Frequent cytoplasmic exchanges between oak species that are not closely related: Quercus suber and Q. ilex in Morocco

Chloroplast (cp) and mitochondrial (mt) DNA variation were studied in 97 populations of cork oak (Quercus suber) in Morocco; in 31 of these populations, holm oak (Quercus ilex), a clearly distinct species, also occurred and was compared with Q. suber. Three cpDNA and one mtDNA primer pairs were used in the survey, each in combination with one restriction enzyme. Six haplotypes belonging to two very divergent lineages were detected; one lineage predominates in each species, and is probably ancestral, as inferred from comparisons with other oak species. In the mixed-species populations, cytoplasmic genomes were frequently shared across species, as indicated by an introgression ratio of 0.63. This index is a new measure of the propensity of species to share locally genetic markers, varying from zero (complete differentiation) to one (no differentiation). By contrast, more closely related deciduous oak species (Q. robur, Q. petraea and Q. pubescens) have introgression ratios varying from 0.82 to 0.97. The introgression events appear to have been more frequent in the direction Q. ilex (female) x Q. suber (male), a finding which seems attributable to the flowering phenology of these two species. This asymmetry may have favoured immigration of Q. suber beyond its main range, in regions already colonized by Q. ilex. There, rare hybridization and further introgression through long distance pollen flow have established populations that are morphologically indistinguishable from Q. suber but that have cytoplasmic genomes originating from the local Q. ilex populations.     

Natural hybridisation between kermes (Quercus coccifera L.) and holm oaks (Q. ilex L.) revealed by microsatellite markers

Hybridisation between species of the genus Quercus is a common phenomenon as a result of weak reproductive isolation mechanisms between phylogenetically close species that frequently co-occur in mixed stands. In this study, we use microsatellite markers to analyse introgression between kermes ( Quercus coccifera L.) and holm ( Q. ilex L.) oak, two closely related taxa that frequently dominate the landscape in extensive areas in the Mediterranean region. All tested microsatellites amplified and were polymorphic in both kermes and holm oaks. Bayesian admixture analyses showed a good correspondence between each species and one of the two inferred genetic clusters. Five sampled individuals were a priori tentatively identified as hybrids on the basis of intermediate morphological characteristics, and it was confirmed that they also presented mixed genotypes. However, we also detected different levels of genetic introgression among morphologically pure individuals, suggesting that successful backcrossing and/or reduced phenotypic expression of genetic variance in certain individuals may have resulted in strong convergence towards a single species phenotype.     

Allozyme variation in cork oak (Quercus suber L.): the role of phylogeography and genetic introgression by other Mediterranean oak species and human activities

 Genetic variation in the cork oak (Quercus suber L.) was investigated using 11 loci from seven enzyme systems in 40 populations sampled over the entire distribution of this species in the western Mediterranean Basin. Mean heterozygosity values over the polymorphic loci (Ho=0.283), the percentage of polymorphic populations (M=0.76), and the total genetic diversity (Ht=0.31) from which 11% was accounted for among-population variation, are among the highest recorded in oak species. In contrast to previous results in holm oak (Q. ilex L.), another evergreen species in the same area, cork oak possessed a smaller allele pool and a lower average number of alleles per locus and per population (A=2.0). More particularly, very few low-frequency alleles were observed in cork oak except for eight populations in which allozyme polymorphism at locus Pgi 1, diagnostic between both species, indicates that these low-frequency alleles are introgressed from holm oak. On the basis of the genetic distance estimated from allozyme frequencies, 32 of the 40 cork oak populations studied were classified into two very distinct sets which also corresponded to distinct geographic areas. One set gathered together the 18 populations from the Iberian peninsula and two adjacent areas in France, i.e. the centre of origin of cork oak, according to paleobotanical data. This set was characterized by a larger allele pool, a higher within-population genetic diversity and a lower differentiation between populations than was observed in the other set, which comprised the populations from North Africa, Sicily, Sardinia, Corsica, continental Italy and the region of Provence (southeastern France). In these more southern and eastern disjunct areas, cork oak migration from Iberia may have occurred at different periods since the end of the Tertiary. The possible effect of human activity on cork oak genetic structure, i.e. the selection of good-quality cork, acorn over-use for animal food, and even human nutrition, is discussed. Received: 3 March 1998 / Accepted: 19 March 1998     

