New microsatellite markers for the endemic Mediterranean seagrass Posidonia oceanica

The seagrass Posidonia oceanica is endemic to the Mediterranean Sea, where it plays an important role in coastal ecosystem dynamics. Because of this important role and concerns about the observed regression of some meadows, population genetic studies of this species have been promoted. However, the markers used until now were not polymorphic enough to efficiently assess the level and spatial pattern of genetic variability. Hypervariable molecular markers were obtained by screening a genomic library enriched for microsatellite dinucleotide repeats. Among 25 primer pairs defined, eight amplified polymorphic microsatellites with an encouraging level of variability at the two geographical scales sampled.     

Mediterranean warming triggers seagrass (Posidonia oceanica) shoot mortality

Rapid warming of the Mediterranean Sea threatens marine biodiversity, particularly key ecosystems already stressed by other impacts such as Posidonia oceanica meadows. A 6‐year monitoring of seawater temperature and annual P. oceanica shoot demography at Cabrera Archipelago National Park (Balearic Islands, Western Mediterranean) allowed us to determine if warming influenced shoot mortality and recruitment rates of seagrasses growing in relative pristine environments. The average annual maximum temperature for 2002–2006 was 1 °C above temperatures recorded in 1988–1999 (26.6 °C), two heat waves impacted the region (with seawater warming up to 28.83 °C in 2003 and to 28.54 °C in 2006) and the cumulative temperature anomaly, above the 1988–1999 mean annual maximum temperature, during the growing season (i.e. degree‐days) ranged between 0 °C in 2002 and 70 °C in 2003. Median annual P. oceanica shoot mortality rates varied from 0.067 year^?1 in 2002 to 0.123 year^?1 in 2003, and exceeded recruitment rates in all stations and years except in shallow stations for year 2004. Interannual fluctuations in shoot recruitment were independent of seawater warming (P>0.05). P. oceanica meadows experienced a decline throughout the study period at an average rate of ?0.050±0.020 year^?1. Interannual variability in P. oceanica shoot mortality was coupled (R²>0.40) to seawater warming variability and increasing water depth: shoot mortality rates increased by 0.022 year^?1 (i.e. an additional 2% year^?1) for each additional degree of annual maximum temperature and by 0.001 year^?1 (i.e. 0.1% year^?1) for each accumulated degree water temperature remained above 26.6 °C during the growing season. These results demonstrate that P. oceanica meadows are highly vulnerable to warming, which can induce steep declines in shoot abundance as well indicating that climate change poses a significant threat to this important habitat.     

Properties of ion channels in the protoplasts of the Mediterranean seagrass Posidonia oceanica

Posidonia oceanica (L) Delile, a seagrass endemic of the Mediterranean sea, provides food and shelter to marine organisms. As environment contamination and variation in physico‐chemical parameters may compromise the survival of the few Posidonia genotypes living in the Mediterranean, comprehending the molecular mechanisms controlling Posidonia growth and development is increasingly important. In the present study the properties of ion channels in P. oceanica plasma membranes studied by the patch‐clamp technique in protoplasts obtained from the young non‐photosynthetic leaves were investigated. In protoplasts that were presumably originated from sheath cells surrounding the vascular bundles of the leaves, an outward‐rectifying time‐dependent channel with a single channel conductance of 58 ± 2 pS which did not inactivate, was selective for potassium and impermeable to monovalent cations such as Na⁺, Li⁺ and Cs⁺ was identified. In the same protoplasts, an inward‐rectifying channel that has a time‐dependent component with single channel conductance of the order of 10 pS, a marked selectivity for potassium and no permeation to sodium was also identified, as was a third type of channel that did not display any ionic selectivity and was reversibly inhibited by tetraethylammonium and lanthanum. A comparison of Posidonia channel characteristics with channels identified in terrestrial plants and other halophytic plants is included.     

