High variability of chloroplast DNA in three Mediterranean evergreen oaks indicates complex evolutionary history

Chloroplast DNA variation was studied in three evergreen Quercus species (Q. suber L., Q. ilex L. and Q. coccifera L.) from the Western Mediterranean Basin using PCR-RFLP. We studied five primer pair/enzyme combinations, four of them previously used in other European Quercus, obtaining a large number of haplotypes (81) grouped in three main types (suber type, ilex-coccifera I type and ilex-coccifera II type). Such level of haplotype diversity is higher than previously reported for the genus. Remarkable differences in haplotype richness between species have been found. Q. ilex and Q. coccifera usually share the same haplotypes, while a number of Q. suber populations possesses variants of the ilex-coccifera I type. This fact is interpreted as a result of genetic introgression between Q. suber and Q. ilex. Reproductive factors that could determine this exchange are discussed, as well as the influence of different species histories on the present structure of evergreen Quercus in the Western Mediterranean Basin.     

Contrasting nuclear and cytoplasmic exchanges between phylogenetically distant oak species (Quercus suber L. and Q. ilex L.) in Southern France: inferring crosses and dynamics

Gene flow is particularly frequent in the genus Quercus (oaks), especially between closely related species. We focus here on Quercus ilex and the cork-producing Quercus suber , which occasionally hybridize although they are phylogenetically markedly separated. Morphological observations were combined with both allozymic and chloroplastic diagnostic markers to characterize hybridization and introgression and to infer their dynamics in two French regions (French Catalonia and Provence), which are separated by several hundred kilometres. Some hybrids were found in both regions, indicating recent hybridization events. As expected from previous studies, most hybrids resulted from ♀ Q. ilex  × ♂ Q. suber crosses, but our data showed that the reciprocal cross is also possible. Partial independence between nuclear and chloroplastic introgression was observed in the two species. Nuclear introgression was limited in both species and both regions, with no preferred direction. In Provence, chloroplastic introgression was very rare in both species. Conversely, all Q. suber individuals from French Catalonia were introgressed by Q. ilex chlorotypes. This might be explained by introgression in the Iberian Peninsula antedating the first occurrence of the two species in French Catalonia. We also observed a new chlorotype that was created locally, and was exchanged between the two species. However, the two species still remain genetically differentiated. The dynamics and complexity of exchanges and the factors determining them (including human management of Q. suber ) are discussed.     

Molecular and palaeoecological evidence for multiple glacial refugia for evergreen oaks on the Iberian Peninsula

Aim  A multiple glacial refugia hypothesis for Mediterranean plant species was tested with the evergreen Quercus complex ( Quercus suber L., Quercus ilex L. and Quercus coccifera L.) from the Iberian Peninsula, using molecular and palaeobotanical data.
Location  The Iberian Peninsula, which is an ecologically and physiographically complex area located on the western edge of the Mediterranean Basin.
Methods  We sampled 1522 individuals from 164 populations of Q. suber , Q. ilex and Q. coccifera . A review of the recent literature on fossil pollen and charcoal records and a nested clade analysis on chloroplast DNA polymerase chain reaction-restriction fragment length polymorphism was carried out to infer demographic and historical processes.
Results  The analysis indicates at least one glacial refugium for Q. suber in south-western Iberia. Extensive introgression of Q. suber with Q. ilex indicates several potential refugia in eastern Iberia. Past fragmentation was followed by a restricted range flow/range expansion, suggesting multiple refugia for Q. ilex–Q. coccifera elsewhere in central and northern Iberia and multiple areas of secondary contact. This finding is consistent with fossil records.
Main conclusions  The predicted multiple refugia during glacial periods indicates the existence of secondary post-glaciation contact areas. These areas contained complex diversity patterns resulting mainly from range expansions followed by isolation by distance. To a lesser degree, traces of restricted and long-distance dispersal were also found.     

