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A large fraction of plastic litter found in natural environments is constituted by conventional not biodegradable plastic bags, and their adverse effects via ingestion or entanglement on terrestrial and marine organisms are largely documented. Biodegradable and compostable shoppers have been recently developed as alternative to traditional ones. These bags are specifically designed to degrade in composting facilities and generate a product devoid of toxicity to soils and crops. However, very little is known on the effects of bag leaching, i.e. the transfer of chemicals from plastic into natural environments, on vegetation. Some plant species are highly sensitive to a variety of chemicals, and seedling growth is generally the most affected life history stage. In this study we assessed the potential effects of conventional (high-density polyethylene, HDPE) and compostable (Mater-bi®, MB) bags, when left in natural environments, on water quality and plant development. To this end, seeds of Lepidium sativum L., a terrestrial plant commonly used in phytotoxicity standard tests, were exposed to leachates obtained from different amount of HDPE and MB bags, simulating various pollution degrees occurring in nature, for 72 h. Both not-exposed (or virgin) bags and natural weathering exposed bags were used. Variations of chemical-physical characteristics of extracts were used as indicative of water quality deterioration, while alterations of seed gemination and seedling radicle and hypocotyl length were considered as indicative of phytotoxicity. A chemical qualitative screening of the leachates was also performed to identify the compounds with potential phytotoxicity. Both types of bags affected water characteristics (pH, salinity and total dissolved solids) relevant to plants, and released into water intentionally added chemicals, such as the noxious bisphenol A, and other phytotoxic substances probably generated during bag manufacturing. Leachates from both bag types did not affect seed germination. But, a significant number of seedlings showed developmental abnormalities or reduced seedling growth. The hypocotyl was the most sensible seedling organ to HDPE bag leachates while the radicle was the most vulnerable to MB ones. These findings indicate that plastic bags, including those that meet biodegradability and compostability standards, represent a potential threat to plants, if left in natural environments. Therefore, people and managers should be adequately informed about the potential environmental impact of an incorrect bag disposal. Simple, rapid standard phytotoxicity tests, such as the L. sativum bioassay, applied to bag leachates could be used in the future to select not noxious additives so to develop more eco-friendly bags.

Epiphytes colonizing adult seagrasses highly contribute to seagrass ecosystem functioning and plant growth. Yet, little information exists on epiphytic communities developing on seagrass seedlings. Moreover, for some species our knowledge about seedling performance is limited to early establishment phases, and the role of substrate type in affecting their growth is still unclear. These are considerable knowledge gaps, as seedlings play an important role in meadow expansion and recovery from disturbance. In this study, seedlings of Posidonia oceanica, a keystone species of the Mediterranean, were grown in a shallow (1.5 m deep) coastal area along the Tuscany coast (Italy). After five years of growth (July 2009), seedlings were collected and, through multivariate analysis, we examined whether the epiphytic communities of leaves (both internal and external side) and rhizomes, as well as the growth characteristics differed between rock and sand substrate. The epiphytic communities of seedlings largely reflected those found on adult shoots. Epiphyte cover was similar between the two leaf sides, and it was higher on seedlings grown on rock than on sand, with encrusting algae dominating the community. No differences in epiphyte cover and community structure on rhizomes were found between substrates. Seedling growth characteristics did not differ between substrates, apart from the number of standing leaves being higher on rock than on sand. No correlation was found among epiphyte communities and seedling growth variables (i.e., leaf area, maximum leaf length, number of leaves, total number of leaves produced, rhizome length, total biomass, and root to shoot biomass ratio). Results indicate that epiphytes successfully colonize P. oceanica seedlings, and the surrounding micro-environment (i.e., substrate type) can influence the leaf epiphytic community. This study provides new valuable insights on the biological interactions occurring in seagrass ecosystems and highlights the need for better understanding the effects of seedling epiphytes and substrate on the formation of new meadows.

Plastics are found in marine environments worldwide, and their effects on macrophytes (seagrasses and macroalgae) colonizing sandy bottoms are still poorly known. Seagrass meadows are valuable but declining ecosystems due to local and global-change related stressors, including sediment disturbance and introduced macroalgae. Understanding whether plastics pose a further threat to seagrasses is critically important. In two simultaneous additive experiments performed in an aquaculture tank, we examined the individual and combined effects of macroplastics (non-biodegradable high-density polyethylene and biodegradable starch-based) and sedimentation (no and repeated sedimentation) on the performance (in terms of biomass and architectural variables) of a native Mediterranean seagrass (Cymodocea nodosa) and an introduced macroalga (Caulerpa cylindracea), and on the intensity of their interactions. Macroplastics were still present in sediments after 18 months. Cymodocea nodosa produced a greater biomass and longer horizontal rhizome internodes forming clones with more spaced shoots probably to escape from plastics. Plastics prevented C. nodosa to react to sedimentation by increasing vertical rhizome growth. Under C. cylindracea invasion, C. nodosa allocated more biomass to roots, particularly to fine roots. In the presence of C. nodosa, C. cylindracea performance was reduced. High-density polyethylene (HDPE) plastic and sedimentation shifted species interactions from competitive to neutral. These results suggest that both HDPE and biodegradable starch-based macroplastics, if deposited on marine bottoms, could make seagrasses vulnerable to sedimentation and reduce plant cover within meadows. HDPE plastic and sedimentation could contribute to the decline of seagrass habitats by facilitating the spread of non-native macroalgae within meadows. Overall, the study highlights the urgent need to implement more effective post-marketing management actions to prevent a further entering of plastics in natural environments in the future, as well as to establish to conservation measures specifically tailored to protect seagrass habitats from plastic pollution.