Patch dynamics of the Mediterranean seagrass Posidonia oceanica: Implications for recolonisation process

Patch dynamics of the Mediterranean slow-growing seagrass Posidonia oceanica was studied in two shallow sites (3–10 m) of the Balearic Archipelago (Spain) through repeated censuses (1–2 year⁻¹). In the sheltered site of Es Port Bay (Cabrera Island), initial patch density (October 2001) was low: 0.05 patches m⁻², and the patch size (number of shoots) distribution was bimodal: most of the patches had less than 6 shoots or between 20 and 50 shoots. Mean patch recruitment in Es Port Bay (0.006 ± 0.002 patches m⁻² year⁻¹) exceeded mean patch loss (0.001 ± 0.001 patches m⁻² year⁻¹), yielding positive net patch recruitment (0.004 ± 0.003 patches m⁻² year⁻¹) and a slightly increased patch density 3 years later (July 2004, 0.06 patches m⁻²). In the exposed site of S’Estanyol, the initial patch density was higher (1.38 patches m⁻², August 2003), and patch size frequency decreased exponentially with size. Patch recruitment (0.26 patches m⁻² year⁻¹) and loss (0.24 patches m⁻² year⁻¹) were high, yielding a slightly increased patch density in the area 1 year later (October 2004, 1.40 patches m⁻²). Most recruited patches consisted of rooting vegetative fragments of 1–2 shoots. Seedling recruitment was observed in Summer 2004 at both sites. Episodic, seedling recruitment comprised 30% and 25% of total patch recruitment in Es Port Bay and S’Estanyol, respectively. Patch survival increased with patch size and no direct removal was observed among patches of 5 shoots or more. Most patches grew along the study, shifting patch distribution towards larger sizes. Within the size range studied (1–150 shoots), absolute shoot recruitment (shoots year⁻¹) increased linearly with patch size (R² = 0.64, p < 4 × 10⁻⁵, N = 125), while specific shoot recruitment was constant (about 0.25 ± 0.05 year⁻¹), although its variance was large for small patches. Given the slow growth rate and the high survival of patches with 5 or more shoots, even the low patch recruitment rates reported here could play a significant role in the colonisation process of P. oceanica.     

Spatial patterns of genetic diversity in Posidonia oceanica, an endemic Mediterranean seagrass

Posidonia oceanica is an endemic seagrass species in the Mediterranean Sea. In order to assess levels of genetic structure in this species, the microsatellite polymorphism was analysed from meadows collected in several localities, along the coasts of the Tyrrhenian Sea (Mediterranean Sea). The existence of single population units and the recruitment of seedlings collected in some localities were investigated. Moreover, genetic structure at different spatial scales and biogeographic relationships among populations were also assessed. Our analysis showed the existence of clear patterns of genetic structure in P. oceanica in the area considered in the analysis. P. oceanica, in fact, is present in separate meadows that represent discrete populations, characterized by low genetic diversity. Comparable levels of genetic variability between mature meadows and seedlings were found. Patterns of genetic relatedness among populations seem to be in accord with direction of dominant current flux in the whole area, separating South Tyrrhenian from North Tyrrhenian populations. Moderate levels of gene flow between populations and genetic substructure within populations, together with the finding of the limited role of sexual reproduction in increasing genetic variability, should be a cause for concern for the persistence of this essential resource in the Mediterranean basin.     

Are lulworthioid fungi dark septate endophytes of the dominant Mediterranean seagrass Posidonia oceanica?

