Gone with the waves: the role of sea currents as key dispersal mechanism for Mediterranean coastal and inland plant species.

Thalassochory, the dispersal of propagules through marine currents, is a key long-distance dispersal (LDD) mechanism with implications for global biogeography and particularly for island colonization. The propagules of coastal plant species are generally assumed to be better adapted for sea dispersal than those of inland plants, but this hypothesis remains largely untested. We conducted experiments on four genera (Juniperus, Daucus, Ferula, and Pancratium) and compared traits among nine species with different habitats and distributions. Our results showed that Juniperus spp. and P. maritimum have strong thalassochorous potential within the Mediterranean Basin. Interestingly, we did not find a clear association on the thalassochorous potential of coastal versus inland species within all the tested genera, apart from P. maritimum compared with the endemic inland P. illyricum. These findings suggest that thalassochory may be a more common dispersal mechanism than previously assumed. The apparently weak link of dispersal syndrome with species ecology broadens the possibility of dispersal by the sea also for inland plants, although considered to be poorly salt-tolerant. Moreover, our results reveal significant differences in sea dispersal between endemic and widespread species, but do not rule out an important role of thalassochory in shaping the distribution patterns of archipelago endemic flora. The presented method is largely replicable and could be used for further studies with a larger set of species to better delineate trends of sea dispersal syndrome among species with different ecology or dispersal traits.

Is vegetation an indicator for evaluating the impact of tourism on the conservation status of Mediterranean coastal dunes?

Mediterranean coastal dunes are threatened by several factors; particularly, tourism causes modifications to the vegetation and the disappearance of endemic species. Understanding the dunes' conservation status is crucial for preserving these vulnerable environments through appropriate management strategies. This study was conducted on 17 Sardinian coastal dunes, with different levels of touristic pressure. We focused on endemic plant species and developed a new endemicity index (EI). Our study aimed: 1) to assess the conservation status by applying the diversity indices; 2) to verify if the study sites would reveal a general pattern based on different degrees of human disturbance and 3) to test the effectiveness of the EI index. Four m2 plots (2 × 2 m) were placed along orthogonal transects to the coastline (446 plots in total), in which all plant species were identified, and their relative abundance was estimated. We found significant differences among the sites for Hdune and EI values but no statistically significant differences in the N values. The EI showed the high naturalistic value of Sardinian coastal dunes and allowed us to distinguish the sites with higher anthropic pressure. We found significant differences in the indices among the degrees of human disturbance in the coastal systems. The Hdune values were positively related to a medium level of human disturbance, and the EI allowed us to distinguish the sites with varying levels of human disturbance, although it differentiated better those with the highest anthropic pressure. A medium level of human disturbance was positively related to the plant richness and cover, and human trampling could be tolerated by psammophilous vascular plants. Results showed a satisfactory conservation status of Sardinian dune systems and highlighted diversity indices as valuable support for implementing a conservation strategy, compatible with the tourism purposes and the integrated management of the Mediterranean coastal dune systems.

Seed traits and germination behaviour of four Sardinian populations of Helichrysum microphyllum subsp. tyrrhenicum (Asteraceae) along an altitudinal gradient.

Helichrysum microphyllum subsp. tyrrhenicum (Asteraceae) is an endemic taxon of Sardinia and Corsica, where it grows at different altitudes. The objective of this study was to investigate the seed traits and germination behaviour of four Sardinian populations of this taxon located at different altitudes. Seed traits were evaluated, and germination tests were carried out by incubating seeds at a range of constant (5-30 °C) and alternating (25/10 °C) temperatures. The dry after-ripening (DAR) pre-treatment was also applied by storing seed in dry conditions for 3 months at 25 °C. Seed traits and germination behaviour data were statistically analysed to identify if there was a correlation with altitude. Differences in seed size, area and mass among populations were recorded, however, no relationship was found with altitude. High germination percentages were obtained in all populations, both in untreated and DAR seeds, and were positively affected by alternating temperatures. The final germination percentage and time required to reach 50% final germination (T50 ) showed no relationship with altitude. The differences in seed traits and germination detected among the studied populations of H. microphyllum subsp. tyrrhenicum were not correlated with altitude. This study provides new and important knowledge for this taxon. H. microphyllum subsp. tyrrhenicum is characterised by high germination percentages and low T50 values and does not seem to require any dormancy-breaking treatment. This species represents a high-potential native plant species that should be considered within environmental management plans.