Marine litter, that is solid materials deliberately or accidentally released into marine and coastal environments, is a growing issue both at local and global scale. In particular, litter accumulation on beaches represents a relevant problem in touristic coastal areas. The DPSIR framework, already applied to tackle other environmental issues, can help in analysing the origin, impact, and possible solution of material stranded on the beach allowing a holistic approach in the “one health” perspective. It was applied in a popular sandy bathing location in the North-Western Tuscany (Italy). Information and data on origin and fate of beach-stranded material as well as existing local strategies were retrieved from a stakeholders’ network. In summer 2020, a monitoring campaign was performed to evaluate the sanitary state of the shoreline and the impact of this material on coastal habitat. In a 7-year period (2014-2020), a mean amount of 1.548,3 tons of beach-stranded material have been collected and treated to separate sand (55%), organic green fraction (31%), and anthropogenic debris (2%), while 12% weight-loss was attributable to evaporation. Analysis of historical environmental data revealed that shoreline accumulation of this material was associated with low atmospheric pressure, medium-strong winds, and storms. Preliminary monitoring data showed that the green fraction was mainly represented by wood and terrestrial plants with little quantity of the seagrass Posidonia oceanica, while most of the anthropogenic debris was rubber and metal. Microbial pollution of beached material was at least one order of magnitude higher than the surrounding environment (sand and seawater) for total coliforms, Escherichia coli, intestinal enterococci, and total fungal load. Overall, our results indicate a potential impact of beach-stranded material in this coastal area, with possible health risk for human population. The instruments already in place for the management of beached material, although consistent, could be further improved.

Marine plastic litter is a global concern. Carrier bags manufactured from non-biodegradable polymers constitute a large component of this litter. Because of their adverse impact on marine life, non-biodegradable bags have recently been replaced by biodegradable ones. However, growing evidence shows that these latter are not readily degradable in marine sediments and can alter benthic assemblages. The potential impact of biodegradable bags on seagrasses inhabiting sandy bottoms, which are the most widespread and productive ecosystems of the coastal zones, has been ignored. Mesocosm experiments were conducted to assess the effect of a commercialized biodegradable bag on a common seagrass species of the Mediterranean, Cymodocea nodosa, both at the level of individual plant (clonal growth) and of plant community (plant-plant relationships), under three culture regimes (plant alone, in combination with a neighbour of the same species or of the co-existing seagrass Zostera noltei) simulating different natural conditions (bare substrate, monospecific meadows or mixed meadows). The bag behaviour in marine sediment and sediment physical/chemical variables were also examined. After six months of sediment exposure, the bag retained considerable mass (85% initial weight) and reduced sediment pore-water oxygen concentration and pH. In the presence of bag, C. nodosa root spread and vegetative recruitment increased compared to controls, both intra- and interspecific interactions shifted from neutral to competitive, and the growth form changed from guerrilla (loosely arranged group of widely spaced ramets) to phalanx form (compact structure of closed spaced ramets) but only with Z. noltei. These findings suggest that biodegradable bags altering sediment geochemistry could promote the spatial segregation of seagrass clones and influence species coexistence.

Non-biodegradable and compostable plastic bags on beaches negatively affect the development of coastal dune vegetation by drastically reducing seedling emergence and growth.

The presence of plastic bags on coastal dunes worldwide is well documented. Plastic bags contain additives that during rainfall events can leach out from bags into sand dune and be absorbed by seeds and roots of plants. Dune plants play a fundamental role in dune system formation, yet the possible impact of bag leaching on their establishment and development has been neglected. We assessed in laboratory whether (i) not biodegradable bags (high-density polyethylene, PE) and new generation of compostable bags (Mater-bi®, MB) would influence via leaching water chemical/physical properties and (ii) leachates would affect germination and seedling growth using Thinopyrum junceum and Glaucium flavum as models. Leachates were obtained from different amounts of not-exposed and bags exposed to beach or marine conditions simulating various pollution degrees (none, low, intermediate and high pollution). All water variables were affected by leaching. The magnitude of these alterations depended on bag type and environmental exposure. Seeds of T. junceum treated with the high concentration of marine-exposed MB bag leachate germinated later than controls while those of G. flavum treated with the remaining leachates germinated earlier. For both species, leachates from the low concentration of PE and MB marine-exposed bags increased seed germinability. A short radicle was observed in T. junceum seedlings treated with not-exposed MB bag leachates. Glaucium flavum seedlings treated with beach- and marine-exposed PE bags and not-exposed MB bags leachates showed a greater below-aboveground length ratio and those grown with the low concentration of not-exposed PE bag leachate had a longer hypocotyl compared to controls. Leachates from the high concentration of PE and MB bag caused seedling anomalies in both species. These findings indicate that not biodegradable and compostable bags may interact with abiotic/biotic factors and affect via leaching germination phenology, seedling establishment and plant interactions with consequences on dune community structure.