• A previous study from Sicily, Italy, indicated that the dominant Mediterranean seagrass Posidonia oceanica forms a dark septate endophytic (DSE) association with a lulworthioid fungus (‘Lulwoana sp.’). This conflicts with several other studies from the NW Mediterranean Sea that point at the recently described pleosporalean fungus Posidoniomyces atricolor.
• I collected P. oceanica roots at eight sites around Sicily and checked them for fungal colonization using light microscopy. At three sites, root fungal symbionts (mycobionts) were isolated into pure cultures and identified using ITS rDNA sequences.
• Posidoniomyces atricolor represented the most frequent mycobiont (56 isolates), closely followed by lulworthioid fungi (51). The obtained mycobiont spectrum also included Cladosporium (2), Alternaria (1), Corollospora (1), Fusarium (1), Penicillium (1) and Vishniacozyma (1) isolates. The characteristic DSE root colonization, similar to those occurring in terrestrial plants but not known from any other seagrass, was found in all investigated P. oceanica individuals. The microscopy screening suggests that P. atricolor is responsible for the observed DSE colonization.
• This study extends the known range of Pos. atricolor and the DSE association characteristic for P. oceanica to the southern Tyrrhenian Sea/Sicily. While lulworthioid fungi regularly occur in P. oceanica tissues, including terminal fine roots, their significance and functioning (e.g. parasitic, pathogenic, endophytic) are unknown and require further investigation. However, there is currently no proof that they belong among dark septate endophytes of this seagrass.

     

Consequences of Mediterranean warming events in seagrass (Posidonia oceanica) flowering records

Posidonia oceanica, a seagrass endemic to the Mediterranean forms extended and extremely persistent meadows. It is a clonal plant with an apparently irregular pattern of flowering events. An extensive bibliographic review allowed the reconstruction of past flowering events of this species around the Mediterranean, with a high degree of confidence for the last 30 years. The data series on annual flowering prevalence (FP, flowering records per total records) and flowering intensity (FI, fraction of flowering shoots) produced have been compared with four series on Sea Surface annual Temperature maxima (SST_max) obtained for the NW Mediterranean (averaged from the local data series of l'Estartit and Villefranche: 1957–2005) and for the Eastern, Western basin and the whole Mediterranean sea (extracted from NCEP Reynolds interpolated SST maps: 1982–2005). Significant warming trends are detected in the Mediterranean SST_max series, at a rate of (mean+SE) 0.04±0.01°C yr⁻¹ (R²=0.24, P<0.01, N=24 years), in the Eastern basin series (0.06±0.01°C yr⁻¹, R²=0.43, P<0.001, N=24 years) and in the long SST_max series of the NW Mediterranean (0.02±0.01 C yr⁻¹, R²=0.12, P<0.02, N=49 years). The magnitudes of the SST_max anomalies around the absolute warming trend do not increase with time in any SST_max series. Peaks of FP and FI in the Mediterranean seem to occur each 9–11 years, and coincide with peaks of annual SST_max. Annual FP and FI increase with the residuals of annual SST_max warming trend in all Mediterranean basins (FP_MED: R²=0.27, P<0.01, N=23; FP_NW: R²=0.34, P<0.01, N=31; FP_E: R²=0.20; P<0.10, N=23). An outstanding event of P. oceanica flowering across the Mediterranean has been registered in Autumn 2003; 1 month after the highest annual SST_max recorded in the series. The hypothesis of flowering induction by thermal stress as the possible cause of this relationship is discussed, as well as the potential use of P. oceanica flowering record as early indicator of biological change induced by global sea warming in Mediterranean marine ecosystems.     

Genetic structure in the Mediterranean seagrass Posidonia oceanica: disentangling past vicariance events from contemporary patterns of gene flow

The Mediterranean Sea is a two‐basin system, with the boundary zone restricted to the Strait of Sicily and the narrow Strait of Messina. Two main population groups are recognized in the Mediterranean endemic seagrass Posidonia oceanica, corresponding to the Western and the Eastern basins. To address the nature of the East–West cleavage in P. oceanica, the main aims of this study were: (i) to define the genetic structure within the potential contact zone (i.e. the Strait of Sicily) and clarify the extent of gene flow between the two population groups, and (ii) to investigate the role of present water circulation patterns vs. past evolutionary events on the observed genetic pattern. To achieve these goals, we utilized SSR markers and we simulated, with respect to current regime, the possible present‐day dispersal pattern of Posidonia floating fruits using 28‐day numerical Lagrangian trajectories. The results obtained confirm the presence of the two main population groups, without any indices of reproductive isolation, with the break zone located at the level of the Southern tip of Calabria. The populations in the Strait of Sicily showed higher affinity with Western than with Eastern populations. This pattern of genetic structure probably reflects historical avenues of recolonization from relict glacial areas and past vicariance events, but seems to persist as a result of the low connectivity among populations via marine currents, as suggested by our dispersal simulation analysis.     

