Morphological and functional seed traits of the wild medicinal plant Dioscorea strydomiana, the most threatened yam in the world.

Morphological and functional seed traits have important roles in characterising the species regeneration niche and help to understand the reproductive biology of rare and threatened plants, which can thus support appropriate plant conservation measures. Seed morphometric and dispersal kinetics of the critically endangered Dioscorea strydomiana were measured and compared with those of four other Dioscorea species, and seed germination response under constant temperatures (5-35 °C) was compared with that of the congeneric and widespread D. sylvatica. Seed mass of D. strydomiana (ca. 14 mg) was twice that of D. sylvatica, but similar to or smaller than the other species examined. Seeds of D. strydomiana have the lowest speed of descent and lowest variability in most of the morphological traits considered, suggesting lower phenotypic plasticity but higher variance in the wing-loading value. Seeds of D. strydomiana reached maximum germination at 15 °C (ca. 47%), which decreased slightly to ca. 37% at 25 °C and was completely inhibited at 35 °C. D. sylvatica seeds started to germinate at 10 °C (ca. 3%), reached 75-80% germination at 15-20 °C and maximum (ca. 90%) at 25-30 °C. Base temperatures for germination (Tb ) were 9.3 and 5.7 °C, for D. strydomiana and D. sylvatica, respectively. Due to the higher germination percentages of D. sylvatica, ceiling and optimum temperatures could also be modelled for this species, suggesting higher sensitivity to high temperature for seeds of D. strydomiana. The detected poor seed lot quality of D. strydomiana suggests difficulties in reproduction from seed, highlighting the need for further investigation and conservation actions for this threatened yam species.

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.

Understanding biological and ecological factors affecting seed germination of the multipurpose tree Anogeissus leiocarpa.

Anogeissus leiocarpa (DC.) Guill. & Perr. (Combretaceae) has important economic and cultural value in West Africa as source of wood, dye and medicine. Although this tree is in high demand by local communities, its planting remains limited due to its very low propagation via seed. In this study, X-rays were used to select filled fruits in order to characterise their morphology and seed germination responses to treatment with sulphuric acid and different incubation temperatures. Morphological observations highlighted a straight orthotropous seed structure. The increase in mass detected for both intact and scarified fruits through imbibition tests, as well as morphological observations of fruits soaked in methylene blue solution, confirmed that they are water-permeable, although acid-scarified fruits reached significantly higher mass increment values than intact ones. Acid scarification (10 min soaking in 98% H2 SO4 ) positively affected seed germination rate but not final germination proportions. When intact fruits where incubated at a range of temperatures, no seeds germinated at 10 °C, while maximum seed germination (ca. 80%) was reached at 20 °C. T50 values ranged from a minimum of ca. 12 days at 25 °C to a maximum of ca. 34 days at 15 and 35 °C. A theoretical base temperature for germination (Tb ) of ca. 10 °C and a thermal requirement for 50% germination (S) of ca. 195 °Cd were also identified for intact fruits. The results of this study revealed the seed germination characteristics driven by fruit and seed morphology of this species, which will help in its wider propagation in plantations.

Integration of genetic and seed fitness data to the conservation of isolated subpopulations of the Mediterranean plant Malcolmia littorea.

Plant autoecology and population genetics provide a perspective on the likelihood of natural regeneration, which is critical when designing conservation strategies for endangered species. The threatened coastal plant Malcolmia littorea (Brassicaceae) was sampled across its European distribution and studied for genetic diversity and seed fitness, with the aim of providing information for the conservation of isolated and declining populations. Nine microsatellite markers (five chloroplast and four nuclear) were analysed to assess population genetic diversity and structure and to conduct a spatial analysis using the software DIVA-GIS. Germination percentages and rates were assessed by incubating the seeds under eight constant temperatures (0-27 °C). The genetic diversity was found to be similar among subpopulations (chloroplast H = 0.04-0.17; nuclear Ho  = 0.20-0.37), with no correlation between subpopulation diversity and the area of occupancy (AOO). The subpopulations were found to be clustered in three genetic groups, and three of them were identified as conservation priorities due to their unique genetic composition. The germination study revealed a significant influence of the maternal environment and AOO on seed germination, with the smaller subpopulations showing lower germination percentages (P < 0.05). These results highlight the importance of obtaining information on isolated subpopulations through different experimental approaches (e.g. seed germination plus population genetics) to enable planning of effective conservation actions. For M. littorea, seed collection for both in situ and ex situ conservation should take into account the local adaptation of the subpopulation and the genetic structure of the species.

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.

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.

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.

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.

Dependency of seed dormancy types on embryo traits and environmental conditions in Ribes species.

The hypothesis that seed dormancy may be dependent on environmental conditions and seed morphological traits was tested for six Ribes species, across an altitudinal gradient of 1300 m and a longitudinal separation of 120°. Embryo measurements and seed germination experiments were conducted for R. alpinum L., R. hudsonianum Richardson var. petiolare (Douglas) Jancz., R. nevadaense Kellogg, R. roezlii Regel var. cruentum (Greene) Rehder and R. speciosum Pursh, and data taken from the literature for R. multiflorum Kit. ex Schult. ssp. sandalioticum Arrigoni. Germination was compared with seed viability to reveal proportional seed dormancy, which was then correlated to seed/embryo morphological traits and these traits related to the seed provenance environment. The embryos of all the investigated species are linear underdeveloped and all had a morphological component of seed dormancy (MD). Seeds of R. roezlii, R. hudsonianum and R. nevadaense required a temperature and/or hormone pre-treatment in order to germinate, highlighting morphophysiological seed dormancy (MPD). Seed dormancy was found to be strongly negatively correlated with embryo length, but not with embryo to seed (E:S) ratio or seed mass. Initial embryo length was positively related to mean annual temperature. Seed dormancy in the investigated Ribes species could be quantified and predicted by the interaction of embryo traits and environmental conditions. This approach may be helpful in assessing and predicting seed dormancy in the Ribes genus and in other genera and families with underdeveloped embryos.

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.