Molecular differentiation and diversity among the California red oaks (Fagaceae; Quercus section Lobatae)

A recent epidemic of Phytopthora (Sudden Oak Death) in coastal woodlands of California is causing severe mortality in some oak species belonging to the red oak (Lobatae) group. To predict the risks of spread of this disease, an understanding of the relationships among California's red oak species and of their population genetic structure is needed. We focus here on relationships among the four species of red oak. Whereas morphological distinction of Quercus wislizeni and Quercus parvula can pose problems, Quercus kelloggii and Quercus agrifolia in pure forms are easily distinguishable from one another and from Q. wislizeni and Q. parvula in the field. However, hybrids among all species combinations are known to occur in nature and these can confound data from ecological studies. Our results revealed greatest differentiation of the deciduous Q. kelloggii, with only weak AFLP fragment differentiation of the three remaining evergreen species. The molecular data suggest a closer affinity of Q. agrifolia with Q. wislizeni and Q. parvula contrary to earlier suggestions that its origins are likely to have been with northern deciduous oaks probably through a common ancestor with Q. kelloggii. Interior and coastal populations of Q. wislizeni separated in dendrograms based on phenetic and genetic distances suggesting probable isolation in different glacial refugia. The position of Q. parvula remains ambiguous, having a closer affinity with interior populations of Q. wislizeni and with Q. agrifolia, than with coastal populations of Q. wislizeni. Mean population differentiation in Q. wislizeni was 0.18, which is somewhat higher than the average for other oak species, suggesting that range fragmentation has occurred in the past, resulting in a metapopulation structure. Our results provide evidence that introgression among these species may be causing reticulation, further confounding species separation. Whereas Phytopthora has been reported on Q. agrifolia, Q. parvula and Q. kelloggii, it has not yet been detected in natural populations of Q. wislizeni. The species relationships that our molecular data show suggest that this is more likely a result of escape due to ecological tolerances than to genetic differences.     

Comparative anatomical analysis of the cotyledonary region in three Mediterranean Basin Quercus (Fagaceae)

Anatomical changes at the cotyledonary node from the embryo to the seedling stage in Quercus coccifera, Q. ilex, and Q. humilis were investigated by light and scanning electron microscopy techniques. Mature embryos of Q. humilis possess 2-3 pairs of leaf primordia and a pair of cotyledonary buds, whereas in Q. coccifera and Q. ilex there are two incipient primordia, and cotyledonary buds are not observed until 1 wk after germination. In all three species the cotyledonary buds multiply, forming bud clusters, and a vascular connection is well established within 5-6 wk after germination. As development proceeds, the cotyledonary region becomes woody, but buds, which are exogenous in origin, never become embedded in the periderm. In comparison with Q. suber, another native Mediterranean Basin oak, the cotyledonary node is short and axillary buds are not present below the insertion of cotyledons. In addition, starch accumulation in the cotyledonary region is not observed from histological analysis in the three oaks. Therefore, in Q. coccifera, Q. ilex, and Q. humilis seedlings the cotyledonary node can be considered to be an important regenerative structure enabling them to resprout after the elimination of the shoot above the cotyledons, despite the absence of a lignotuberous structure.     

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.     

Differences in fine-scale genetic structure and dispersal in Quercus ilex L. and Q. suber L.: consequences for regeneration of mediterranean open woods

Cork oak (Quercus suber L.) and holm oak (Q. ilex L.) are among the most important tree species (economically and ecologically) in the Western Mediterranean region, where they define unique open woods (created and maintained by man) known as 'dehesas' in Spain. However, these formations are under increasing threat due to the lack of regeneration. We have analysed spatial genetic structure in a mixed parkland; inferences about gene dispersal have also been performed, according to the isolation by distance model. Noticeable differences have been detected between the species, despite their similar ecological roles. Restricted effective dispersal leads to kin structures in cork oak, up to 70 m, while no genetic structure is observed in holm oak. Our results suggest a very effective dispersal for the latter, with a local historical gene flow estimated between 55 and 95 m. This is the first time regeneration of Mediterranean oak parklands has been assessed from a genetic perspective. Effective gene flow detected for holm oaks allows us to discount the risk of inbreeding over successive generations. Thus, regeneration of Q. ilex dehesas will just require action directed to help the settlement of the saplings (such as limiting grazing). However, in those cases where densities are too low, more intense forestation (such as plantation and/or establishment of appropriate shelter) will be needed. The 'density threshold' for initiating regeneration will probably be higher for cork oak, due to its more limited dispersal and minor full-light tolerance.     

