Molecular characterization and genetic relationships among most common identified morphotypes of critically endangered rare Moroccan species Argania spinosa (Sapotaceae) using RAPD and SSR markers

? The objective of this work is the molecular characterization of most common identified morphotypes of critically endangered rare Moroccan species Argania spinosa.

? Eighteen RAPD markers and twenty SSR markers have been assayed in 38 argan tree accessions from the three most commonly identified morphotypes: oval, spherical and spindle fruit types.

? A total of 140 polymorphic RAPD bands were detected out of 146 bands. The number of presumed alleles revealed by the SSR analysis ranged from 1 to 5 alleles per locus with a total number of 32 alleles identified. Results demonstrated an extensive genetic variability within the tested argan accessions. RAPDs presented a high level of polymorphism and greater information content than SSRs.

? Our results could indicate that Vitellaria paradoxa is genetically closer to argan than Manilkara huberi. In addition, the correlation between the clustering based on RAPD and SSR markers were in general low. The observed clustering could be better explained according to geographic proximity than morphotype. For this reason the traditional morphological characterization of the argan accessions in morphotypes (according to fruit phenotype) seem not to be in correlation to the real genetic background (genotype) of this specie. The implications of these results in the creation of effective germplasm core collection in argan have been also discussed.

     

Genetic structure of Tunisian natural carob tree (Ceratonia siliqua L.) populations inferred from RAPD markers

? Seven RAPD markers were used to assess the genetic diversity and structure of ten Tunisian natural Ceratonia siliqua L. populations from different geographic and bioclimatic zones.

? The species maintain a high diversity within population as estimated by the percentage of polymorphic loci and Shannon’s index (P% = 76.31, $\bar H'_{pop} = 0.569$ ). The range of variation between populations was large. Populations from the upper semi-arid bioclimates, with more continuous distribution area showed the highest level of variation.

? A high genetic differentiation among populations (Φ_ST = 0.250 and $\bar G_{ST} = 0.347$ ), as a result of population isolation was revealed. Nevertheless, the genetic structure is in accordance with bioclimate indicating that ecological factors also should influence differentiation. Populations from the sub-humid, upper semi-arid and mean semi-arid zones clustered together and were distinct from those of the lower semi-arid ones.

? Conservation strategy should be made according to the level of polymorphism within population and bioclimate.

     

Genetic diversity in Tunisian Crataegus azarolus L. var. aronia L. populations assessed using RAPD markers

? The genetic diversity of nine wild Tunisian Crataegus azarolus var. aronia L. populations from different bioclimates was assessed using RAPD markers.

? Eight selected primers generated a total of 105 bands, 81 of which were polymorphic. Shannon’s index (H′) ranged from 0.222 to 0.278 according to a population with an average of 0.245. The genetic variation within the species (H _SP = 0.423) was relatively low. A high differentiation (G _ST = 0.421) among populations coupled with a low level of gene flow (N _m = 0.472) were observed. The analysis of molecular variance (AMOVA) revealed also significant differentiation among populations (Φ_ST = 0.371), even at a low scale space. The majority of variation occurred within populations (63.31%). The Mantel test performed on genetic (Φ_ST) and geographic distance matrices among population pairs did not reveal an isolation by distance.

? Interpretation of Neighbour-joining tree based on Nei’s and Li’s genetic distance among individuals showed distinct population groupings. The UPGMA dendrogram based on Φ_ST values revealed two population sub-clusters, each including populations from different bioclimates and/or geographic regions.

? The low level of genetic diversity and the high genetic structure of populations resulted from genetic drift caused both by habitat fragmentation and the low size of populations.

? The high differentiation among populations and the similar low level of diversity within populations suggest that in situ conservation should interest all populations. The ex situ conservation should be based on the collection of seeds rather within than among populations because of the maximum of variation was revealed within populations.