Propagule pressure is considered a major driver of plant invasion success. Great propagule pressure would enable invasive species to colonize new areas overcoming the resistance of native species. Many highly invasive aquatic macrophytes regenerate from vegetative propagules, but few studies have experimentally investigated the importance of propagule pressure and biotic resistance, and their interaction, in determining invasion success. By manipulating both recipient habitat and the input of vegetative propagules of the invasive seaweed Caulerpa cylindracea in mesocosm, we examined whether higher propagule pressure would overcome the resistance of a native congeneric (Caulerpa prolifera) and influence its performance. With the native, C. cylindracea population frond number decreased irrespectively of pressure level. High propagule pressure did not increase stolon length and single plant size decreased due to the effects of intra- and interspecific competition. Native biomass decreased with increasing C. cylindracea propagule pressure. These results indicate that higher propagule pressure may fail in enhancing C. cylindracea invasion success in habitats colonized by the native species, and they suggest that biotic resistance and propagule pressure co-regulate the invasion process. These findings emphasize the need to preserve/restore native seaweed populations and may help to design effective management actions to prevent further C. cylindracea spread.

Most researches on the plastisphere in coastal environments deal with plastics floating in seawater. Comparatively smaller attention has been devoted to the plastisphere of plastics buried in marine sediments, and very little is known on that of plastics on coastal sand dunes. Yet, limited information is available on the impact of plastics, especially biodegradable plastics, on microbial organisms in their surroundings. Nevertheless, a large amount of plastics sink on the seabed or is deposited on beach-dune systems. We investigated the succession of plastisphere microbial community on two biodegradable composites based on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and seagrass fibres (PHBV/PO), buried in seabed and dune sediments over a 27 months period in mesocosm. PHBV is regarded as a valuable alternative to conventional plastics and PHBV/PO has recently been designed for applications in coastal habitat restoration. We also examined the degradation rate and impact of these plastics on the microbial communities of surrounding sediments. Microbial communities of the surface of PHBV and PHBV/PO in seabed and dune sand differ from those of surrounding sediments, displaying a lower richness. Plastics colonization occurs largely from bacteria present in surrounding sediments, although the contribution from the water column bacterial pool could be not negligible for plastics in the seabed. No significant differences were detected between the communities of the two plastics and no significant impact of plastics on microbial community of the surrounding sediments was detected. The exceptional long duration of this study allowed to gain information on the succession of a plastisphere community over a previously unexplored time scale. Succession appears highly dynamic in dune sand even after two years, while the community structure in seabed seems to reach stability after one year. These findings highlight the importance of performing long-term studies when trying to characterize composition and dynamics of plastisphere bacterial communities.

Traditional revegetation techniques employed to restore seagrass meadows and coastal dunes have recently been criticized for their impact on donor populations as well as for the installation of plant anchoring structures made of non-biodegradable or not natural materials in recipient habitats. To improve the ecological sustainability of restoration practices, a novel plantable biodegradable container made of beach-cast seagrass wrack and a bio-based polymer was produced. The long-term performance of two seagrasses, Cymodocea nodosa and Zostera noltei, and two dune plants, Euphorbia paralias and Thinopyrum junceum, grown in nurseries from seeds using the bio-container or a non-biodegradable container of equal size/form made of a conventional plastic (control) was also examined. In addition, the development of bio-container-raised C. nodosa plants in the field was compared to that of plants removed from control containers at the installation and anchored with a traditional system. The bio-container degraded slowly in seawater and in sand and lost its functionality after about three years. In nurseries, all the tested species grown in bio-containers performed as well as, or better than, those raised in non-biodegradable ones. Six months after transplanting into the field, 80% of the C. nodosa nursery-raised plants installed with their bio-container have colonized the surrounding substrate while most of those planted with the traditional system was lost. These results indicate that the new bio-container may support plant growth, and it may also provide protection and anchorage to plants in the field. The use of this bio-container in combination with nursery techniques could improve the environmental sustainability of coastal restoration interventions by providing large plant stocks from seed, thus reducing the impact of collection on donor populations. This approach would also limit the introduction of extraneous materials in recipient habitats and offer an opportunity for valorizing seagrass beach-cast material.

Root morphology and root system architecture of young Posidonia oceanica plants established on two contrasting substrate types, sand and rock, were examined to provide insights into the strategy of adaptation of seagrasses to their environment. After germination, seedlings were planted on sandy patches and on rock within the same area, and survived plants were collected five years later for measurements of the size of the entire root complex and analysis of individual morphological and architectural root traits. Collected plants exhibited up to nine highly intermingled root systems and approx. 2.5 m of total root length. Maximum horizontal extension, total biomass and total length of roots were not significantly affected by substrate. However, on sand roots grew vertically reaching up to 13 cm, while on rock they extended more horizontally and did not penetrate deeper than 5–7 cm leading to the formation of a shallow, densely packed root complex. On rock, the number and the length of second-order laterals on an individual root system were reduced and the topological index higher than on sand (0.8 vs. 0.7) reflecting a more simple (herringbone) branching pattern. Again, root diameter was greater than on sand. The results suggest that P. oceanica can adjust root traits early during plant development according to substrate typology to maximize anchorage and substrate exploration efficiency. This plasticity enables the species to establish and persist also on rocky bottoms which generally prevent establishment of the majority of seagrasses.