Metal Tolerance Capability of Helichrysum microphyllum Cambess. subsp. tyrrhenicum Bacch., Brullo & Giusso: A Candidate for Phytostabilization in Abandoned Mine Sites.

Sardinia was known as an important mine pole in Europe during his history. Still after decades from mine closure, 75.000.000 m3 of mine waste, rich in heavy metals, were left abandoned causing a huge environmental legacy on the mine district area. Consequently, cost effective remediation is required. In this frame, phytoremediation is considered a feasible candidate. This research was focused on Helichrysum microphyllum subsp. tyrrhenicum, which is pioneer in xeric soils with low-functions, like mine tailings. The aim of this study was to evaluate its ability to extract heavy metals from mine soils and accumulate them in plant tissues and its suitability for phytostabilization. Sundry samples of soil, roots and epigean organ were collected through field sampling and analysed in order to obtain metals concentration and mineralogical characteristics. Our results indicate that this species tolerates high concentration of zinc, lead and cadmium, behaving as a species suitable for phytostabilization.

Inter- and intra-variability of seed germination traits of Carpobrotus edulis N.E.Br. and its hybrid C. affine acinaciformis.

Invasions by alien Carpobrotus spp. have been recognised as one of the most severe threats to Mediterranean climate coastal ecosystems, and Carpobrotus is considered one of the most widespread invasive alien genera in the Mediterranean Basin. The aims of this study were to characterise seed germination of both C. edulis and its hybrid C. aff. acinaciformis, in terms of photoperiod, temperature and salinity. Inter- and intra-specific variability in the responses to photoperiod (12/12 h light and total darkness), constant temperatures (5, 10, 15, 20, 25 °C) and an alternating temperature regime (25/10 °C), salt stress (0, 125, 250, 500 mm NaCl) and the recovery of seed germination were evaluated for two seed lots of C. edulis and two of its hybrid C. aff. acinaciformis. All the tested seed lots achieved higher germination percentages in the light, with respect to total darkness. In relation to temperature, the two C. edulis seed lots did not show a preference, while the two C. aff. acinaciformis seed lots differed in their germination response, one germinating more at the lowest temperatures (5 and 10 °C) and one at the highest (20 and 25 °C). For all seed lots, highest germination occurred without NaCl (0 mm) and germination decreased with increasing salinity. Different germination requirements in a saline substrate were not detected for C. edulis, while they were observed for C. aff. acinaciformis. Marked differences were detected in recovery responses between the two taxa. C. edulis has the ability to germinate over a wide time window throughout the year. This study identified significant differences in seed production, seed mass, germination requirements (temperature) and salinity tolerance for both C. edulis and C. aff. acinaciformis. Our results indicate the extreme versatility of the hybrid forms to germinate in a wide range of natural conditions and habitats.

Dissecting seed dormancy and germination in Aquilegia barbaricina, through thermal kinetics of embryo growth.