Random amplified polymorphic DNA assessment of diversity in western Mediterranean populations of the seagrass Posidonia oceanica

Posidonia oceanica is an endemic Mediterranean seagrass species that has often been assumed to contain low levels of genetic diversity. Random amplified polymorfic DNA (RAPD) markers were used to assess genetic diversity among five populations from three geographical regions (north, central, and south) of the western Mediterranean Sea. Stranded germinating seeds from one of the central populations were also included in the analysis. Forty-one putative genets were identified among 76 ramets based on 28 RAPD markers. Genotypic diversity strongly depended on the spatial structure, age, and maturity of the meadows. The lowest clonal diversity was found in the less structured and youngest prairies. Conversely, a high genotypic diversity was found in the highly structured meadows. The genotypic diversity in these meadows was at the same level as in P. australis and higher than previously reported data for P. oceanica populations in the Tyrrhenian Sea near the coast of Italy.     

Mercury and non-protein thiol compounds in the seagrass Posidonia oceanica

Mercury concentrations, non-protein thiol levels and the enzyme activities of glutathione-S-transferase (GST) were measured in the blades and sheaths of the marine phanerogam Posidonia oceanica. The seagrass was collected in January and June and at three sites: the Bay of Rosignano (Italy) known for its mercury contamination, the north of the Lérins islands (Bay of Cannes, France), the Bay of Tonnara (Corsica, France). The two latter sites are considered as free of any known industrial inputs. Mercury concentrations and GST activities in both tissues were always higher in samples from Rosignano, particularly in June. Non-protein thiol levels were significantly higher in the blades than in the sheaths of P. oceanica from Tonnara and Lérins. In contrast, at Rosignano, the sheaths presented a significantly higher non-protein thiol concentration than the blades, particularly in June. Levels in the sheaths appeared to increase with the degree of pollution. Western Blot performed on sheaths of P. oceanica collected in June at Rosignano and Lérins revealed a characteristic band of GSTs at 31 kDa, proving the presence of the GST enzyme in this tissue. Mercury seemed to exert an influence upon non-protein thiol metabolism, including GST induction, in P. oceanica collected from the NW Mediterranean.     

Unexpected response of the seagrass Posidonia oceanica to a warm-water episode in the north western Mediterranean Sea

The response of Posidonia oceanica (Linnaeus) Delile to the warm-water episode of summer 1999 was studied by means of the technique of lepidochronology. Study sites include three sites affected by the mass mortality event of benthic invertebrates and one not affected. The results showed a significant decline in some parameters (number of leaves and/or rhizome growth) for the three sites affected by the mass mortality event for the year following the warm-water episode (1999-2000). A similar decline was not observed for the unaffected site. The fact that high temperatures could have a negative impact on deep Posidonia oceanica near its cold limit of distribution is an unexpected result.     

On the unusual flowering of plagiotropic shoots in the seagrass Posidonia oceanica

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.     