The effects of climate change are likely to be dependent on local settings. Nonetheless, the compounded effects of global and regional stressors remain poorly understood. Here, we used CO2 vents to assess how the effects of ocean acidification on the seagrass, Posidonia oceanica, and the associated epiphytic community can be modified by enhanced nutrient loading. P. oceanica at ambient and low pH sites was exposed to three nutrient levels for 16 months. The response of P. oceanica to experimental conditions was assessed by combining analyses of gene expression, plant growth, photosynthetic pigments and epiphyte loading. At low pH, nutrient addition fostered plant growth and the synthesis of photosynthetic pigments. Overexpression of nitrogen transporter genes following nutrient additions at low pH suggests enhanced nutrient uptake by the plant. In addition, enhanced nutrient levels reduced the expression of selected antioxidant genes in plants exposed to low pH and increased epiphyte cover at both ambient and low pH. Our results show that the effects of ocean acidification on P. oceanica depend upon local nutrient concentration. More generally, our findings suggest that taking into account local environmental settings will be crucial to advance our understanding of the effects of global stressors on marine systems.

Seagrasses are declining globally, and deeper understanding is needed on the recruitment potential and distribution of new populations for many threatened species to support conservation planning in the face of climate change. Recruitment of Posidonia oceanica, a threatened seagrass endemic to the Mediterranean, has long been considered rare due to infrequent flowering, but mounting evidence demonstrates that the species is responding to a changing climate through greater reproductive effort. Due to the fragmentary information on recruit occurrence and distribution, little is known about reproductive success in the species and its contribution to persistence. We assembled P. oceanica recruitment data from published and unpublished sources, including our own, to examine the frequency and extent of recruitment events (establishment of seedlings in a site), seedling growth potential and habitat characteristics at recruitment sites. Results show that at least one recruitment event has occurred about every three years, and eighteen localities were colonized at least one time since the first seedling record in 1986. Notably, consistently high seedling inputs were observed in four localities of the Western Mediterranean. Seedlings established mainly on unoccupied substrate areas along the coasts of islands, in sheltered sites and at shallower depths (

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.

Sediment fertilization is recommended for improving seagrass restoration efforts, but few studies have evaluated the efficacy of such practice. Increasing storm frequency due to global change could lead to greater sediment mobilization. Understanding how this alteration will interact with fertilization to affect transplants is essential for future restoration planning. We examined the individual and combined effects of nutrients (ambient vs. repeated addition) and burial (control vs. increased frequency and intensity) on the performance and biomass partitioning of transplants of the seagrass Cymodocea nodosa at two sites within a north-western Mediterranean meadow. Fertilization stimulated the production of shoots, total biomass, and branching. Burial increased leaf sheath length in one site while reduced shoot number, leaf number, leaf sheath length, total biomass, net shoot gain, and root-to-shoot ratio in the other site. Regardless of the site, fertilization and burial interaction reduced the length of vertical internodes and horizontal rhizomes, and the net shoot gain. Our research demonstrates that sediment fertilization ensures rapid colonization of restoration sites, providing C. nodosa plants up to eight times larger than controls in one growing season. However, it also indicates that interaction of increased burial and nutrients reduced the gain in terms of vegetative expansion and depressed vertical growth, making plants more vulnerable to subsequent disturbances. Therefore, seagrass restoration practitioners should account for changes in sediment elevation at transplanting sites when planning restoration programs and carefully evaluate the opportunity of applying fertilizers in sites subjected to greater sediment accumulation to avoid failure.

M.M. van Katwijk et al., 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. 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. Journal of Applied Ecology, A.T. was funded by Greater Caribbean Energy and Environment Foundation grants. N.M. was supported by a Gledden Fellowship from the Institute of Advanced Studies of the University of Western Australia. N.M. C.M.D and A.C. were supported by Biomares contract number LIFE06 NAT/PT/000192. N.M. and C.M.D. were supported by Opera (FP7, contract number 308393). C.P. and the Cornell Cooperative Extension Marine Program are funded in part by County Executive Steve Bellone and the Suffolk County Legislature, Hauppauge, New York. E.B. and C.L. were funded by University of Pisa (Lardicci 308/ex60%2010). M.L.C and G.A.K were supported by ARC Linkage Grants (LP130100155, LP0454138)

Exogenous application of plant growth regulators (PGRs) may be an effective technique for increasing the rooting ability and the growth of vegetative fragments (cuttings) of plants used in dune restoration programs. Various concentrations (0, 50 and 100 mg l −1 ) of two auxins, alpha-naphtaleneacetic acid (NAA) and indole-3-butyric acid (IBA), and two cytokinins, 6-furfurylaminopurine (Kinetin) and 6-benzylaminopurine (BAP), were applied separately to cuttings of two widely used species for restoration, Ammophila arenaria and Sporobuls virginicus . Root development and production of new buds in cuttings were examined under laboratory conditions one month after application. Cuttings were also examined one year after transplanting into a sandy substratum under natural conditions, to test for possible long term effects of PGRs on plant establishment success and growth. The response of the two study species to PGRs differed substantially. In A. arenaria the auxin NAA at 100 mg l −1 reduced the time for root initiation and increased the rooting capacity of cuttings, while the cytokinin Kinetin at 50 mg l −1 facilitated root growth. No auxin had effect on rooting or growth of S. virginicus cuttings, but treatment with 100 mg l −1 Kinetin resulted in higher rooting success than the control. One year after planting, the cuttings of A. arenaria treated with 100 mg l −1 NAA showed a higher establishment success (90% vs. 55%) and produced more culms and longer roots than the control; those treated with cytokinins did not differ in the establishment success from the control, but had longer roots, more culms and rhizomes. On the other hand, the cuttings of S. virginicus treated with 100 mg l −1 Kinetin showed a higher establishment success (75% vs. 35%) and had more culms than the control. Therefore, in restoration activities that involved A. arenaria, a pre-treatment of cuttings with NAA would be beneficial, as it allows the production of a higher number of well-developed plants with high survival potential and greater area cover. In contrast, a pre-treatment of cuttings of S. virginicus with Kinetin would achieve more acceptable plant survival rates. This easy and low cost-effective technique may be extended to other dune plant species and applied on a large scale to improve the chance of dune restoration success.