Threshold-based thermal time models provide insight into the physiological switch from the dormant to the non-dormant germinating seed. This approach was used to quantify the different growth responses of the embryo of seeds purported to have morphophysiological dormancy (MPD) through the complex phases of dormancy release and germination. Aquilegia barbaricina seeds were incubated at constant temperatures (10-25 °C) and 25/10 °C, without pre-treatment, after warm+cold stratification (W+C) and GA3 treatment. Embryo growth was assessed and the time of testa and endosperm rupture scored. Base temperatures (Tb ) and thermal times for 50% (θ50 ) of embryo growth and seed germination were calculated. W+C enabled slow embryo growth. W+C and GA3 promoted rapid embryo growth and subsequent radicle emergence. The embryo internal growth base temperature (Tbe ) was ca. 5 °C for W+C and GA3 -treated seeds. GA3 treatment also resulted in similar Tb estimates for radicle emergence. The thermal times for embryo growth (θe50 ) and germination (θg50 ) were four- to six-fold longer in the presence of GA3 compared to W+C. A. barbaricina is characterised by a multi-step seed germination. The slow embryo growth during W+C reflects continuation of the maternal programme of development, whilst the thermal kinetics of both embryo and radicle growth after the removal of physiological dormancy are distinctly different. The effects of W+C on the multiphasic germination response in MPD seeds are only partially mimicked by 250 mg·l-1 GA3 . The thermal time approach could be a valid tool to model thermal kinetics of embryo growth and radicle protrusion.

Confirmed mixed bird-insect pollination system of Scrophularia trifoliata L., a Tyrrhenian species with corolla spots.

Both bird and mixed vertebrate-insect (MVI) pollination systems are very rare in Europe and the Mediterranean region. Because MVI can ensure reproduction over a wider range of environmental conditions than when insects are the sole pollinators, under certain circumstances such systems are highly advantageous to plants. Here, we investigated the pollination and some reproductive traits of the Tyrrhenian Scrophularia trifoliata, the only species of the genus possessing two showy dark spots inside the corolla, for which MVI pollination system had been inferred on the basis of limited censuses. We conducted field experiments to study MVI pollination and some reproductive traits and elucidate the role of corolla spots, analysing their ultraviolet pattern, histology and pigments versus the rest of the corolla. The primary pollinators were wasps and passerine birds. Corolla spots absorb UV light, present abundant anthocyanins and are histologically identical to the rest of the corolla. Control flowers had higher visitation frequency than flowers without spots. S. trifoliata is self-compatible, with efficient intrafloral protogyny and herkogamy that prevent self-pollination but not geitonogamy. We confirmed the existence of a mixed bird-insect pollination system in S. trifoliata. This system is found in three other Scrophularia species with large, showy flowers - two Mediterranean (S. grandiflora and S. sambucifolia) and one Macaronesian (S. calliantha). Unlike those species, S. trifoliata has two large spots inside the corolla. These likely operate as nectar guides and their dark colouration is related to abundant anthocyanin content.

Morpho-colorimetric characterisation of Malva alliance taxa by seed image analysis.

Seed morphometric and -colorimetric features describing shape, size and textural seed traits of 28 taxa belonging to the genera Lavatera L. and Malva L., were recorded by means of computer vision techniques. The data were statistically analysed to contribute to the taxonomical treatment of the Malva alliance and to assess some doubtful systematic positions. A clear differentiation between taxa traditionally attributed to Lavatera or Malva was highlighted. Furthermore, the identification system proposed here was able to discriminate among the Lavatera sections, confirming the taxonomic organization of this genus. The results obtained for Malva, both at the species level and among sections, supported this analytical tool as diagnostic for systematic purposes.

Bioaugmentation-Assisted Phytostabilisation of Abandoned Mine Sites in South West Sardinia.

Bioaugmentation-assisted phytoremediation implies the administration of selected plant growth promoting bacteria, which significantly improve plant growth and sequestration of heavy metals. In this work, 184 bacterial strains associated with roots of Pistacia lentiscus were isolated from plants spontaneously growing in the abandoned Sardinian mining areas (SW Sardinia, Italy) and phylogenetically characterised. Twenty-one bacterial isolates were assayed for properties relevant for plant growth promotion and metal tolerance. Five different strains, belonging to the genera Novosphingobium, Variovorax, Streptomyces, Amycolatopsis, Pseudomonas, were selected based on their properties for the greenhouse phytoremediation tests. Among the tested inocula, the strain Variovorax sp. RA128A, able to produce ACC deaminase and siderophore, was able to significantly enhance germination and increase length and weight of shoots and roots. Irrespective of the applied treatment, mastic shrub was able to accumulate Cd, Pb and Zn especially in roots.