     

Genetic variation in architectural seedling traits of Patagonian cypress natural populations from the extremes of a precipitation range

? Species distributed along wide environmental ranges are expected to be either plastic or adapted to local optima. The elucidation of which of these alternatives prevails, is crucial in planning breeding and conservation strategies for not yet domesticated species. Austrocedrus chilensis (Cupressaceae) is the most commercially important conifer of the temperate forests of Argentina and the target of a domestication program. A steep precipitation gradient characterizes its Argentinean range.

? Variation within and differentiation among four natural populations of this Patagonian cypress representative of two contrasting precipitation regimes (>1 300 and 330 mm per y) were assessed by analyzing several morpho/architectural traits in one-year-old seedlings grown in a greenhouse.

? Progenies from one of the two xeric populations did not differ from those corresponding to both humid-site populations. The two most variable populations in terms of additive genetic variance belonged to contrasting precipitation regimes. Differentiation among populations was low as measured by each and every variable (average Q _st = 0.088). Morphological traits related to the main axis size would have a dubious adaptive meaning.

? The results suggest that the Patagonian cypress would have not evolved genetic pools adapted to local optima, and thus it appears to be a phenotypically plastic species, at least regarding growth at juvenile age.

     

Genetic structure and variability of phenological forms in the European beech (Fagus sylvatica L.)

? Microsatellite markers were used to describe the genetic structure and variability of early, intermediate and late phenological forms of European beech (Fagus sylvatica L.). Two hundred and seventy individuals from three populations located in southern Poland were divided into three forms according to the phenological criterion — bud burst, and analyzed for allelic variation at five highly polymorphic microsatellite loci.

? Population differentiation was moderate and differed significantly among phenological forms. Average values of F _ST and R _ST decreased across phenological forms and amounted to F _ST values of 0.135, 0.110 and 0.108 and R _ST values of 0.365, 0.231 and 0.098 for early, intermediate and late forms of beech, respectively.

? Analysis of Molecular Variance (AMOVA) revealed different genetic structures characteristic of respective phenological forms of beech. The amount of within-population variability increased with the delay of the beginning of vegetation and amounted to 64%, 77% and 90% of total variability, depending on phenological form. A similar trend was found in average pairwise genetic distance between individuals belonging to a given phenological form (11.78, 11.85 and 12.22, from early to late forms).

? Our results demonstrate the importance of late spring frosts as a factor influencing the genetic structure of beech, and as a cause of the decrease in genetic variability as well as the increase in population differentiation proportional to the degree of phenological earliness.

     

Genetic similarity of natural populations and plantations of Pinus roxburghii Sarg. in Nepal

? Genetic structures of five population pairs each consisting of one natural population and one neighbouring plantation of Pinus roxburghii Sarg. in Nepal were assessed using nuclear (nSSRs) and chloroplast microsatellite loci (cpSSRs).

? The mean number of alleles at nSSRs loci in natural populations was 5.0 compared to 4.93 in plantations while the average observed heterozygosities were the same in both groups (H ₀ = 0.50). Most of the alleles were common to all the populations, indicating that the populations correspond to a single genetic entity. Similarly forty-seven haplotypes were observed in natural populations compared to fifty haplotypes in plantations. Mean haplotype diversities of natural populations (0.953) and plantations (0.955) were very similar. Genetic diversity of Pinus roxburghii was relatively high with low or no evidence of inbreeding while genetic differentiation among all populations was very low (about 1%).

? The very low differentiation among natural populations indicates efficient long-distance gene flow among populations resulting in homogeneous genetic structures at least at selectively neutral loci. Even though the harvest and production of seeds and seedlings was largely uncontrolled, genetic structures of most plantations show no signs of reduced variation, inbreeding or other negative effects compromising the adaptedness or adaptability of planted forests.

     

Phenotypic variation in the phenology of ascospore production between European populations of oak powdery mildew

? Oak powdery mildew severity (Erysiphe alphitoides) is usually mild in Europe because epidemics start late in spring, at the end of the first oak growth unit maturation. However, the disease can occasionally be very severe when strong infection occurs early during the development of the first growth unit, suggesting that host-pathogen synchrony in spring could be a critical factor in disease severity.