Monoterpene synthase activities in leaves of Picea abies (L.) Karst. and Quercus ilex L

In addition to direct ecological functions in the interaction of plants with the environment, the emission of monoterpenes, especially from the foliage of evergreen trees, is of great importance for the production of ozone and photochemical oxidants in the troposphere. In the present work, we established a reproducible non-radioactive standard enzyme assay and characterized monoterpene synthase activities in needles of Norway spruce (Picea abies (L.) Karst.) and in leaves of holm oak (Quercus ilex L.). In Norway spruce, the dominant monoterpenes formed were alpha-pinene, camphene, and to a lesser extent beta-pinene and limonene. In holm oak, alpha-pinene, sabinene, and beta-pinene were the main products, while limonene was a minor component. Under optimum conditions, in both Norway spruce and holm oak, monoterpene formation remained constant up to 180 min and 90 min, respectively, and varied with the buffer and Mg2+ and Mn2+ concentrations used. Optimum temperature for monoterpene synthase activity was 40 degrees C in both species; optimal pH ranged between 6.5 and 7.5 in both species. Apparent Michaelis-constants for the substrate GDP were ca. 17.9 +/- 5.1 microM for Norway spruce and ca. 69.4 +/- 22.1 microM for holm oak. Molecular weight determination by FPLC indicated that the monoterpene synthases in Norway spruce and holm oak have native molecular weights of ca. 59 and 50 kDa, respectively.     

Isolation and functional analysis of a cDNA encoding a myrcene synthase from holm oak (Quercus ilex L.).

An 859-bp cDNA segment of a terpene synthase gene was amplified by PCR from the evergreen sclerophyllous holm oak (Quercus ilex L.) using heterologous primers for conserved regions of terpene synthase genes (TPS) in dicotyledonous plants. Based on the sequence of this segment, homologous primers were designed for amplification by RACE-PCR of a cDNA segment carrying the monoterpene synthase gene myrS. The gene encodes a protein of 597 amino acids including an N-terminal putative plastid transit peptide. The gene without the segment encoding the transit peptide was cloned by PCR into a bacterial expression vector. Expression in Escherichia coli yielded an active monoterpene synthase, which converted geranyl diphosphate (GDP) predominantly into the acyclic monoterpene myrcene and to a very small extent into cyclic monoterpenes. Sequence comparison with previously cloned monoterpene synthases revealed that the myrcene synthase from Q. ilex belongs to the TPSb subfamily.     

The Balearic Islands: a reservoir of cpDNA genetic variation for evergreen oaks

Aim To analyse the role of the Balearic Islands as a refuge area for evergreen Quercus (cork oak: Quercus suber L., holm oak: Q. ilex L., kermes oak: Q. coccifera L.), by using molecular, historical and palaeobotanical data. Location The Western Mediterranean Basin (Balearic Islands, eastern Iberia, Provence, Sardinia, Corsica, Sicily, Malta, Italy, Northern Africa). Methods We sampled 108 populations and used the PCR‐RFLP technique with five universal cpDNA primers to define haplotypes in the sampled populations. Diversity, differentiation parameters and spatial analysis of the populations, using a spatial version of amova , were linked to the geological history of the Western Mediterranean Basin in order to explain the present spatial pattern of the evergreen Quercus populations in the Balearics. Results Evergreen Quercus cpDNA shows a complex structure, with remnants of ancient diversity in the Balearics. Balearic populations of holm oak are related to Iberian populations, while for cork and kermes oaks, we found both Tyrrhenian and Iberian haplotypes. Main conclusions The complex spatial patterns of cpDNA in Balearic evergreen Quercus appears explicable in terms of a combination of physical (vicariance and long distance dispersal) and biological (introgressive hybridization) factors. The Balearics constitute a glacial refuge area and a reservoir of genetic variation with traces of ancient diversity from Messinian–Pliocene stages.     