The use of fragments detached naturally from Posidonia oceanica , a threatened seagrass protected by European legislation, as non-destructive alternative to removing cuttings from donor meadows for restoration efforts has been recently proposed. A 4-year study was performed to investigate whether storm-generated fragments deposited on beaches can be salvaged and used as transplant source. Specifically, we examined in outdoor tanks (i) how long fragments survive after detachment from parents, (ii) whether fragment survival and regeneration ability are influenced by morphological characteristics, i.e., initial fragment growth form and size, and (iii) whether a single fragment can be divided artificially to obtain more transplants. Finally, we monitored the first year survival of fragments lodged between rubbles of a submersed artificial reef 3 years after storage in tanks to test for their capacity to re-establish in the field. Four months after collection, fragment survival was high (80–96%). Half of the fragments regenerated and survived as long as 3 years in tanks. Initial growth form and size were not predictors of fragment survival or regeneration probability. Division of fragments allowed doubling transplant number. One year after transplanting, 50% of the fragments on the artificial reef were still present and ca. half of them produced new shoots. This indicates that fragments retain the capacity to re-establish once reintroduced into the field and thus could represent a potentially important transplant source. The use of this material could have major advantages over traditional restoration techniques, including large availability with zero impact on existing populations and low collection efforts.

In this study the occurrence of twin-like embryos and abnormal embryos in Posidonia oceanica , a rare phenomenon in seagrasses, was documented. The ability of additional embryos to develop seedlings was also demonstrated for the first time in seagrasses. Approximately 1750 fruits collected at three localities of the north-western Mediterranean on 2 years (1994 and 2004) were screened for abnormal embryo morphology. The frequency of embryo anomalies varied from 1.9 to 7.1% among localities, and no differences between years were detected within the same locality. Twin-like embryos were usually in contact and germinated to produce complete twin seedlings that could be separated. Abnormal embryos showed additional plumules but had a common hypocotyl and a single primary root; these embryos germinated and grew like “Siamese” seedlings.

A recruitment event of Posidonia oceanica was recorded in the Gulf of Ventilegne (Corsica, Western Mediterranean) in June 2005. One-year seedlings (alive and dead) were found at shallow depth (2–3 m) on a mix of rock and sand substrates. Pattern in the distribution of seedlings was examined over spatial scales ranging from centimetres to hundreds of metres. We also examined (a) whether establishment was preferentially associated with a specific substrate type; (b) the relation between proportional mortality and the density of established seedlings; and (c) the growth characteristics of seedlings. The average density of seedlings varied from 19 to 189 seedlings m−2, with a maximum of 480 seedlings m−2. While significant variation was detected at the scale of area (i.e., tens of metres), no difference was found in abundance of seedlings at the scale of sites (i.e., hundreds of metres). Most of the overall variability was, however, at on the smallest scale examined (i.e., among sampling units tens of centimetres apart). No differences were also observed in the establishment probability and seedling abundance between sand and rock substrates. The fine scale analysis of spatial patterns on rock and sand substrates showed a clumped distribution of seedlings. These results indicated that the spatial distribution of seedlings was not random, but organized at small spatial scales within the study sites. Hydrodynamics and micro-scale bottom features appeared to account for such patterning. Proportional seedling mortality was highly variable (ranging from 0.3 to 0.8), and tended to increase with increasing settlement density. A total of 128,000 seedlings were recoded over the 1600 m2 surface area investigated. Such event reflected conspicuous seed supply and particularly favourable local conditions. This finding contradicts the current statement of low recruitment ability of P. oceanica and indicates that the species can recruit both on consolidated and unconsolidated substrates, at least in the study area.

The intensive reconstructive sampling (1957-2004, 39 localities), a systematic direct observation (1992-2004, 1 locality) and particular direct observations (66 localities) of Posidonia oceanica meadows were analysed together with temporal series of flowering available in the literature (19 localities). This allowed the examination of temporal and spatial variability in annual flowering prevalence (FP, the fraction of meadows flowering in a given year) and of annual meadow flowering intensity (FI, number of inflorescences per shoot) for the period 1979-2004 across the Western Mediterranean, as well as spatial variability of flowering frequency (FF, the fraction of years that a given meadow has flowered) and shoot flowering probability (Pf, fraction of flowering stalks appeared per annual segment). Each year, on an average 17% of the investigated meadows flowered, ranging from 3 to 86% of meadows among the years. The highest annual FP and FI values were obtained in 2003 (FP = 0.86 and mean FI = 0.23 ± 0.03 inflorescences shoot-1). A secondary peak of FP and mean FI occurred 9 years earlier, in 1994 (FP = 0.44 and mean FI = 0.08 ± 0.02). Both peaks of flowering occurred after hot summers. Flowering synchrony in particular years across the Western Mediterranean and clines of increased meadow flowering frequency towards the North and East, suggests the existence of large-scale environmental mechanisms controlling the floral induction. On the other hand, meadow FF and Pf were highly heterogeneous among and within the meadows, indicating that local factors also may play a significant role in flowering induction. When flowering, the Western Mediterranean meadows showed an average 0.11 ± 0.02 inflorescences shoot-1, but FI greatly varied among and along the series (from 0.002 to 0.54 inflorescences shoot-1) and decreased significantly with depth but was independent of meadow shoot density and meadow latitude or longitude. The shoot flowering probability was quite low (0.007 ± 0.002 inflorescences shoot-1 year-1) and exponentially increased with shoot age. © Springer-Verlag 2005.

