Promoting mixed stands through conversion treatments. Effect of holm oak coppices thinning on black pine regeneration

The effects of conversion treatments, depending on ecological factors and silvicultural parameters (thinning intensity, thinning type and rotation, among others) have been studied during the last fifteen years in an experimental trial in Central Spain. The general climate is continental Mediterranean; soils are low depth and limy; vegetation is an homogeneous dense coppices of Quercus ilex with isolated Pinus nigra trees. The experimental design (three locations) includes different thinning intensities (from 0 to 100% of extracted basal area). Inventories have been carried out in 1994 and 2010; thinning treatments were done in 1995 and 2011. Analysis of the effects of the conversion treatment show the increment of diameter and height growth rates, the canopy recovery and the stand resprouting, finding differences in these effects between thinning treatments. Besides the induced changes at holm oak stand, the application of conversion treatment clearly changed the woodland dynamics. Fifteen years after the thinnings, floristic composition varied and an abundant pine regeneration was installed in the woodland. In this work we describe the changes between inventories in tree species composition and diameter distribution, specially in the case of black pine. The conversion treatment caused changes in forest dynamics in the short term, increasing biodiversity and diversifying the forest structure. The fast installation of Pinus regeneration suggests the potential of the zone for the establishment of multipurpose mixed Quercus-Pinus stands in wide areas where Quercus species were favoured by human populations for firewood production. Conversion treatment of coppices, with the creation of mixed stands, constitutes a good management alternative for extensive areas and an interesting technique to adaptation to global change.

Leftovers in seed dispersal: ecological implications of partial seed consumption for oak regeneration

1. Successful seed dispersal by animals is assumed to occur when undamaged seeds arrive at a favourable microsite. Most seed removal and dispersal studies consider only two possible seed fates, predation or escape intact. Whether partial consumption of seeds has ecological implications for natural regeneration is unclear. We studied partial consumption of seeds in a rodent-dispersed oak species. 2. Fifteen percent of dispersed acorns were found partially eaten in a field experiment. Most damage affected only the basal portion of the seeds, resulting in no embryo damage. Partially eaten acorns had no differences in dispersal distance compared to intact acorns but were recovered at farther distances than completely consumed acorns. 3. Partially eaten acorns were found under shrub cover unlike intact acorns that were mostly dispersed to open microhabitats. 4. Partially eaten acorns were not found buried proportionally more often than intact acorns, leading to desiccation and exposure to biotic agents (predators, bacteria and fungi). However, partial consumption caused more rapid germination, which enables the acorns to tolerate the negative effects of exposure. 5. Re-caching and shrub cover as microhabitat of destination promote partial seed consumption. Larger acorns escaped predation more often and had higher uneaten cotyledon mass. Satiation at seed level is the most plausible explanation for partial consumption. 6. Partial consumption caused no differences in root biomass when acorns experienced only small cotyledon loss. However, root biomass was lower when acorns experienced heavy loss of tissue but, surprisingly, they produced longer roots, which allow the seeds to gain access sooner to deeper resources. 7.Synthesis. Partial consumption of acorns is an important event in the oak regeneration process, both quantitatively and qualitatively. Most acorns were damaged non-lethally, without decreasing both dispersal distances and the probability of successful establishment. Faster germination and production of longer roots allow partially eaten seeds to tolerate better the exposure disadvantages caused by the removal of the pericarp and the non-buried deposition. Consequently, partially consumed seeds can contribute significantly to natural regeneration and must be considered in future seed dispersal studies.