? We studied the timing of ascospore production in a given environment for four E. alphitoides populations sampled from SW France to the Czech Republic to determine whether this trait shows variation within Europe.

? Timing of ascospore production was clearly influenced by environmental factors as chasmothecia from a single origin showed very different dates of optimal ascospore production when transferred for overwintering in locations with different climate. In common garden experiments, no differences were observed between populations for the date of optimal ascospore production.

? Results suggest little genetic differentiation for timing of ascospore production for E. alphitoides populations across Europe and therefore a lack of local adaptation to their host phenology. Availability in ascospore inoculum is limited during host budburst, explaining the low infection usually observed on the first oak growth unit.

     

Fecundity and larval development of Monochamus galloprovincialis (Coleoptera Cerambycidae) in experimental breeding

?The study aimed at clarifying basic life traits (fecundity, mortality and development) of Monochamus galloprovincialis, the vector of Bursaphelenchus xylophilus, the causative agent of the Pine Wilt Disease, in Portugal.

? Mating and oviposition experiments were conducted on P. sylvestris logs under laboratory conditions during two years. Larval development was followed outdoors.

? Fecundity was high, with 138.2 eggs per female.

? For the first time, the number of larval instars (4) in M. galloprovincialis was recognized and instars were described.

? Informations are provided on the shape and size of the larval galleries. Boring of galleries in sapwood and heartwood started at the third instar. All instars could overwinter but adults emerged simultaneously resulting in a univoltine life cycle for the majority of the individuals.

? An obligatory diapause in the forth instar was assumed. However, 8.1% of the insects had a two year development.

? Important differences in mortality were noted between the two experimental years, which could be explained by differences on the size of the breeding logs.

? There is a high biological proximity between M. galloprovincialis populations in France and those in Portugal and Northern Europe. However, a higher fecundity and longevity was observed in France, as well as a higher percentage of insects with a two year development, compared with portuguese populations.

     

Using SSR markers to study the mechanism of 2n pollen formation in Populus × euramericana (Dode) Guinier and P. × popularis

? Using cytological methods and SSR DNA marker analysis, this study revealed the formation mechanisms and the genetic constitutions of the 2n pollen in Populus × euramericana (Dode) Guinier and P. × popularis.

? In P. × euramericana (Dode) Guinier, four abnormalities in microsporogenesis were observed: parallel spindle, fused spindle, tripolar spindle and premature cytokinesis. The first three can lead to first-division restitution (FDR) 2n pollen formation and the last one can form second-division restitution (SDR) 2n pollen. The SSR marker analysis results of parents and their tetraploidy filial generation confirmed that the genetic constitution of 2n pollen produced by P. × euramericana (Dode) Guinier was FDR.

? In P. × popularis, three of these abnormalities were observed: parallel spindle, fused spindle and premature cytokinesis. The SSR marker analysis results showed the genetic constitution of 2n pollen produced by P. × popularis was SDR. Natural 2n female gametes in P. × euramericana (Dode) Guinier are reported for the first time. SSR analysis indicated that natural 2n female gametes of P. × euramericana (Dode) Guinier did exist and were fertile, which could be FDR genetic constitution.

? The results from this study showed a great potential for using 2n gametes to produce polyploid poplar clones, which can be used effectively for polyploid breeding for poplar species in the section Aigeiros.

     

Physiological variation among western redcedar (Thuja plicata Donn ex D. Don) populations in response to short-term drought

? Variation in the ability of western redcedar (Thuja plicata Donn ex D. Don) populations to withstand water stress may exist because this species is found in coastal and interior biogeoclimatic subzones representing the full range of precipitation regimes in British Columbia, Canada.

? Seven western redcedar populations from locations in British Columbia, representing a wide range of habitat types, were assessed for their gas exchange and water relations response to controlled drought.