Potential host plants of Corythucha arcuata (Het., Tingidae) in Europe: a laboratory study

Abstract:  The oak lace bug Corythucha arcuata (Say) (Het., Tingidae), native to North America, was found in Europe on Quercus robur L. and other oaks in the spring of 2000. The potential host plant range of this species in Europe and its development time were investigated in a laboratory study. An assay was performed on leaf cuts of different plant species. On the deciduous European oaks ( Q. robur , Quercus pubescens Willd, Quercus petraea (Mattuschka) Liebl., Quercus cerris L.), as well as Rubus ulmifolius Schott. and Rubus idaeus L., most of the lace bugs (>50%) reached the adult stage; on Castanea sativa Mill., Rubus caesius L. and Rosa canina L., a reduced number of individuals (<25%) reached the adult stage. No nymphs survived on Quercus rubra L. (mentioned in literature as a host plant), on the evergreen oaks Quercus suber L. and Quercus ilex L., on Malus domestica Borkh. and four tested maple species. On plant species where the lace bug reached the adult stage, the development time varied from 13 to 27 days. On European deciduous oak species, the development time was longer on leaves taken in late summer (September) than on those of late spring (June); on the contrary, such differences were not observed on Rubus species, and Castanea sativa .     

Representational difference analysis reveals genomic differences between Q. robur and Q. suber: implications for the study of genome evolution in the genus Quercus

Very similar genome sizes, similar karyotypes and heterochromatin organisation, and identical number/position of ribosomal loci characterise the common oak (Q. robur) and the cork oak (Q. suber), two distantly related oak species. Representational Difference Analysis (RDA) was used to subtract the genome of Q. suber from the genome of Q. robur in order to search for genome differentiation. A library of 400 clones (bearing RDA fragments) representing genome differences between the two species was obtained. Seven Q. robur-specific DNA sequences were analysed with respect to their molecular and chromosome organisation. All belong to the dispersed repetitive component of the genome, as revealed by Southern hybridisation and in situ hybridisation. They are present in the Q. robur genome in between 100 and 700 copies, and are distributed along the length of almost all chromosomes. A search for homologies between RDA fragments and sequences in Genbank revealed similarities of all RDA fragments with known retrotransposons. The RDA fragments were also tested for their presence/absence in the genomes of six additional oak species belonging to different phylogenetic groups, in order to examine the evolutionary dynamics of these DNA sequences.     

Species distinction in Irish populations of Quercus petraea and Q. robur: morphological versus molecular analyses

BACKGROUND AND AIMS: Populations of oak (Quercus petraea and Q. robur) were investigated using morphological and molecular (AFLP) analyses to assess species distinction. The study aimed to describe species distinction in Irish oak populations and to situate this in a European context. METHODS: Populations were sampled from across the range of the island of Ireland. Leaf morphological characters were analysed through clustering and ordination methods. Putative neutral molecular markers (AFLPs) were used to analyse the molecular variation. Cluster and ordination analyses were also performed on the AFLP markers in addition to calculations of genetic diversity and F-statisitcs. KEY RESULTS: A notable divergence was uncovered between the morphological and molecular analyses. The morphological analysis clearly differentiated individuals into their respective species, whereas the molecular analysis did not. Twenty species-specific AFLP markers were observed from 123 plants in 24 populations but none of these was species-diagnostic. Principal Coordinate Analysis of the AFLP data revealed a clustering, across the first two axes, of individuals according to population rather than according to species. High F(ST) values calculated from AFLP markers also indicated population differentiation (F(ST) = 0.271). Species differentiation accounted for only 13 % of the variation in diversity compared with population differentiation, which accounted for 27 %. CONCLUSIONS: The results show that neutral molecular variation is partitioned more strongly between populations than between species. Although this could indicate that the populations of Q. petraea and Q. robur studied may not be distinct species at a molecular level, it is proposed that the difficulty in distinguishing the species in Irish oak populations using AFLP markers is due to population differentiation masking species differences. This could result from non-random mating in small, fragmented woodland populations. Hybridization and introgression between the species could also have a significant role.     