Effects of NaCl stress on seed germination and seedling development of Brassica insularis Moris (Brassicaceae).

Brassica insularis is a protected plant that grows on both coastal and inland cliffs in the western Mediterranean Basin. The objective of this study was to test if any variability exists in the salt stress response during seed germination and seedling development in this species relative to its provenance habitat. Variability among three populations in the salt stress effects on seed germination and recovery under different temperatures was evaluated. The effect of nebulisation of a salt solution on seedling development was evaluated between populations growing at different distances from the sea. Seeds of B. insularis could germinate at NaCl concentrations up to 200 mm. Seed viability was negatively affected by salt, and recovery ability decreased with increasing temperature or salinity. Inter-population variability was detected in salt response during the seed germination phase, as well as in seedling salt spray tolerance. The inland population seedlings had drastically decreased survival and life span and failed to survive to the end of the experiment. In contrast, at least 90% of the coastal seedlings survived, even when sprayed at the highest frequency with salt solution. This study allowed investigation of two natural factors, soil salinity and marine aerosols, widely present in the B. insularis habitat, and provided the first insights into ecology of this protected species and its distribution in the Mediterranean. These results might be useful in understanding the actual distributions of other species with the same ecology that experience these same abiotic parameters.

Inter- and intraspecific diversity in Cistus L. (Cistaceae) seeds, analysed with computer vision techniques.

This work aims to discriminate among different species of the genus Cistus, using seed parameters and following the scientific plant names included as accepted in The Plant List. Also, the intraspecific phenotypic differentiation of C. creticus, through comparison with three subspecies (C. creticus subsp. creticus, C. c. subsp. eriocephalus and C. c. subsp. corsicus), as well as the interpopulation variability among five C. creticus subsp. eriocephalus populations was evaluated. Seed mean weight and 137 morphocolorimetric quantitative variables, describing shape, size, colour and textural seed traits, were measured using image analysis techniques. Measured data were analysed applying step-wise linear discriminant analysis. An overall cross-validated classification performance of 80.6% was recorded at species level. With regard to C. creticus, as case study, percentages of correct discrimination of 96.7% and 99.6% were achieved at intraspecific and interpopulation levels, respectively. In this classification model, the relevance of the colorimetric and textural descriptive features was highlighted, as well as the seed mean weight, which was the most discriminant feature at specific and intraspecific level. These achievements proved the ability of the image analysis system as highly diagnostic for systematic purposes and confirm that seeds in the genus Cistus have important diagnostic value.

Conservation genetics of two island endemic Ribes spp. (Grossulariaceae) of Sardinia: survival or extinction?

Measuring levels of population genetic diversity is an important step for assessing the conservation status of rare or endangered plant species and implementing appropriate conservation strategies. Populations of Ribes multiflorum subsp. sandalioticum and R. sardoum, two endangered endemic species from Sardinia, representing the whole genus on the island, were investigated using ISSR and SSR markers to determine levels and structure of genetic variability in their natural populations. Results indicated medium to low genetic diversity at the population level: Nei's gene diversity for ISSR markers ranged from 0.0840 to 0.1316; the expected heterozygosity (HE ) for SSR ranged from 0.4281 to 0.7012. In addition, only one remnant population of R. sardoum showed a high level of inbreeding, in accordance with its very small size. Regarding the structure of the six R. sandalioticum populations, both principal coordinates analysis (PCoA) and STRUCTURE analysis of ISSR and SSR data highlighted low population structure, although two populations appeared to be clearly distinct from the others. The genetic pattern of the two taxa associated with their different ecological positions indicated resilience of R. sandalioticum populations in fresh and humid habitats and uncertain future resistance for the residual R. sardoum population in xeric calcareous stands. Hence, this study highlights the importance of an integrated conservation approach (genetic plus in situ and ex situ conservation studies/measures) for activating management programmes in these endemic and threatened taxa that can be considered as crop wild relatives of cultivated Ribes species.

Use of native plants for the remediation of abandoned mine sites in Mediterranean semiarid environments.