Seed germination and seedling performance of two Mediterranean tree species,holm oak (shape Quercus ilex L.) and Aleppo pine (shape Pinus halepensis Mill.): a multifactor experimental approach

A two-level multifactor experimental approach was used to compare seed germination and seedling performance of two Mediterranean tree species: the early successional Aleppo pine (Pinus halepensis Mill.) and the late successional holm oak (Quercus ilex L.). In a first experiment germination rate was evaluated under the combined effects of shade, nitrogen availability, and pine or holm oak leaf litter. In a second experiment we tested for the effects of shade, nutrient availability, and litter type on seedling survival, growth and biomass allocation. Holm oak showed higher germination rates under shaded than under unshaded conditions, while Aleppo pine showed no differences between shaded and unshaded conditions. Nitrogen availability and litter type had no significant effect on germination of either species. Both species showed increased RGR, but also higher mortality rates, when grown in an enriched nutrient environment. While Aleppo pine showed no differences in RGR and mortality rate under different shading levels, RGR decreased and mortality increased for holm oak in full light. Increased radiation decreased LAR, SLA and height:diameter ratio, and increased RWR and R/S in both species, although Aleppo pine showed more pronounced changes. Unlike Aleppo pine, holm oak responded to increased nutrient availability by decreasing R/S and increasing LAR. From these results, no seed-seedling conflicts were found in either species, but a trade-off does seem to exist for holm oak between biomass allocation traits deployed in response to increased nutrient availability and radiation. Aleppo pine outperformed holm oak under most environmental conditions tested and showed a wider regeneration niche.     

Development and feeding efficiency of Malacosoma neustrium larvae reared with Quercus spp. leaves

Observations on larval development of Malacosoma neustrium were conducted both in a cork oak stand and in the laboratory by using leaves of different host trees (cork oak, holm oak and downy oak) as food source. Instars were determined using head capsule and frass measurements. In the field the larvae progressed up to the fifth instar before pupating, and the increase in head capsule width followed Dyars Rule with a rate of increase (R.I.) value of 1.74. The same number of instars was determined for the larvae reared with cork oak (R.I. = 1.73) and holm oak (R.I. = 1.70) leaves. The caterpillars reared with downy oak foliage completed larval development in five, six and seven instars and the R.I. values obtained were 1.60, 1.52 and 1.44 respectively. A lower mortality was recorded for the larvae reared on holm oak. Growth and feeding indices were determined for the larvae from the third up to the last instar. The highest leaf consumption was detected for the fifth instar larvae reared on holm oak. For the caterpillars which completed five instars before pupating, the relative consumption rate (RCR) decreased from the third up to the fifth instar: from 4.8 to 1.7 (cork oak), from 7.4 to 3.3 (holm oak) and from 14.3 to 2.1 (downy oak). The relative growth rate (RGR) was highest during the fourth stadium (0.24, 0.27 and 0.33 for larvae reared with cork oak, holm oak and downy oak leaves respectively) and decreased in the fifth instar (0.09, 0.14 and 0.14 for larvae reared with cork oak, holm oak and downy oak leaves respectively), probably because of greater expense of energy due to the approach of maturity. Feeding and growth indices could be useful to define a defoliation prediction model.     

Ecological behavior of Quercus suber and Quercus ilex inferred by topographic wetness index (TWI)

The ecological behaviors of a network of pure evergreen oak stands (Quercus suber L. and Quercus ilex L.) in the Central-Western Mediterranean Basin were investigated toward climatic and edaphic factors implemented with the application of topographic wetness index (TWI). A Categorical Principal Component Analysis (Catpca) using climatic and soil physico-chemical parameters was performed on 23 cork oak and holm oak pure stands with the aim to understand better the effectiveness of TWI for characterizing soil ecology of the two species. Catpca pointed out that, although cork oak and holm oak are able to growth in similar Mediterranean conditions, they show different behaviors in terms of needs and tolerance to soil water content. TWI confirmed such results at local scale, allowing highlighting some interesting features of the species differential ecology. Although both species confirmed to be drought-tolerant, the heliophilous cork oak revealed to dominate the landscape on wettest soils with high TWI values—indicating the capacity to tolerate stresses due to periods of waterlogging—, while the shade-tolerant holm oak prevails for low-medium TWI values—drier and mesophilous sites. Despite the application of TWI to vegetation science and ecology is relatively recent, results are encouraging and suggest considering this user-friendly and synthetic index in ecological investigations and modeling.     