Flying vs. climbing: Factors controlling arboreal seed removal in oak-beech forests

Nuts are heavy and nutritious seeds that need animals to be successfully dispersed. Most studies address nut removal by a single animal species once seeds fall onto the ground. However, nuts are also accessible before the seed drop and usually to a wide guild of seed foragers. This study examines the factorscontrollingarborealseedremoval in oak–beechforests within the whole guild of nut foragers. We found that seed-dispersing rodents (Apodemus sylvaticus) were the main acorn removers in the oaks (up to 3.75 m height), with a rapid seed encounter and a high removal rate. However, rodents did not climb the beech trees, probably due to their smoother bark in comparison to oak bark and/or the lower nutritional value of beechnuts with regard to acorns. Jays (Garrulus glandarius) were more abundant in oak stands (both dense and scattered) and clearly preferred acorns to beechnuts whereas nuthatches (Sitta europaea) were more abundant in beech stands and preferred beechnuts to acorns. Non-storing birds such as great tits (Parus major) also removed acorns and beechnuts, especially in the stands where oaks are dominant. Jays and rodents preferred sound seeds over insect-infested seeds but such a preference was not found for nuthatches. This study highlights that pure beech stands showed a reduced guild of arboreal nut foragers in comparison to oak stands. This different guild could probably affect the spatial patterns of seed dispersal, with a proportionally higher number of long dispersal events for acorns (mostly jay-dispersed) than for beechnuts (mostly nuthatch-dispersed). Long-distance dispersal of beechnuts (by jays) is determined by the presence of other preferred species (oaks) and their frequency of non-mast years. Seed location in different habitats strongly determines the contribution of different arboreal removers (including climbing rodents) and their removal speed, leading to a differential seed fate that will eventually affect tree regeneration. As nuthatches are sedentary birds, it is important to maintain old and dead trees where they can breed (crevices), forage (arthropods) and store seeds in order to favor beechnut dispersal and gene flow. By maintaining or favoring oak trees within beech stands we will ensure a wider guild of arboreal nut dispersers.

Embryogenic suspensions of adult cork oak: the first step towards mass propagation.

Abstract Protocols have been established to clone adult cork oak trees by somatic embryogenesis using semisolid medium. However, for economically viable mass propagation, embryogenic cultures in liquid medium need to be developed. In this study, suspension cultures were initiated from embryo clusters obtained by secondary embryogenesis on a gelled medium lacking plant growth regulators. After 6 days of culture, these embryo clusters generated high cell density suspensions that also contained small organized structures (embryos and embryogenic clumps). As the culture duration increased, tissue necrosis and fewer embryogenic structures were observed and the establishment of suspension cultures failed. An alternative method was found adequate for initiation of embryogenic suspensions: embryo clusters from gelled medium were briefly shaken in liquid medium and detached cells and embryogenic masses of 41?800 lm were used as inoculum. Maintenance of embryogenic suspensions was achieved using a low-density inoculum (43 mg l-1) by subculturing four embryogenic clumps of 0.8?1.2 mm per 70 ml of medium. Proliferation ability was maintained for almost 1 year through ten consecutive subcultures. The initiation and maintenance protocols first developed for a single genotype were effective when tested on 11 cork oak genotypes.

Allocation of 14C assimilated in late spring to tissue and biochemical stem components of cork oak (Quercus suber L.) over the seasons

Carbon distribution in the stem of 2-year-old cork oak plants was studied by 14CO2 pulse labeling in late spring in order to trace the allocation of photoassimilates to tissue and biochemical stem components of cork oak. The fate of 14C photoassimilated carbon was followed during two periods: the first 72 h (short-term study) and the first 52 weeks (long-term study) after the 14CO2 photosynthetic assimilation. The results showed that 14C allocation to stem tissues was dependent on the time passed since photoassimilation and on the season of the year. In the first 3 h all 14C was found in the polar extractives. After 3 h, it started to be allocated to other stem fractions. In 1 day, 14C was allocated mostly to vascular cambium and, to a lesser extent, to primary phloem; no presence of 14C was recorded for the periderm. However, translocation of 14C to phellem was observed from 1 week after 14CO2 pulse labeling. The phellogen was not completely active in its entire circumference at labeling, unlike the vascular cambium; this was the tissue that accumulated most photoassimilated 14C at the earliest sampling. The fraction of leaf-assimilated 14C that was used by the stem peaked at 57% 1 week after 14CO2 plant exposure. The time lag between C photoassimilation and suberin accumulation was ∼8 h, but the most active period for suberin accumulation was between 3 and 7 days. Suberin, which represented only 1.77% of the stem weight, acted as a highly effective sink for the carbon photoassimilated in late spring since suberin specific radioactivity was much higher than for any other stem component as early as only 1 week after 14C plant labeling. This trend was maintained throughout the whole experiment. The examination of microautoradiographs taken over 1 year provided a new method for quantifying xylem growth. Using this approach it was found that there was more secondary xylem growth in late spring than in other times of the year, because the calculated average cell division time was much shorter.

