construction activities led to poorer restoration success than, for instance, dredging, local direct impact and natural causes. Proximity to and recovery of donor beds were positively correlated with trial performance. Planting techniques can influence restoration success. The meta-analysis shows that both trial survival and seagrass population growth rate in trials that survived are positively affected by the number of plants or seeds initially transplanted. This relationship between restoration scale and restoration success was not related to trial characteristics of the initial restoration. The majority of the seagrass restoration trials have been very small, which may explain the low overall trial survival rate (i.e. estimated 37%). Successful regrowth of the foundation seagrass species appears to require crossing a minimum threshold of reintroduced individuals. Our study provides the first global field evidence for the requirement of a critical mass for recovery, which may also hold for other foundation species showing strong positive feedback to a dynamic environment.Synthesis and applications. For effective restoration of seagrass foundation species in its typically dynamic, stressful environment, introduction of large numbers is seen to be beneficial and probably serves two purposes. First, a large-scale planting increases trial survival - large numbers ensure the spread of risks, which is needed to overcome high natural variability. Secondly, a large-scale trial increases population growth rate by enhancing self-sustaining feedback, which is generally found in foundation species in stressful environments such as seagrass beds. Thus, by careful site selection and applying appropriate techniques, spreading of risks and enhancing self-sustaining feedback in concert increase success of seagrass restoration.

IN VITRO GERMINATION AND SEEDLING DEVELOPMENT OF POSIDONIA OCEANICA

Abstract The effects of culture media and fruit storage on germination, development and survival of seedlings of Posidonia oceanica (L.) Delile (Posidoniaceae) were investigated under axenic conditions. Seeds germinated within 15–18 days both on solid media and in liquid over solid media as well as on filter-paper bridges over liquid media. The results showed a significant effect of the culture medium on germination and survival of seedlings among the solid media and the liquid over solid media tested. The highest mean germination (96.2%) was found in the solid artificial seawater medium (ASW). Only solid or liquid/solid ASW medium, however, allowed continuous growth of seedlings. Addition of nutrients and inclusion of the plant growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D) inhibited development of seedlings. After 10 months, the highest mean survival (75%) was reached on solidified ASW following addition of liquid medium. No significant effect of the culture medium on seed germination and seedling survival was seen using the paper-filter bridge technique. With the latter method, germination and survival rates ranged from 54 to 85% and from 31 to 58%, respectively. All seedlings showed continuous growth. Addition of nutrients, however, reduced the production of adventitious roots. The results also showed significant effect of fruit storage treatment on the fruit surface-sterilization success, but not on seed germination or seedling survival. In vitro raised seedlings of P. oceanica appear to be useful for conservation and propagation of this species.

Experimental transplanting of Posidonia oceanica (L.) Delile into a disturbed habitat in the Mediterranean Sea

A pilot transplanting experiment was conducted to evaluate the feasibility of a restoration project in a disturbed coastal area (Vada, Livorno) of the Ligurian Sea, Italy. The effects of morphological type, geographical and depth origin on survival and growth of Posidonia oceanica transplants were investigated over approximately a three year period (1994-1997). Despite the frequent high turbidity level of the water and the high degree of exposure at the site, transplants survived and persisted. Plagiotropic rhizomes showed significantly higher mean survival (76.4 ± 5.7%), rhizome growth increase (70.6 ± 25.7%) and ramification increase (87 ± 61.6%) compared to orthotropic rhizomes (survival, 59.5 ± 3.8%; rhizome length increase, 22.2 ± 10.1%; ramification increase, 37.3 ± 16.7%). Survival did not differ between transplants from different origins and depths. In contrast, rhizome growth and ramification were higher in transplants obtained from distant meadows compared to those from the local meadow. These results suggest that the site, which historically supported a P. oceanica meadow, is now suitable for restoration purposes. Results also show a different growth response of populations.