Sulfur cycling and seagrass (Posidonia oceanica) status in carbonate sediments

Sulfur cycling was investigated in carbonate-rich and iron-poor sediments vegetated with Posidonia oceanica in oligotrophic Mediterranean around Mallorca Island, Spain, to quantify sulfate reduction and pools of sulfide in seagrass sediments. The oxygen penetration depth was low (< 4.5 mm) and sulfate reduction rates were relatively high (0.7–12 mmol m^–2d^–1). The total pools of reduced sulfides were remarkably low (< 5 mol S m^–2) indicating a fast turnover of reduced sulfides in these iron-poor sediments. The sulfate reduction rates were generally higher in vegetated compared to bare sediments possible due to enhanced sedimentation of sestonic material inside the seagrass meadows. The sulfate reduction rates were positively correlated with the seasonal variation in water temperature and negatively correlated with the shoot density indicating that the microbial activity was controlled by temperature and release of oxygen from the roots. The pools of reduced sulfides were low in these iron-poor sediments leading to high oxygen consumption for reoxidation. The sediments were highly anoxic as shown by relatively low oxygen penetration depths (< 4.5 mm) in these low organic sediments. The net shoot recruitment rate was negative in sediments enriched with organic matter, suggesting that organic matter enrichment may be an important factor for seagrass status in these iron-depleted carbonate sediments.     

Vicariance patterns in the Mediterranean Sea: east–west cleavage and low dispersal in the endemic seagrass Posidonia oceanica

Aim The seagrass, Posidonia oceanica is a clonal angiosperm endemic to the Mediterranean Sea. Previous studies have suggested that clonal growth is far greater than sexual recruitment and thus leads to low clonal diversity within meadows. However, recently developed microsatellite markers indicate that there are many different genotypes, and therefore many distinct clones present. The low resolution of markers used in the past limited our ability to estimate clonality and assess the individual level. New high‐resolution dinucleotide microsatellites now allow genetically distinct individuals to be identified, enabling more reliable estimation of population genetic parameters across the Mediterranean Basin. We investigated the biogeography and dispersal of P. oceanica at various spatial scales in order to assess the influence of different evolutionary factors shaping the distribution of genetic diversity in this species. Location The Mediterranean. Methods We used seven hypervariable microsatellite markers, in addition to the five previously existing markers, to describe the spatial distribution of genetic variability in 34 meadows spread throughout the Mediterranean, on the basis of an average of 35.6 (± 6.3) ramets sampled. Results At the scale of the Mediterranean Sea as a whole, a strong east–west cleavage was detected (amova) . These results are in line with those obtained using previous markers. The new results showed the presence of a putative secondary contact zone at the Siculo‐Tunisian Strait, which exhibited high allelic richness and shared alleles absent from the eastern and western basins. F statistics (pairwise θ ranges between 0.09 and 0.71) revealed high genetic structure between meadows, both at a small scale (about 2 to 200 km) and at a medium scale within the eastern and western basins, independent of geographical distance. At the intrameadow scale, significant spatial autocorrelation in six out of 15 locations revealed that dispersal can be restricted to the scale of a few metres. Main conclusions A stochastic pattern of effective migration due to low population size, turnover and seed survival is the most likely explanation for this pattern of highly restricted gene flow, despite the importance of an a priori seed dispersal potential. The east–west cleavage probably represents the outline of vicariance caused by the last Pleistocene ice age and maintained to this day by low gene flow. These results emphasize the diversity of evolutionary processes shaping the genetic structure at different spatial scales.     

Genetic Polymorphism and Transplantation Success in the Mediterranean Seagrass Posidonia oceanica

We studied the role of genetic variability of donor beds in establishing transplantation criteria for the Mediterranean seagrass Posidonia oceanica. Horizontal rhizomes, collected from three geographically distinct populations, were transplanted into a common bed at a highly human‐impacted locality. The transplantation site was located near one of the donor populations. After three years, the shoots collected in the population adjacent to the transplanting site showed the lowest growth performance. Genetic variability, assessed through the analysis of hypervariable microsatellite regions, and growth performance followed a similar trend. The shoots growing and branching at the highest rate were those collected from populations with the highest heterozygosity values, despite greater geographic distance. No genetic differences were found between the transplanted shoots and shoots from donor meadows, as expected due to the low rate of sexual reproduction in P. oceanica and the short time that had passed since the transplants. The problem of affecting the local gene pool by the introduction of foreign genotypes could arise, but introduction of new alleles could balance the degradation of genetic variability caused by human impact. In general our study suggests that the genetic variability of source material is an important aspect to consider in the development of seagrass restoration strategies.