The seagrass Posidonia oceanica (L.) Delile can produce plagiotropic (horizontally oriented) and orthotropic (vertically oriented) axes. These axes differ in various morphological and growth characteristics and ecological functions. Only orthotropic shoots are likely to support flowering and seed production. To asses whether flowering may occur on plagiotropic shoots, we examined plagiotropic axes that grew at the edge of a meadow during the flowering of 2003. We identified and analysed 10 rhizome fragments that bore at least one reproductive plagiotropic shoot. We then investigated whether reproductive plagiotropic shoots differed from orthotropic ones in morphological characteristics and investment in flowering (i.e., number of hermaphroditic flowers per inflorescence). Multivariate analysis did not show any significant difference between plagiotropic and orthotropic shoots with regard to the characteristics examined and investment in flowering. Further studies are necessary to ascertain if the observed pattern of flowering is a common or an exceptional event in P. oceanica.

Urban and industrial wastes have been claimed to negatively affect Posidonia oceanica meadows, but few studies have addressed this issue by comparing disturbed locations with replicated reference locations. Here, we examined the general proposition that patterns of growth and morphology of P. oceanica exposed to urban and industrial effluents were different from those observed in reference meadows. Hypotheses were both on differences in mean values of response variables and on variation of these measures at a hierarchy of spatial scales (from centimetres to hundreds of metres). Results indicated a significant reduction in mean number of intermediate leaves at the outfall compared to reference locations, whereas the opposite pattern occurred for juvenile leaves. There were significant, though temporally variable differences in growth of rhizomes between disturbed and reference locations, with reduced growth at the outfall in 2 out of 3 years analysed. Measures of spatial variance in number of juvenile leaves and length of adult leaves at the scale of shoots were significantly larger at the outfall compared to reference locations. At the same scale, measures of spatial variance in length of juvenile and intermediate leaves were significantly lower at the putatively impacted location. Spatial variance in number of intermediate leaves was reduced at the outfall compared to reference locations at the scale of quadrats. Past values of spatial variance in number of leaves per shoot were lower at the outfall than at the two reference locations at the scale of shoots, whereas the opposite occurred at the scale of areas. None of the structural variables examined showed any difference between the putatively impacted location and the two reference locations, either in terms of mean response or as changes in spatial variance. These results indicated that integrating methods to examine present and past events of disturbance, including analyses to detect changes in spatial variance of response variables, may provide a powerful approach to the analysis of environmental impacts on P. oceanica.

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...

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.

In seagrasses, elongation of horizontal rhizomes, and hence clone expansion, is supported by physiological integration. However, horizontal rhizomes are subjected to breakage by physical disturbances and to burial, often simultaneously. Little is known about the combined impact of such factors on the growth of apical rhizome regions. We factorially manipulated connections on runners of Cymodocea nodosa and sediment height for 1 mo at 2 sites within a meadow to test the hypothesis that breakage of interconnections close (10 cm) to the rhizome apex causing premature loss of physiological integration would negatively affect the performance of runners and reduce their ability to tolerate burial (60% of shoot height). Shoot survival, length of vertical internodes and horizontal rhizome network, biomass production, partitioning, and costs were unaffected by the investigated factors. Rhizome breakage interacted with burial for some characteristics. Breakage reduced branch length and rhizome diameter only in unburied runners, while burial reduced branch length only in intact runners. Moreover, burial stimulated leaf sheath elongation and greater branching, irrespective of the status of connections. These findings indicate that breakage only delays lateral spread and increases the risk of fragmentation by subsequent disturbances, and, contrary to our hypothesis, has no substantial influence on the capacity of runners to withstand transitory and moderate burial. The findings also reveal that runners may escape from burial not only vertically, but also horizontally, changing growth pattern and direction from predominantly unidirectional to multidirectional. This additional strategy could allow buried runners to accelerate space occupation even if disconnected from clones.

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. © 2001 Elsevier Science B.V. All rights reserved.

Two pilot transplant experiments were conducted in a damaged coastal area of the Ligurian Sea (Italy) to examine the feasibility of a large-scale restoration project. Two sites were selected in the area and two-month-old aquarium-grown Posidonia oceanica (L.) Delile seedlings were transplanted at each site on different substrata. Survival and development of seedlings were followed every month. The effects of substratum type (`matte' vs. pebbles) and herbivore exclosure on seedling success were tested approximately after three years. Survival and growth of natural P. oceanica seeds germinated in the study area were also investigated. Sites did not differ significantly in the survival and development of seedlings. The highest survival and development was observed for seedlings growing on `matte', with survival rates of about 70% for transplanted and 66% for natural seedlings. Growth characteristics of natural and transplanted seedling populations were similar. Herbivores did not affect the survival and development of transplanted seedlings. These results indicate that P. oceanica seedlings may be successfully used in the study area for restoration purposes. Furthermore, our data support the hypothesis that sexual recruitment may occur in P. oceanica but could be limited by postsettlement factors relating to substratum type, in the study area.

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.