Endemic North African Quercus afares Pomel originates from hybridisation between two genetically very distant oak species (Q. suber L. and Q. canariensis Willd.): evidence from nuclear and cytoplasmic markers

Hybridisation is a potent force in plant evolution, although there are few reported examples of stabilised species that have been created through homoploid hybridisation. We focus here on Quercus afares, an endemic North African species that combines morphological, physiological and ecological traits of both Q. suber and Q. canariensis, two phylogenetically distant species. These two species are sympatric with Q. afares over most of its distribution. We studied two Q. afares populations (one from Algeria and one from Tunisia), as well as several populations of both Q. suber and Q. canariensis sampled both within and outside areas where these species overlap with Q. afares. A genetic analysis was conducted using both nuclear (allozymes) and chloroplastic markers, which shows that Q. afares originates from a Q. suber x Q. canariensis hybridisation. At most loci, Q. afares predominantly possesses alleles from Q. suber, suggesting that the initial cross between Q. suber and Q. canariensis was followed by backcrossing with Q. suber. Other hypotheses that can account for this result, including genetic drift, gene silencing, gene conversion and selection, are discussed. A single Q. suber chlorotype was detected, and all Q. afares individuals displayed this chlorotype, indicating that Q. suber was the maternal parent. Q. afares is genetically, morphologically and ecologically differentiated from its parental species, and can therefore be considered as a stabilised hybrid species.     

Diversity and specificity of ectomycorrhizal fungi retrieved from an old-growth Mediterranean forest dominated by Quercus ilex

We analysed the ectomycorrhizal (ECM) fungal diversity in a Mediterranean old-growth Quercus ilex forest stand from Corsica (France), where Arbutus unedo was the only other ECM host. On a 6400 m2 stand, we investigated whether oak age and host species shaped below-ground ECM diversity. Ectomycorrhizas were collected under Q. ilex individuals of various ages (1 yr seedlings; 3-10 yr saplings; old trees) and A. unedo. They were typed by ITS-RFLP analysis and identified by match to RFLP patterns of fruitbodies, or by sequencing. A diversity of 140 taxa was found among 558 ectomycorrhizas, with many rare taxa. Cenococcum geophilum dominated (35% of ECMs), as well as Russulaceae, Cortinariaceae and Thelephoraceae. Fungal species richness was comparable above and below ground, but the two levels exhibited < 20% overlap in fungal species composition. Quercus ilex age did not strongly shape ECM diversity. The two ECM hosts, A. unedo and Q. ilex, tended to share few ECM species (< 15% of the ECM diversity). Implications for oak forest dynamics are discussed.     

Geographic structure of chloroplast DNA polymorphisms in European oaks

Chloroplast DNA polymorphisms have been detected by the conventional Southern-blotting hybridization method in four species of European oaks (Quercus petraea, Q. robur, Q. pubescens and Q. pyrenaica). Three polymorphisms, shared by at least three of these species, can be scored directly in ethidium bromidestained gels and were used in a broad survey of the level of differentiation of the oak species and of their pattern of genetic structure in western Europe. The highly significant geographic variation and the high genetic differentiation (Gst=0.895, S_Gst=0.025) indicate a low level of cytoplasmic gene flow. We conclude that cytoplasmic genomes are well suited for the reconstruction of past migrational routes of such a complex of species.     