Abandoned tailing dumps from mining industry represent important sources of metal contamination in the surrounding environments. This study evaluates the potential of two Mediterranean native plants, Pistacia lentiscus and Phragmites australis, for phytoremediation of two Sardinian contaminated mine sites. A 6 months study has been conducted at greenhouse-controlled conditions with the aim of investigating the plant capability to tolerate high metal concentrations and to extract or immobilize them within the roots. The possibility to mitigate stress on the plants and improve treatment efficiency by adding compost as amendment was also evaluated. Both species were able to restrict accumulation of Cd, Pb and Zn to the root tissues exhibiting a metal concentration ratio of plant roots to soil bioavailable fraction higher than two (four in the case of Zn). However, the two species showed different adaptation responses, being the survival of P. australis after 6 months in contaminated soil lower (25 %-58 %) than that observed for P. lentiscus (77 %-100 %). Compost addition resulted in a lower metal uptake in tissues of both plants and a higher survival of P. australis, whilst almost no effect was observed as regard the growth of both species. The two tested species appear to be promising candidates for phytostabilization, P. lentiscus exhibiting a greater adaptability to heavy metal contaminated matrices than P. australis.

Morpho-colorimetric analysis and seed germination of Brassica insularis Moris (Brassicaceae) populations.

Brassica insularis is a perennial plant growing on both coastal and inland cliffs. Three seed lots from Sardinia were analysed using an image analysis system to detect differences in seed morphology, both within and among populations. Germination requirements at constant (5-25 °C) and alternating temperatures (25/10 °C), both in light and in darkness, were evaluated for all populations. In addition, the effect of a dry after-ripening period (90 days at 25 °C) was also investigated. Morpho-colorimetric analysis clearly identified seeds from different populations and discriminated three chromatic categories for seeds belonging to the Isola dei Cavoli coastal population, but not for the inland Masùa and the coastal Planu Sartu. Inter-population variability was also observed in germination behaviour. B. insularis seeds germinated, with percentages up to 60%, in a wide range of temperatures (5-25 °C), and neither light nor dry after-ripening affected final germination percentages. Moisture content measurements were made for seeds of each colour, but there were no particular differences among colours. Inter-populational variability in germination behaviour may be a survival strategy for species growing under unpredictable environmental conditions, such as under Mediterranean climate, while heteromorphy may be due to independent evolutionary divergence processes of the Isola dei Cavoli population.

The genetic diversity and spatial genetic structure of the Corso-Sardinian endemic Ferula arrigonii Bocchieri (Apiaceae).

Corsica and Sardinia represent major hotspots of plant diversity in the Mediterranean area and are priority regions for conservation due to their high number of endemic plant species. However, information supporting human decision-making on the conservation of these species is still scarce, especially at the genetic level. In this work, the first assessment is reported of the species-wide spatial genetic structure and diversity of Ferula arrigonii Bocchieri, a Corso-Sardinian endemic located in a few coastal sites and on small islands. Nine populations covering the entire natural range of the species were investigated by means of AFLP (amplified fragment length polymorphism) markers. Results indicate that this species is characterised by high levels of genetic polymorphism (92% polymorphic fragments) and of genetic diversity (H(w) = 0.317) and by relatively low differentiation among populations (F(st) = 0.057). PCoA, Bayesian analysis and neighbour-joining clustering were also employed to investigate the genetic structure of this species. Three genetically distinct groups were detected, although with considerable overlap between populations.

Spatial genetic structure of Aquilegia taxa endemic to the island of Sardinia.