Dune plants are subjected to disturbance and environmental stresses, but little is known about the possible combined effects of such factors on growth and spatial colonization. We investigated how clones of Sporobolus virginicus , a widespread dune species, responded to the independent and interactive effects of breakage of rhizomes, breakage position and burial regime. Horizontal rhizomes were severed at three different internode positions relative to the apex to span the range of damage by disturbance naturally observed or left intact, and apical portions exposed to two burial scenarios (ambient vs. increased frequency) for three months in the field. The performance of both parts of severed rhizomes, the apical portion and the remaining basal portion connected to clone containing four consecutive ramets, was compared with that of equivalent parts in intact rhizomes. Apical portions severed proximal to the third internode did not survive and their removal did not enhance branching on their respective basal portions. Severing the sixth or twelfth internode did not affect survival and rhizome extension of apical portions, but suppressed ramet production and reduced total biomass and specific shoot length. Their removal enhanced branching and ramet production on basal portions and changed the original rhizome growth trajectory. However, the gain in number of ramets in basal portions never compensated for the reduction in ramet number in apical portions. Recurrent burial increased biomass allocation to root tissues. Burial also stimulated rhizome extension only in intact rhizomes, indicating that disturbance interacts with, and counteracts, the positive burial effect. These results suggest that disturbance and recurrent burial in combination reduces the regeneration success and spread capacity of S . virginucus . Since global change leads to increasingly severe or frequent storms, the impact of disturbance and burial on clones could be greater in future and possibly prevent colonization of mobile dunes by the species.

SummaryThe genome of Posidonia oceanica (Potamogetonaceae) was studied at cytophotometric and biochemical level. P. oceanica is an important species for food web in the Mediterranean basin where it occupies very large prairies. It is also an important biological indicator of mercury contamination. Cytophotometric analyses showed that this species has a basic (4C) genome size of 11.16 pg DNA. Biochemical analyses were made to establish genome composition in terms of highly and medium repeated and unique sequences. Melting profiles and CsCl density gradient of DNA extracted from leaves indicated the presence of a large A-T rich satellite DNA.

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.

Two mesocosm experiments were performed to examine the effects of seed density and small-scale nutrient heterogeneity on seedling recruitment and early development of patches of Cymodocea nodosa. In the first experiment, seeds were sown at 4 densities selected after determining mean natural density of seeds in the field, and the emergence and survival rates of seedlings were monitored for 1 yr. In the second experiment, seedlings were grown individually for 2 yr, either on substrate made heterogeneous or homogeneous with the addition of a fertiliser or on unfertilised substrate. The emergence rate and proportional survival of seedlings decreased significantly with increasing seed density. Seedlings grown on fertilised substrates were able to form patches, while those grown on unfertilised substrate failed to branch, providing experimental evidence of the role of nutrient limitation as a constraint for patch initiation. The size of seed-generated patches was significantly larger (in terms of total length of the rhizome network and number of shoots) on heterogeneous substrate than on homogeneous substrate at the same overall level of nutrient supply. Apparently, no preferential proliferation of shoots and branches occurred in the nutrient-rich half of the heterogeneous substrate. The results support our hypothesis that negative density dependence at the seed-seedling stage plays an important role in regulating population size. They also reveal that seedlings experiencing nutrient heterogeneity are capable of exploiting resources more efficiently than those in homogeneous environments, but are probably unable to escape from unfavourable patches or to select favourable ones, at least at the scale examined here.

We investigated the effects of body size, feeding strategy and depth distribution on the trophic resource partitioning among the 26 dominant fish consumers in a fish assemblage on the central Mediterranean shelf-break. The fish assemblage was structured in two major trophic guilds: epibenthic and zooplanktonic feeders, according to the position of each predator along the benthos–plankton gradient. Within each main guild, the species were segregated along a prey-size or fish-size gradient into five further guilds. Fish size and prey size were strongly correlated, indicating that the prey-size niche can be well explained by predator size. Fish consumers showed a significant negative correlation between the similarity in prey type and the similarity in depth distribution; most species with similar trophic preferences segregated along the depth dimension. The only predators overlapping in both food and depth preferences were those with a more specialist trophic behavior. These results suggest that fish body size and depth preferences are the two main niche dimensions, explaining a large part of the coexistence between the Mediterranean shelf-break fish consumers.

A method for the isolation of protoplasts from Posidonia oceanica (L.) Delile was established. Axenic tissues from young and mature leaves were used as the starting material. By using commercial enzymes (cellulase, pectinase and driselase), two distinct types of protoplasts were obtained from the youngest areas of leaves: normal spherical-shaped protoplasts from mesophyll cells and irregular-shaped protoplasts from epidermal cells.

We studied nutritional constraints on growth of Posidonia oceanica seedlings, morphometry and elemental content of mature seeds, and seedlings stranded on a beach of the northwestern Mediterranean Sea on 3 dates (May 2004, May 2005 and December 2005). The mean total nitrogen (N) and phosphorus (P) content of P. oceanica seeds was high compared with closely related species, Seeds varied considerably in mass, and their nutrient and carbon (C) content. increased linearly with mass. This indicates substantial variation in the amount of reserves initially available for seedling growth. Stored reserves in seeds were used by seedlings within 6 to 8 mo after germination: P was retrieved with greater efficiency (86%) than was N (69%) and C (20%). From May to December of the 2nd year (2005), the tissue nutrient concentrations of seedlings declined due to fast utilization and translocation of nutrients from the internal pools to support summer growth. Instead the C concentration in roots and rhizomes increased, indicating an accumulation of C. Seedling tissue N and P concentrations were above the critical levels reported for seagrasses, and were also lower than those of plagiotropic rhizomes of P. oceanica collected in the same period. These results suggest that P may play a major role in constraining growth of P. oceanica seedlings.