? Before drought, population variation occurred in stomatal conductance, net CO₂ assimilation rate and intrinsic water use efficiency and the relative water content at turgor loss point. During drought, populations had different responses of net CO₂ assimilation to decreasing predawn shoot water potential. After drought, populations differed in stomatal conductance and intrinsic water use efficiency, plus osmotic potential at turgor loss point, osmotic potential at saturation and apparent cuticular transpiration. Western redcedar populations from drier-inland habitats had a lower osmotic potential at turgor loss point, lower relative water content at turgor loss point and lower apparent cuticular transpiration in response to drought than populations from coastal origin with temperate maritime habitat.

? Reduction of cuticular water loss and adjustments of cellular water relations in response to drought was found to occur among seven western redcedar populations originating along a precipitation gradient while; there were minimal population differences in the gas exchange response to drought.

     

Intraspecific variation in growth and allocation patterns in seedlings of Pinus pinaster Ait. submitted to contrasting watering regimes: can water availability explain regional variation?

? Interpopulation variation in key functional traits of Pinus pinaster Ait. is well recognized. However, the relative importance of drought tolerance to explain this regional variation in the species remains elusive.

? Here, we raise the question whether water availability constitutes a likely driver of regional variation in biomass allocation, growth and morphological traits of ten populations that cover the distribution range of P. pinaster. We carried out an experiment where seedlings of five families per population were submitted to two contrasting watering treatments.

? The effects of water availability and population were significant for relative diameter and height growth rate, biomass allocation and number of lateral stems and dwarf shoots. Total dry mass significantly differed between watering treatments but it did not among populations. Populations could be clustered into four main groups. Root mass fraction explained most of the variation and significantly correlated to altitude but not to aridity.

? The geographical pattern of genetic variation found in morphology and biomass allocation did not translate into population differences in drought tolerance or phenotypic plasticity to water availability, indicating that water availability is not a likely driver of the regional variation observed in the studied traits of P. pinaster at the seedling stage.

     

Pedigree and mating system analyses in a western larch (Larix occidentalis Nutt.) experimental population

?The mating pattern and gene flow in a western larch (Larix occidentalis Nutt.) experimental population was studied with the aid of microsatellite markers and a combination of paternity-mating system analysis. The commonly difficult to assess, male gametic contribution was determined with 95% confidence and its impact on genetic gain and diversity was determined.

? Male fertility success rate ranged between 0 and 11%. Male reproductive output parental imbalance was observed with 50% of the pollen being produced by the top 5% of males while the lower 39% males only produced 10% of the pollen.

? A significant difference was observed between male effective population size (genetic diversity) estimates from paternity assignment compared to those based on population’s census number (21 vs. 41); however, this difference did not affect estimates of genetic gain.

? A total of 221 full-fib families were identified (sample size range: 1–8) and were nested among the studied 14 seed-donors.

? A combination of paternity-mating system analysis is recommended to provide a better insight into seed orchards’ mating dynamics. While pollen flow tends to inflate mating system’s outcrossing rate, the paternity analysis effectively determined the rate and magnitude of contamination across receptive females.

     

Genetic correlations between pulpwood and solid-wood selection and objective traits in Eucalyptus globulus

? If selective breeding is to be successful, significant genetic variation must be present in the traits targeted for improvement (i.e. “objective traits”).

? This study aimed to quantify genetic variation in Eucalyptus globulus pulpwood and sawn-timber objective traits (rotation-age whole-tree volume, survival, whole-tree basic density, sawn-board Janka hardness and sawn-board internal checking) and estimate additive genetic correlations between these and inexpensively-assessed “selection traits”.

? Significant genetic variation was identified in all objective traits at the subrace and/or family within subrace level.