Plant regeneration from an endangered valuable cork oak tree by somatic embryogenesis.

Among the several applications of in vitro tissue culture techniques, the conservation of plant germplasm is one of the most widely used. The cork oak is one of the principal tree species in the Western Mediterranean región. Within this área, the Balearic Islands are considered to be a glacial refuge, and therefore a reservoir of genetic resources. A singular tree has been found in the small Minorca Island population. The haplotype of this tree is of Tyrrhenian origin, showing a past link between Minorca and Sardinia. Moreover, this tree do not bear a deletion within an ITS from ribosimic nuclear DNA, which is fairly common in many populations of this species, and indicates that ir may be the descendant of a very ancient population. This tree is currently in a precarious condition, and it has not produced acorns in the last years. Hence there is a clear need of vegetative propagation to conserve this genotype. We have previously developed methods to clone adult cork oak tres by somatic embryogenesis, and therefore the aim of the present work was to clone this singular tree. There braches from the corwn were collected in November 2004, and methods previously described were carried out. By February 2005 somatic embryogenesis was obtained from leaves of the tree with percentages on induction ranging from 17 to 54% depending on the branch, which may show a novel source of variation that requires further study. Spontaneously matured somatic embryos germinated at 46% in average, and the first somatic seedlings from the Alfavaret's cork oak tree were obtained. Therefore, this study shows one of the most relevant applications of somatic embryogenesis: the plant regeneration of valuable genotypes for the in situ and ex situ conservation of forest genetic resources.

Implementation of a module for risk of ozone impacts assessment to vegetation in the integrated assessment modelling system for the Iberian peninsula. Evaluation for wheat and holm oak

A module to estimate risks of ozone damage to vegetation has been implemented in the Integrated Assessment Modelling system for the Iberian Peninsula. It was applied to compute three different indexes for wheat and Holm oak; daylight AOT40 (cumulative ozone concentration over 40 ppb), cumulative ozone exposure index according to the Directive 2008/50/EC (AOT40-D) and PODY (Phytotoxic Ozone Dose over a given threshold of Y nmol m−2 s−1). The use of these indexes led to remarkable differences in spatial patterns of relative ozone risks on vegetation. Ozone critical levels were exceeded in most of the modelling domain and soil moisture content was found to have a significant impact on the results. According to the outputs of the model, daylight AOT40 constitutes a more conservative index than the AOT40-D. Additionally, flux-based estimations indicate high risk areas in Portugal for both wheat and Holm oak that are not identified by AOT-based methods.

Spatial distribution of the soil carbon pool in a Holm oak dehesa in Spain

Aims Dehesas are agroforestry systems characterized by scattered trees among pastures, crops and/or fallows. A study at a Spanish dehesa has been carried out to estimate the spatial distribution of the soil organic carbon stock and to assess the influence of the tree cover. Methods The soil organic carbon stock was estimated from the five uppermost cm of themineral soil with high spatial resolution at two plots with different grazing intensities. The Universal Kriging technique was used to assess the spatial distribution of the soil organic carbon stocks, using tree coverage within a buffering area as an auxiliary variable. Results A significant positive correlation between tree presence and soil organic carbon stocks up to distances of around 8 m from the trees was found. The tree crown cover within a buffer up to a distance similar to the crown radius around the point absorbed 30 % of the variance in the model for both grazing intensities, but residual variance showed stronger spatial autocorrelation under regular grazing conditions. Conclusions Tree cover increases soil organic carbon stocks, and can be satisfactorily estimated by means of crown parameters. However, other factors are involved in the spatial pattern of the soil organic carbon distribution. Livestock plays an interactive role together with tree presence in soil organic carbon distribution.