Seed-mass effects in four Mediterranean Quercus species (Fagaceae) growing in contrasting light environments

Three hypotheses have been proposed to explain the functional relationship between seed mass and seedling performance: the reserve effect (larger seeds retain a larger proportion of reserves after germinating), the metabolic effect (seedlings from larger seeds have slower relative growth rates), and the seedling-size effect (larger seeds produce larger seedlings). We tested these hypotheses by growing four Mediterranean Quercus species under different light conditions (3, 27, and 100% of available radiation). We found evidence for two of the three hypotheses, but none of the four species complied with all three hypotheses at the same time. The reserve effect was not found in any species, the metabolic effect was found in three species (Q. ilex, Q. pyrenaica, and Q. suber), and the seedling-size effect in all species. Light availability significantly affected the relationships between seed size and seedling traits. For Q. ilex and Q. canariensis, a seedling-size effect was found under all three light conditions, but only under the lowest light (3%) for Q. suber and Q. pyrenaica. In all species, the correlation between seed mass and seedling mass increased with a decrease in light, suggesting that seedlings growing in low light depend more upon their seed reserves. A causal model integrates the three hypotheses, suggesting that larger seeds generally produced larger seedlings.     

Past distribution and ecology of the cork oak (Quercus suber) in the Iberian Peninsula: a pollen-analytical approach

Abstract.  This study presents pollen-analytical data from continental and offshore Iberian Peninsula sites that include pollen curves of Quercus suber , to provide information on the past distribution and ecology of the cork oak ( Q. suber ). Results centre on a new pollen record of Navarrés (Valencia, eastern Spain), which shows that the cork oak survived regionally during the Upper Pleistocene and was important during a mid-Holocene replacement of a local pine forest by Quercus -dominated communities. This phenomenon appears linked to the recurrence of fire and reinforces the value of the cork oak for reforestation programmes in fire-prone areas. In addition to Navarrés, other Late Quaternary pollen sequences (Sobrestany, Casablanca-Almenara, Padul, SU 8103, SU8113, 8057B) suggest last glacial survival of the cork oak in southern and coastal areas of the Peninsula and North Africa. Important developments also occur from the Late Glacial to the middle Holocene, not only in the west but also in the eastern Peninsula. It is suggested that, in the absence of human influence, Q. suber would develop in non-monospecific forests, sharing the arboreal stratum both with other sclerophyllous and deciduous Quercus and Pinus species.     

Foliar trichornes of the evergreen and semideciduous species of the genus Quercus (Fagaceae) in the Iberian Peninsula

The leaf trichomes of the evergreen and semi-deciduous species of the genus Quercus living in the Iberian Peninsula were studied by means of SEM and LM. The SEM has been particularly useful because of the high quality of the illustrations and the possibility of recognizing the structures under I,M or the stereomicroscope afterwards. The studied species were: Quercus coccifera, Q ilex , Q rotundifolia, Q suber, Q canariensis, Q faginea, Q brateroi and Q lusitanica . All species show some exclusive particularities that permit their identification using these characters. The abaxial surface of the leaf always has more trichomes than the adaxial and hence provides more characters for identification. Quercus alpestris Boiss. shows the same features in the leaf trichomes as Q faginea Lam. A key for identification using these characters is provided.     

Seasonal changes of root hydraulic conductance (KRL) in four forest trees: an ecological interpretation

This paper deals with the possibility of relating root hydraulic parameters to an ecological index describing the continentality/oceanicity of four forest trees. Root hydraulic conductance ($K_R$) of seedlings of Fagus sylvatica L., Quercus ilex L., Quercus suber L. and Quercus pubescens Willd. was measured in May, August and November 1996. $K_R$ was calculated in terms of the relation of the water flow through intact root systems in situ measured with the pressure chamber, and the pressure driving the flow. The sufficiency of the root system to supply the foliage was estimated by dividing $K_R$ by the seedlings leaf surface area ($A_L$) thus obtaining $K_RL$. In the spring, $K_RL$ was largest in F. sylvatica and smallest in Q. pubescens with intermediate values recorded in Q. ilex and Q. suber. All the species studied showed a large decline in $K_RL$ just prior to the winter rest except for Q. suber which mantained $K_RL$ approximately constant through the period of study. In most cases, $K_RL$ changed in accordance with analogous changes in the flow. When the total seedlings' leaf surface area ($A_L$) was plotted versus $K_RL$, it appeared that $K_RL$ of Q. pubescens increased with $A_L$, proportionally, while $K_RL$ of F. sylvatica was inversely related to $A_L$. This, together with the largest $K_RL$ recorded in the summer in Q. pubescens, was interpreted as advantageous to this species (which is adapted to semi-arid environments) in that: (a) roots could supply water to foliage efficiently even during the adverse season and (b) the foliage growth could be sustained even in summer.No statistically significant relation of $K_RL$ to the continentality index calculated for the four species studied on the basis of their European distribution, was found to exist. Nonetheless, our data appear to be encouraging for future research aimed at better interpreting the typical distribution areas of plant species.     