BACKGROUND AND AIMS: The Mediterranean Basin is one of the most important regions for the Earth's plant biodiversity; however, the scarcity of studies on fine scale patterns of genetic variation in this region is striking. Here, an assessment is made of the spatial genetic structure of all known locations of the three Sardinian endemic species of Aquilegia in order to determine the relative roles of gene flow and genetic drift as underlying evolutionary forces canalizing the divergence of Sardinian Aquilegia taxa, and to see if the spatial genetic structure found fits the current taxonomic differentiation of these taxa. METHODS: DNA from 89 individuals from all known locations of Aquilegia across Sardinia was analysed by means of amplified fragment length polymorphism (AFLP) markers. Both principal co-ordinates analysis (PCoA) and Bayesian clustering analyses were used to determine the spatial genetic structure irrespective of any taxonomic affiliation. Historical effects of gene flow and genetic drift were assessed by checking for the existence of isolation-by-distance patterns. KEY RESULTS: STRUCTURE and PCoA analyses revealed a pattern of genetic variation geographically structured into four spatial genetic groups. No migration-drift equilibrium was detected for Aquilegia in Sardinia, when analysed either as a whole or in individual groups. The scenario approached a Case III pattern sensu Hutchinson and Templeton, which is associated with extreme isolation conditions where genetic drift has historically played a dominant role over gene flow. CONCLUSIONS: The pattern of genetic variation of Sardinian taxa of Aquilegia indicates that genetic drift has been historically more influential than gene flow on population structure of Sardinian species of Aquilegia. Limited seed dispersal and divergent selection imposed by habitat conditions have been probably the main causes reinforcing post-Pleistocene geographical isolation of Aquilegia populations. The spatial genetic structure found here is not fully compatible with current taxonomic affiliations of Sardinian Aquilegia taxa. This is probably a consequence of the uncoupling between morphological and genetic patterns of differentiation frequently found in recently radiated taxa.

Interchangeable effects of gibberellic acid and temperature on embryo growth, seed germination and epicotyl emergence in Ribes multiflorum ssp. sandalioticum (Grossulariaceae).

Morphophysiological dormancy was investigated in seeds of Ribes multiflorum Kit ex Roem et Schult. ssp. sandalioticum Arrigoni, a rare mountain species endemic to Sardinia (Italy). There were no differences in imbibition rates between intact and scarified seeds, suggesting a lack of physical dormancy, while methylene blue solution (0.5%) highlighted a preferential pathway for solution entrance through the raphe. Embryos were small at seed dispersal, with an initial embryo:seed ratio (E:S) of ca. 0.2 (embryo length, ca. 0.5 mm), whereas the critical E:S ratio for germination was three times longer (ca. 0.6). Gibberellic acid (GA(3), 250 mg · l(-1)) and warm stratification (25 °C for 3 months) followed by low temperature (<15 °C) enhanced embryo growth rate (maximum of ca. 0.04 mm · day(-1) at 10 °C) and subsequent seed germination (radicle emergence; ca. 80% at 10 °C). Low germination occurred at warmer temperatures, and cold stratification (5 °C for 3 months) induced secondary dormancy. After radicle emergence, epicotyl emergence was delayed for ca. 2 months for seeds from three different populations. Mean time of epicotyl emergence was affected by GA(3) . Seeds of this species showed non-deep simple (root) - non-deep simple (epicotyl) morphophysiological dormancy, highlighting a high synchronisation with Mediterranean seasonality in all the investigated populations.

[Esophageal function remains unchanged in systemic sclerosis at four years of follow-up]. / La funzionalità esofagea rimane inalterata nella sclerosi sistematica ad un follow-up di 4 anni.

AIM: Esophageal involvement is one of the earliest gastrointestinal manifestations of systemic sclerosis. Reduced lower esophageal sphincter pressure and reduced esophageal motility can both be measured by manometry. METHODS: 15 patients with systemic sclerosis (6 with altered manometric values and 9 with symptoms of pyrosis and/or dysphagia but with normal manometric values) were reassessed after a follow-up period of over 46 months. RESULTS: Low though still within normal range values in lower esophageal pressure were found on manometry. Distal esophageal amplitude remained substantially unchanged. Only in one patient was the amplitude significantly reduced. In all patients, the symptom scores for pyrosis and dysphagia had worsened slightly. CONCLUSIONS: Esophageal involvement does not necessarily occur in all patients with systemic sclerosis; more time may be required before its manifestations can be assessed by esophageal manometry.