Identifying signatures of natural selection in cork oak (Quercus suber L.) genes through SNP analysis

Cork oak (Quercus suber L.) is an evergreen tree species endemic to the western Mediterranean Basin with a major economical, social and ecological relevance, associated with cork extraction and exploitation. In the last years, cork oak stands have been facing a significant decline, which may be aggravated by the climate changes that are predicted to occur within cork oak distribution range during this century. Under this scenario, the assessment of adaptive genetic variation is essential to understand how cork oak may cope with these threats and to delineate strategies for the management of its genetic resources. In this study, six candidate genes possibly significant for environmental adaptation were analysed in cork oak populations from its entire distribution range. Signatures of natural selection were investigated using population genetic statistics and environmental association tests under alternative scenarios of population genetic structure. Signals of balancing selection were detected in the putative non-expressor of pathogenesis-related gene 1 (NPR1), involved in plant defence response against pathogens, in auxin response factor 16 (ARF16), a gene implicated in root development, in RAN3, also involved in developmental processes, and in glutamine synthetase nodule isozyme (GS), involved in nitrogen fixation. Furthermore, for ARF16, a class I heat shock protein (sHSP) and GS, associations were found between SNP allele and haplotype frequencies and several spatial and climatic variables, suggesting that these genes may have a role on cork oak local adaptation. In this study, the first steps were taken into gathering information on cork oak adaptation to environmental conditions.     

Realized gene flow within mixed stands of Quercus robur L. and Q. petraea (Matt.) L. revealed at the stage of naturally established seedling

The estimates of contemporary gene flow assessed based on naturally established seedlings provide information much needed for understanding the abilities of forest tree populations to persist under global changes through migration and/or adaptation facilitated by gene exchange among populations. Here, we investigated pollen‐ and seed‐mediated gene flow in two mixed‐oak forest stands (consisting of Quercus robur L. and Q. petraea [Matt.] Liebl.). The gene flow parameters were estimated based on microsatellite multilocus genotypes of seedlings and adults and their spatial locations within the sample plots using models that attempt to reconstruct the genealogy of the seedling cohorts. Pollen and seed dispersal were modelled using the standard seedling neighbourhood model and a modification—the 2‐component seedling neighbourhood model, with the later allowing separation of the dispersal process into local and long‐distance components. The 2‐component model fitted the data substantially better than the standard model and provided estimates of mean seed and pollen dispersal distances accounting for long‐distance propagule dispersal. The mean distance of effective pollen dispersal was found to be 298 and 463 m, depending on the stand, while the mean distance of effective seed dispersal was only 8.8 and 15.6 m, which is consistent with wind pollination and primarily seed dispersal by gravity in Quercus. Some differences observed between the two stands could be attributed to the differences in the stand structure of the adult populations and the existing understory vegetation. Such a mixture of relatively limited seed dispersal with occasional long distance gene flow seems to be an efficient strategy for colonizing new habitats with subsequent local adaptation, while maintaining genetic diversity within populations.     

Topography modulates climate sensitivity of multidecadal trends of holm oak decline

Forest decline events have increased worldwide over the last decades being holm oak (Quercus ilex L.) one of the tree species with the most worrying trends across Europe. Since this is one of the tree species with the southernmost distribution within the European continent, its vulnerability to climate change is a phenomenon of enormous ecological importance. Previous research identified drought and soil pathogens as the main causes behind holm oak decline. However, despite tree health loss is a multifactorial phenomenon where abiotic and biotic factors interact in time and space, there are some abiotic factors whose influence has been commonly overlooked. Here, we evaluate how land use (forests versus savannas), topography, and climate extremes jointly determine the spatiotemporal patterns of holm oak defoliation trends over almost three decades (1987–2014) in Spain, where holm oak represents the 25% of the national forested area. We found an increasing defoliation trend in 119 out of the total 134 holm oak plots evaluated, being this defoliation trend significantly higher in forests compared with savannas. Moreover, we have detected that the interaction between topography (which covariates with the land use) and summer precipitation anomalies explains trends of holm oak decline across the Mediterranean region. While a higher occurrence of dry summers increases defoliation trends in steeper terrains where forests dominate, an inverse relationship was found in flatter terrains where savannas are mainly located. These opposite relationships suggest different causal mechanisms behind decline. Whereas hydric stress is likely to occur in steeper terrains where soil water holding capacity is limited, soil waterlogging usually occurs in flatter terrains what increases tree vulnerability to soil pathogens. Our results contribute to the growing evidence of the influence of local topography on forest resilience and could assist in the identification of potential tree decline hotspots and its main causes over the Mediterranean region.     