Foliar trichomes of the evergreen and semi-deciduous species of the genus Quercus (Fagaceae) in the Iberian Peninsula

The leaf trichomes of the evergreen and semi-deciduous species of the genus Quercus living in the Iberian Peninsula were studied by means of SEM and LM. The SEM has been particularly useful because of the high quality of the illustrations and the possibility of recognizing the structures under LM or the stereomicroscope afterwards. The studied species were-. Quercus coccifera, Q. ilex, Q. rotundifolia, Q suber, Q canariensis, Q. faginea, Q. broteroi and Q. lusitanica. All species show some exclusive particularities that permit their identification using these characters. The abaxial surface of the leaf always has more trichomes than the adaxial and hence provides more characters for identification. Quercus alpestris Boiss. shows the same features in the leaf trichomes as Q. faginea Lam. A key for identification using these characters is provided.     

Selection and dispersal in a multispecies oak hybrid zone

The four western North American red oak species (Quercus wislizeni, Q. parvula, Q. agrifolia, and Q. kelloggii) are known to produce hybrid products in all interspecific combinations. However, it is unknown whether hybrids are transitory resulting from interspecific gene flow or whether they are maintained through extrinsic selection. Here, we examine cryptic hybrid structure in Q. wislizeni through a broad region including contact and isolation from three other western North American red oaks using amplified fragment length polymorphism molecular markers. All four species were simultaneously detected in the genetic background of individuals morphologically assigned to Q. wislizeni, although the contribution of Q. kelloggii was minor. In some cases, introgression was detected well outside the region of sympatry with one of the parental species. Molecular structure at the individual level indicated this was due to long-distance pollen dispersal and not to local extinction of parental species. Species admixture proportions were correlated with climatic variables and greater proportions of Q. agrifolia and Q. parvula were present in the genetic background of Q. wislizeni in sites with cooler and more humid summers, corresponding with habitat preferences of the parental species. Partial Mantel tests indicated that climate was more important than distance from pollen source in this association. Despite high levels of introgression, species integrity was maintained in some populations in close proximity to the other species, providing further support to environmental selection in determining population genetic structure. Thus, the contribution of species mixtures to population genetic structure varies across the landscape according to availability of pollen, but more importantly to varying environmental selection pressures that produce a complex pattern of hybrid and pure gene pools.     

Molecular phylogeny and genetic diversity of Tunisian Quercus species using chloroplast DNA CAPS markers

Chloroplast DNA variation was studied in five evergreen Quercus species from the Western part of Tunisia using Cleaved Amplified Polymorphic Sequence (CAPS) technique. Five primer pair/endonuclease combinations have been used. Chlorotypes of Quercus species have been identified. The enzyme HinfI was more efficient in detecting polymorphism in oak species than TaqI. The phenogram showed five groups defining the five studied oak species: suber group, afares group, coccifera group, canariensis group and ilex group. The topology of phenogram showed that the classification depends only on species and independently of their geographic origin. The principal component analysis (ACP) corroborated the results of the tree branching and confirmed the existence of five species groups. Our results showed a genetic proximity between Quercus afares and Quercus coccifera species that may be due to temperature tolerance or the demographic history of these species. Nevertheless, a high value of G_ST calculated (G_ST = 1), suggesting that the maximum of variation is maintained among oak species. This result was confirmed by the low value of the genetic diversity within species (h_S = 0), the value obtained of the total genetic diversity (h_T = 0.378) and the absence of gene flow between species (N_m = 0). A high genetic proximity has been registered between Q. afares, Quercus suber and Quercus canariensis. Moreover, Q. afares shared the chlorotype of Q. suber and Q. canariensis which suggests its hybrid origin.