Grazing of Inflorescences of the Seagrass Posidonia oceanica (L.) Delile

Abstract An experiment using cages was designed to demonstrate herbivore consumption of Posidonia oceanica (L.) Delile reproductive structures, in a north-western area of the Mediterranean Sea. The percentages of mature inflorescences and those damaged by herbivores in caged vs. uncaged (control) vs. partially caged plants (artefact control) were compared. Inflorescences damaged by herbivores were 84.3 ± 3.1% in the control and 79.5 ± 3.2 % in partial cages. Significantly more mature inflorescences were found in caged (37.5 ± 2.4 %) vs. uncaged (10.1 ± 2.9 %) and partially caged plants (6.7 ± 0.9 %). The real contribute of herbivores to the loss of fertilised inflorescences of P. oceanica was approximately 27 %, as the differences between mortality in caged and in uncaged plants. These results suggest that herbivores could affect sexual reproduction in P. oceanica, but further investigation is needed to investigate biotic interactions in the meadows.

Germination and Early-Seedling Establishment Capacity of Pancratium maritimum L. (Amaryllidaceae) on Coastal Dunes in the North-Western Mediterranean

Abstract There is a scarcity of data on the seedling establishment success of plants inhabiting the sand coasts of the Mediterranean, despite on increasing interest in the conservation and restoration of dune habitats. A field experiment, in conjunction with a series of laboratory germination tests, was conducted to (1) determine the recruitment capacity of Pancratium maritimum, a perennial dune plant which is declining along the north-western Italian coasts, by natural seed dispersal and (2) test the ability of seeds to tolerate different environmental stress. We planted seeds at different burial depths and found that seeds from greater depths (6, 10 and 15 cm) showed higher emergence (35.0–46.6%) than seeds from a shallower depth (2 cm; 11.6%). On average, less than 18% of seeds remained dormant at the end of the germination period. None of the seedlings that emerged in autumn survived over winter, and 88% of those that emerged in spring died by the start of the second reproductive season as a result of desiccation. Therefore, less than 3% of the seeds resulted in established seedlings. Under laboratory conditions seeds germinated well (87.5–100%) in darkness as well as at 12 h alternating light/dark and showed little dormancy (27–35 days). Germinability of seeds remained up to 70% for at last one year of storage. Temperatures below 10°C or over 30°C inhibited germination; frost and heat were lethal for most seeds. Germination was totally inhibited by salinity and water stress. Recovery of germination occurred in seeds from solutions below 25% seawater or −0.6 MPa. These data provide useful information for conservation and reintroduction of this species.

Interannual Variability in Flowering of Posidonia oceanica in the North-Western Mediterranean Sea, and Relationships among Shoot Age and Flowering

Abstract The frequency of Posidonia oceanica past flowering events in a north-western Mediterranean area was examined using a reconstructive age technique. We also investigated possible relationships among shoot age and occurrence of flowering. Seven flowering events were recorded over the 14-year period examined. Considerable variations in inflorescence frequency occurred among years (0–25.3%), with two years of abundant flowering (1989 and 1993) separated by several years with little or no flowering. Only 2% of the shoots sampled flowered twice; contrary to previous opinion, the interval among two successive flowerings was considerably shorter than 10 years, varying from 1 to 4 years. The minimum reproductive age of shoots was 6 months. The probability of flowering increased with age until 6 years of age, and decreased thereafter. Both the number of leaves and rhizome segments produced by a shoot during a flowering year did not differ significantly from those produced in the years preceding and following a flowering event, suggesting that the cost of flowering could not affect short-term growth of shoots. On the basis of our results we can speculate that the maximum sexual reproductive output for P. oceanica in the area studied was about 340–460 seeds m−2 in 1994.

Global change and response of coastal dune plants to the combined effects of increased sand accretion (burial) and nutrient availability.