? Selection-age diameter at breast height (1.3 m, DBH) was strongly genetically correlated with rotation-age volume (0.78) and survival (0.82). Subrace and additive genetic correlations of selectionage Pilodyn penetration with rotation-age 12-×-12-mm-sample basic density (?0.70 and ?0.75 respectively) and whole-tree basic density (?0.83 and ?0.91 respectively) were also strong.

? No significant subrace or additive genetic correlation between wood-sample gross shrinkage and sawn-board internal checking was detected. However, subrace and additive genetic correlations of sawn-board Janka hardness with Pilodyn penetration (?0.75 and ?0.58 respectively) and sample gross shrinkage (?0.77 and ?0.73 respectively) were significantly different from zero.

? These findings suggest that genetic improvement of the examined objective traits is possible through selective breeding, although none of the assessed selection traits were strongly correlated with internal checking.

     

Variation in the decay resistance of larch to fungi

? Decay resistance of larch (Larix sp.) to fungi was evaluated on heartwood samples belonging to 3 species (L. decidua, L. kaempferi and their hybrid), 3 races of European larch (polonica, sudetica and alpine), 13 wood lots (populations) and 313 trees.

? Larch wood appeared, on average, as moderately durable although a high variability was observed. At the sample level as well as at the mean individual tree level, durability ranged from class 1 to 5 according to EN 350-1 standard. At the population level, larch wood varied from ‘durable’ to ‘slightly durable’. Genetics played a major role in decay resistance at the species, provenance and tree levels. Environmental factors such as the position of heartwood samples and the age of trees were also identified as a source of variability.

? The most durable wood was not necessarily from old native alpine stands of European larch: some young larches from faster growing lowland origins also produced durable wood.

? Genetic improvement of larch wood durability appeared therefore likely by the selection of the best populations for decay resistance as well as from the selection of individuals.

     

Frost damage in Pinus sylvestris L. stems assessed by chlorophyll fluorescence in cortical bark chlorenchyma

? Several techniques were used to investigate the threshold minimum temperature inducing severe frost damage during winter in Scots pine (Pinus sylvestris L.) needles and stems from southern provenances in the Iberian Peninsula.

? Chlorophyll (Chl) fluorescence, electrolyte leakage (EL), visual scoring (VS), and the normalized difference vegetation index (NDVI) were compared.

? Chl fluorescence, and in particular the maximum potential photosystem II (PSII) efficiency — estimated through the dark-acclimated F_V/F_M ratio-, was found to be a simple, non-destructive indicator of freezing tolerance in needles, as expected.

? Moreover, the existence of a Chl-containing tissue, the cortical bark chlorenchyma, allowed us the use of Chl fluorescence as a new non-destructive indicator of frost damage in live tissues of Pinus sylvestris L. stems.

? Freezing tolerance values of southern provenances are compared with those found by other authors in the northern distribution limit of the species.

     

Cold hardiness and transplant response of Juglans nigra seedlings subjected to alternative storage regimes

? Effects of overwinter storage regimes on seedling cold hardiness and physiological vigor are relatively unexplored, particularly for temperate deciduous forest tree species.

? We evaluated influence of storage duration (0, 66, 119, or 175 d) on electrolyte leakage of stem and root collar tissues following exposure to a series of freeze-test temperatures in black walnut (Juglans nigra L.) seedlings sampled from cold (3 °C) or freezer (?2 °C) storage. Seedlings were subsequently transplanted into a controlled growth chamber environment for two months.

? Regardless of storage temperature, mean LT₅₀ was lowest for seedlings stored for 66 d (≤ ?34 °C) and increased dramatically after 119 d (≥ ?13 °C).

? Root collar tissue had lower LT₅₀ than stem tissue after 119 d for cold-stored seedlings, reflecting importance of evaluative tissue type. Days to bud break shortened with increasing storage duration up to 119 d and stabilized thereafter for both storage regimes. Root growth potential was maximized after 119 d of storage, and subsequently declined for cold-stored seedlings. Height growth increased following storage, regardless of duration.