Multiple Recruitment Limitation Causes Arrested Succession in Mediterranean Cork Oak Systems

Lack of tree regeneration and persistency of species-poor shrublands represent a growing problem across Mediterranean evergreen oak forests. What constrains forest regeneration is poorly understood, and restoration attempts have been largely unsuccessful. We assessed the contribution of four different mechanisms of tree recruitment limitation (that is, source, dispersal, germination, and establishment) in a cork oak (Quercus suber) system in southern Portugal. Using a combination of field studies and experiments, we quantified seed production, seed removal and dispersal, seed survival and germination, seedling establishment and survival, as well as cork oak natural regeneration for the three dominant vegetation types in this system (Cistus ladanifer shrubland, oak forest, and oak savanna). We found that all four forms of cork oak recruitment limitation were significantly more severe in shrublands than in oak forests and savannas, so that oak seedling recruitment in shrubland was impeded in multiple ways. Our results explain why transitions from shrublands to oak savannas and forests are extremely difficult, and that the release from arrested succession in this system requires the simultaneous relief of multiple constraints on recruitment limitation in the early life history of oaks. These results have important implications for the restoration and conservation of Mediterranean oak systems.     

Influence of intra- and inter-specific Interference on Terpene Emission by Pinus Halepensis and Quercus Ilex Seedlings

Seedlings of two common Mediterranean trees, Pinus halepensis L. and Quercus ilex L., were grown alone and together with seedlings of the same or of the other species in the same pot during one year to test the effects of intra- and inter-specific interference on terpene emission. Light, nutrients and water were amply supplied. There were higher emission rates in P. halepensis than in Q. ilex. The emission increased when the neighbour was a pine and decreased when the neighbour was a holm oak. Volatile organic compound and terpene emission rates followed inverse trends to foliar biomass or growth, which decreased when the neighbour was a pine. This revised version was published online in July 2006 with corrections to the Cover Date.     

Germination of Quercus ilex and Q. pubescens in a Q. ilex coppice

Quercus ilex L. (holm oak) coppices, widespread around the Mediterranean basin, are probably the result of 5 000 years of prolonged human disturbance of the original Quercus pubescens Willd. (downy oak) forests. Since disturbance has almost ceased in recent years, a question arises as to the development of these coppices: will the Q. pubescens forests return, or will Q. ilex remain the dominant species? To investigate the phenomenon, we analyzed the first stages, i.e. germination of the two species in holm oak coppices. Our experiments show that both species germinated better in coppices than in clearings or clear-cuts. Moreover, Q. pubescens appears to be slightly favored above Q. ilex and it is suggested auto-allelopathy is involved, at least partially inhibiting the germination of Q. ilex.     

Extensive pollen immigration and no evidence of disrupted mating patterns or reproduction in a highly fragmented holm oak stand

Aims Forest fragmentation and reduced tree population densities can potentially have negative impacts on mating patterns, offspring genetic diversity and reproductive performance. The aim of the present study is to test these hypotheses comparing an extremely fragmented, low tree density (~0.02 trees/ha) holm oak (Quercus ilex L.) stand from Central Spain with a nearby high tree density stand (~50 trees/ha).Methods We genotyped adult trees and seeds from the low-density stand (436 seeds from 15 families) and the high-density stand (404 seeds from 11 families) using nine microsatellite markers. With these data, we performed paternity analyses, determined pollen flow, mating patterns and pollen pool structure, and estimated progeny genetic diversity in both stands. We also studied seed set and production and performed a pollen supplementation experiment to determine whether reduced tree density has limited foreign pollen availability.Important findings We have found extensive pollen immigration (>75%) into the low tree density stand and Monte Carlo simulations revealed that pollen moves larger distances than expected from null models of random dispersal. Mating patterns and differentiation of pollen pools were similar in the high-density stand and the low-density stand but we found higher inter-annual differentiation of pollen pools in the former. Progeny genetic diversity and self-fertilization rates did not differ between the low-density stand and the high-density stand. Seed set rates were significantly lower in the low-density stand than in the high-density stand and experimental cross-pollen supplementation evidenced that foreign pollen availability is indeed a limiting factor in the former. However, seed crops did not differ between the low-density stand and the high-density stand, indicating that limitation of foreign pollen is not likely to be of great concern in terms of reduced seed production and potential recruitment. Poor forest regeneration due to other ecological and human factors is probably a more important threat for the persistence of fragmented and low tree density stands than reduced pollen flow and only extremely small and isolated tree populations would be expected to suffer severe loss of genetic diversity in the long term.     