Coastal dune plants are subjected to natural multiple stresses and vulnerable to global change. Some changes associated with global change could interact in their effects on vegetation. As vegetation plays a fundamental role in building and stabilizing dune systems, effective coastal habitat management requires a better understanding of the combined effects of such changes on plant populations. A manipulative experiment was conducted along a Mediterranean dune system to examine the individual and combined effects of increased sediment accretion (burial) and nitrogen enrichment associated with predicted global change on the performance of young clones of Sporobolus virginicus, a widespread dune stabilizing species. Increased burial severity resulted in the production of taller but thinner shoots, while nutrient enrichment stimulated rhizome production. Nutrient enrichment increased total plant biomass up to moderate burial levels (50% of plant height), but it had not effect at the highest burial level (100% of plant height). The effects of such factors on total biomass, shoot biomass and branching were influenced by spatial variation in natural factors at the scale of hundreds of metres. These results indicate that the effects of burial and nutrient enrichment on plant performance were not independent. Their combined effects may not be predicted by knowing the individual effects, at least under the study conditions. Under global change scenarios, increased nutrient input could alleviate nutrient stress in S. virginicus, enhancing clonal expansion and productivity, but this benefit could be offset by increased sand accretion levels equal or exceeding 100% of plant height. Depletion of stored reserves for emerging from sand could increase plant vulnerability to other stresses in the long-term. The results emphasize the need to incorporate statistical designs for detecting non-independent effects of multiple changes and adequate spatial replication in future works to anticipate the impact of global change on dune ecosystem functioning.

Isolation and cell wall regeneration of protoplasts from Posidonia oceanica and Cymodocea nodosa

A method for the isolation of protoplasts from the seagrasses Posidonia oceanica and Cymodocea nodosa is described. Isolation of protoplasts was achieved using a combination of cellulase Onozuka R-10, hemicellulase and pectinase. Purification was carried out by Ficoll gradient centrifugation. A yield of 1.87 (± 0.16 SD) × 10 6 protoplasts per gram of fresh tissue was obtained from mesophyll cells of P. oceanica. The viability of isolated protoplasts was 82.5% (± 10.6) as confirmed by fluorescein diacetate staining. A high percentage of protoplasts of P. oceanica (61.5 ± 14.8%) regenerated the cell wall within 7 days as confirmed by staining with calcofluor white, but only a few protoplasts were able to divide. Callus-like structures were noticed after 20–30 days in culture. A lower yield of protoplasts, 6.9 (± 3.8) × 10 5 protoplasts per gram of fresh tissue, was obtained from mesophyll of C. nodosa. Viability of these protoplasts was 67.9% (±12.6) after isolation. Some possible applications of the method are discussed.

New Bio-Composites Based on Polyhydroxyalkanoates and Posidonia oceanica Fibres for Applications in a Marine Environment

Bio-composites based on polyhydroxyalkanoates (PHAs) and fibres of Posidonia oceanica (PO) were investigated to assess their processability by extrusion, mechanical properties, and potential biodegradability in a natural marine environment. PHAs were successfully compounded with PO fibres up to 20 wt % while, at 30 wt % of fibres, the addition of 10 wt % of polyethylene glycol (PEG 400) was necessary to improve their processability. Thermal, rheological, mechanical, and morphological characterizations of the developed composites were conducted and the degradation of composite films in a natural marine habitat was evaluated in a mesocosm by weight loss measure during an incubation period of six months. The addition of PO fibres led to an increase in stiffness of the composites with tensile modulus values about 80% higher for composites with 30 wt % fibre (2.3 GPa) compared to unfilled material (1.24 GPa). Furthermore, the impact energy markedly increased with the addition of the PO fibres, from 1.63 (unfilled material) to 3.8 kJ/m2 for the composites with 30 wt % PO. The rate of degradation was markedly influenced by seawater temperature and significantly promoted by the presence of PO fibres leading to the total degradation of the film with 30 wt % PO in less than six months. The obtained results showed that the developed composites can be suitable to manufacture items usable in marine environments, for example, in natural engineering interventions, and represent an interesting valorisation of the PO fibrous wastes accumulated in large amounts on coastal beaches.