At the present, the long period (from 3 up to 12 months) to form roots from rhizome cuttings of Posidonia oceanica is the major cause of transplant failure. To promote earlier rooting, the effects of different concentrations (5 and 10 mg/l) of two auxins, namely indole-3-butyric acid (IBA) and α-naphtalen aetic acid (NAA), and the time of collection on rooting of plagiotropic and orthotropic cuttings of P. oceanica were tested. Rooting, survival and growth of cuttings were assessed 1 month after planting in a mesocosm. Results demonstrated that the use of auxin was essential to achieve root initiation within the observation period. Irrespective of plant source and collection time, IBA and NAA (5 mg/l) treatments increased the rooting capacity in cuttings. The highest rooting success (i.e. percentage of the survived cuttings that rooted) obtained was 33%. Results also revealed that the survival rate and percentages of cuttings with leaf growth and emergence of new leaves on their terminal shoot were influenced by the collection time × plant source interaction, but were unaffected by the auxin treatment. Orthotropic cuttings taken in November and February showed the highest survival rate (100%). No differences in survival among months were detected for plagiotropic cuttings. In May and July, more plagiotropic cuttings survived (80±7 and 75±6%) compared to orthotropic cuttings (58±7 and 51±4%). Overall, more plagiotropic cuttings showed leaf elongation compared to orthotropic ones, but the inverse was observed in November. A higher percentage of cuttings with emergence of new leaves was observed in the plagiotropic type as compared to the orthotropic one, and in general February and November were the best months for leaf production (28±4 and 38±7%). Finally, the percentage of cuttings that had changed leaf growth orientation (from orthotropic to plagiotropic) was significantly higher in November (63±13%) compared to the other months and tended to be higher in cuttings treated with the auxins compared to controls. These preliminary findings indicate that exposure to IBA or NAA (5 mg/l) was effective in stimulating rooting in P. oceanica without inhibiting plant growth. Therefore, this simple pre-planting practice should be beneficial in restoration attempts. Based on the trend observed, rhizomes should be prepared preferably in late autumn–winter and then planted into restoration stands to achieve a prompt rooting response and the best survival rate. Clearly, future work is needed to maximize the rooting success of cuttings.

The biomass of reproductive litter from Posidonia oceanica deposited on a 3.5 km stretch of beach in the north-western Mediterranean, as a consequence of a storm in May 2004, was quantified. The damage caused by this storm to the meadow from which fruits originated was evaluated in terms of loss of seed production. Intermediate fruits (i.e., developing fruits) were the most important reproductive component, followed by immature and damaged fruits. No fully mature fruits were found. No significant differences in the average number of fruits and biomass accumulated were detected among beach sections hundreds of metres apart. Extrapolation of the results at four beach sections indicated that about 1 million fruits were deposited on the entire (3.5 km) beach. This was equivalent to the seed production potential of about 313,217 inflorescences, or a flowered area of 1500 m 2 . The organic input to the beach was 224 kg ash-free dry weight (AFDM). These results suggest that storms may provide an unpredictable source of seed mortality in P. oceanica . The reproductive material produced by storms, however, may constitute an important source of allochthonous organic matter to the beach.

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 occurrenceof 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.

Knowledge of the natural pattern of spatial variation of the variables of interest is fundamental for designing effective sampling strategies to be used in surveys and monitoring programs. Unfortunately, little is known on patterns of spatial variation of structural, morphological and dynamic features of Posidonia oceanica, despite their increasing use as indicators of the status and/or trend of meadows. Here, variations in some such features (such as shoot density, leaf biometry, annual rhizome elongation and leaf formation) were examined on a hierarchy of spatial scales, ranging from cm within individual meadows, to meadows separated by 10 km, using independent full-nested analyses of variance. Overall, at least 3 variables showed significant variations in the mean values on each of the scales examined. Patterns of variability differed among individual traits. Most of the total variation, however, was on the smallest spatial scale examined (i.e. among replicates, shoots or quadrats). For some variables, a significant variation was also associated with the largest scale (i.e. among meadows). Collectively, the results indicated that patchiness in density, morphology and growth exists on a very local scale within P. oceanica meadows along the same isobath. This suggests that sampling designs and analyses which incorporate different spatial scales and appropriate replications should at least be considered in the future when planning large-scale and long-term surveys.

Posidonia oceanica Delile is a marine phanerogam, largely spread in the mediterranean basin, where forms large prairies. Since this plant can live on mercury rich sites, this species is used as a biological indicator of mercury contamination. With the aim to study genotoxic effect of mercury on this plant species, we analyzed genomic DNA of plants treated with 1 µM mercury for 15 days in aquarium. At the end of this treatment, plants accumulated elevated mercury concentrations in their leaves. After ultracentrifugation in a Cs 2 SO 4 gradient, P. oceanica genomic DNA profile showed, beside the main peak, a light shoulder containing A-T rich DNA sequences. This shoulder is missing in the profile of DNA isolated from mercury treated plants. Gradient slot-blot- ting and hybridization with these variable DNA families, labelled with digoxi- genin, indicated that these DNA sequences are at least partially lost in mercury treated plants. It was determined, by genomic DNA slot-blotting and hy- bridization, that DNA loss of A-T rich DNA sequences (forming the light shoulder) was around 48%.