The Description of Clones of Quercus robur L. and Q. petraea (Matt.) Liebl. with Microsatellites and AFLP in an Ancient Woodland

Abstract: In densely populated areas autochthonous Quercus robur L. (pedunculate oak) and Q. petraea (Matt.) Liebl. (sessile oak) (Fagaceae) populations have been maintained as ancient devastated woodlands. The continuous cutting, grazing and resprouting of such woodlands has enabled the development of clonal structures. For conservation purposes, an analysis of the actual number, size and spatial distribution of clones is necessary, especially when there is an interest in genetic variation of the population. This study describes for the first time - based on microsatellite and AFLP™ analysis - clones in an autochthonous mixed Q. robur and Q. petraea population that has been coppiced and grazed for several centuries. Based on six microsatellite loci and 69 polymorphic AFLP markers, only 14 unique genotypes were detected in a plot that consisted of 80 trees. Clones were observed for both Q. robur and Q. petraea. The largest clone diameters were observed for Q. robur, with distances up to 5.8 m. The observed clone sizes may indicate the age of the trees.     

Climate response of cork growth in the Mediterranean oak (Quercus suber L.) woodlands of southwestern Portugal

In the Mediterranean climate regions, drought events are expected to affect the growth of forests ecosystems by changing trees growth rates and eventually inducing shifts in their growth patterns. Cork oak (Quercus suber L.) is a strictly western Mediterranean tree species periodically harvested for its bark, the cork. So far, cork oak has received limited attention for dendroclimatological studies due to its typical faint and erratic tree wood rings. Moreover, its distinct cork rings chronologies have been completely neglected. In this study we introduce an approach using cork ring chronologies dated back 9–10 years for climate response. Despite enhancing interannual variability and increasing statistical response to short-term climatic variability, still poorly understood, this study will possibly allow infer long-term climate response. We analyzed the cork ring chronologies of 55 cork samples collected in mature (under exploitation) trees in three distinct locations in southwestern Portugal. Cork growth recorded a high climate signal, with highly significant and coherent responses to the yearly climate-related sources of variation. We successfully assessed trends of cork growth via correlation analysis including selected climate variables among mean monthly temperature, monthly precipitation and, on an annual basis, eight precipitation indices. The high mean sensitivities and inter-series correlations found for cork ring chronologies combined with the significant variance explained by climate variables suggest that climate is likely one dominant signal that affects cork growth, but local environmental stresses can decisively affect this (climate) signal. Assuming cork growth as a proxy for cork oak growth, it seems conceivable that despite the trees being highly resistant to drought stress, cork oak woodlands in southwestern Portugal would have to face lesser growth in a global warming scenario.     

Seasonal pattern of monoterpene synthase activities in leaves of the evergreen tree Quercus ilex

Monoterpene synthase activities were measured in current year and 1-year-old leaves of holm oak ( Quercus ilex L.). The monoterpene synthase activities of the leaves strongly changed with leaf development and leaf age. Enzyme activities increased rapidly in spring after leaf emergence, reaching maximum values in summer, which declined during the following winter period. In the next spring monoterpene synthase activities recovered in the old leaves to about one-third of values in the previous years and showed a similar seasonal variation as in young leaves. In both leaf age classes the pattern of enzymatic monoterpene formation was stable with α-pinene (33%), β-pinene (28%), and myrcene (26%) as prominent compounds followed by minor fractions of sabinene (10%) and limonene (3%). Monoterpene emission correlated with the activity of the synthetizing enzymes, indicating that monoterpene synthase activities in Q. ilex reflect the seasonal monoterpene emission potential of the leaves.