? To promote stress resistance and transplant growth response, we recommend that black walnut seedlings from this genetic source be outplanted after approximately 66–119 d of storage.

     

What sampling is needed for reliable estimations of genetic diversity in Fraxinus excelsior L. (Oleaceae)?

? Sample size is a critical issue for genetic diversity studies and conservation programs. However, sample size evaluation requires previous knowledge of allele frequencies estimated with precision and this is not often the case.

? Here, we evaluated sample size requirements for accurate genetic diversity in adult trees and family arrays in a 12 ha plot of Fraxinus excelsior L. (Oleaceae) in a community forest in central France. Data consisted of 579 adult trees and 480 offspring from 24 families genotyped at four nuclear microsatellites.

? Mean square errors (MSE) estimates performed on Monte Carlo simulations of resampled data indicated that several adult individuals (> 300) are necessary for accurate measures of allele richness. However, expected heterozygosity requires smaller samples (< 30). Seeds captured about 90% of adult allelic diversity requiring a sampling effort roughly 50% larger than that of adult trees (480 seeds vs. 300 adults) suggesting that seed sampling is heavily penalized for allele counts. Nevertheless, gene diversity of seeds was essentially identical to that of the adult population.

? Extrapolation of these results to other ash tree populations appears feasible because of similar levels of diversity reported in the literature but it is not granted for species with significant selfing or high genetic structure.

     

Photosynthetic temperature adaptation of Pinus cembra within the timberline ecotone of the Central Austrian Alps

? Temperature is suggested to determine the upper limit of tree life. Therefore, future climate warming may be of importance for tree distribution within the European Alps, where low temperatures limit carbon metabolism.

? We focused on the effects of air and soil temperature on net photosynthesis (P _n) of Pinus cembra an evergreen climax species of the timberline ecotone of the Central Austrian Alps. Light response and temperature response curves were estimated along an altitudinal gradient ranging from the forest limit up to the krummholz limit in both summer and fall.

? In general, P _n was significantly lower in fall as compared to summer. Nevertheless, independent from season mean P _n values tended to increase with elevation and were positively correlated with root zone temperatures. The specific leaf area by contrast declined with increasing elevation. Furthermore, the temperature optimum of net photosynthesis declined with increasing elevation and was positively correlated with the mean maximum air temperature of the 10 days prior the date of measurement.

? Thus, our findings appear to reflect a long-term adaptation of the photosynthetic apparatus of Pinus cembra to the general temperature conditions with respect to elevation combined with a short term acclimation to the prevailing temperature regime.

     

Compared effects of competition by grasses (Graminoids) and broom (Cytisus scoparius) on growth and functional traits of beech saplings (Fagus sylvatica)

? The effect of two weeds (grasses and broom, Cytisus scoparius) competition on the growth and functional traits of European beech saplings (Fagus sylvatica) was investigated in an experimental plantation in the French Massif central.

? We hypothesized that grasses would have a much more harmful effect than broom on beech growth through strong competition for soil water and nitrogen.

? A randomized block design was used with three separate blocks, each possessing three types of vegetation; grasses (mainly Agrostis capillaris, Holcus mollis and H. lanatus), broom, and bare soil as a control.

? Two years after plantation beech sapling growth was significantly greater in bare soil than with competition from grasses, and was intermediate on broom. Growth was positively correlated with beech leaf nitrogen content (N_m) and xylem water potential (Ψ_x). N and Ψ_x were dependent, with nitrogen uptake probably decreasing with increasing drought. There was no clear correlation between growth and light transmitted 10 cm below the apex of the saplings.

? Beech leaf mass on an area basis (LMA) was correlated with the three resource variables (water, nitrogen and light) and with growth, confirming that LMA is a sensitive functional trait integrating the degree of stress experienced by the saplings.

? The results suggest that broom is less competitive than grasses and, if properly managed on a sufficiently well-watered site, can generate satisfactory conditions for beech establishment and growth.