Effect of the health status and geographical origin on the cork production characteristics of Western Algeria cork oak stands

Results lead to the conclusion that the effect of health status on traumatic phellogen formation and activity is clear but not uniform. Further studies are necessary for a deeper understanding of the effect of stress situations on pore formation and characteristics

Embryogenesis in Oak species. A Review.

Embryogenesis in Oak species. A Review.

Balancing management and preservation of mediterranean scattered oak woodlands (Dehesas) in human-dominated landscapes

Los sistemas agroforestales mediterráneos tienen una gran importancia ecológica y socioeconómica, y mantienen altos valores medioambientales y de diversidad biológica a la vez que producen importantes servicios ecosistémicos. Estos sistemas han sido testigos de diversos cambios rápidos y drásticos en su gestión y aprovechamiento durante el último siglo. La mayor parte de la investigación desarrollada en esta tesis doctoral ha sido llevada a cabo en las dehesas españolas. Esta tesis nos muestra: i) la evidencia de la existencia de un cambio global del estrato arbóreo y del manejo del pastoreo en el todo el área de distribución de la dehesa durante los últimos 60 años; ii) la importancia del papel que juega el arbolado disperso y el adecuado manejo del ganado en la mejora de la producción, calidad y diversidad de las comunidades herbáceas, que a su vez, un pasto herbáceo bien desarrollado es importante para la rentabilidad del sistema, evaluando estos efectos bajo distintos escenarios de clima y calidad de estación; y iii) la evidencia de la falta de regeneración en sistemas agroforestales mediterráneos bajo distintos tipos de manejo del pastoreo, y además se evalúa el crecimiento y desarrollo de las pocas plántulas existentes que serán las que aseguren la viabilidad y persistencia y de estos sistemas. El arbolado disperso de estos sistemas ha experimentado una reducción importante en su densidad arbórea y fracción de cabida cubierta durante el periodo entre 1950-1980 donde tuvieron lugar importantes transformaciones en la actividad agropecuaria. La cabaña ganadera de ovino disminuyó drásticamente en los años 70 en comparación a la de bovino que desde entonces ha aumentado progresivamente hasta la actualidad. Por otro lado, el mismo tipo de manejo del ganado doméstico (especialmente bovino) durante bastante tiempo (mínimo 30 años) provocó una reducción significativa de la densidad de las plántulas. Además la probabilidad de ocurrencia y la intensidad de daños por herbivoría fue mayor bajo pastoreo bovino (con daños más intensos y consistentes) que bajo pastoreo ovino o sin pastoreo doméstico (presencia de ciervos). También el patrón de crecimiento de las plantas jóvenes estuvo afectado por el tipo de manejo, generando plántulas achaparradas en el caso del bovino y plántulas esbeltas favoreciendo el crecimiento en altura en el caso del ovino. La presencia de un arbolado disperso generó una mayor diversidad y variación en la producción de las comunidades herbáceas según las condiciones de disponibilidad de agua. Especialmente, el ecotono como microhábitat sostuvo altos valores de diversidad herbácea. La presencia del ganado bajo pastoreo continuo de intensidad moderada a alta, especialmente el bovino, incrementó los rendimientos de producción y diversidad del estrato herbáceo. Los resultados de esta tesis nos muestran la importancia que tiene la existencia de un equilibrio entre la producción y la conservación de los sistemas agroforestales mediterráneos para obtener una producción sostenible de servicios ecosistémicos mientras se asegura la perpetuación del sistema a largo plazo. Es crucial diseñar planes de gestión incorporando objetivos de conservación que integren técnicas silvopastorales apropiadas para poder aplicar en los sistemas agroforestales mediterráneos. ABSTRACT Mediterranean scattered oak woodlands have great ecological and socio-economic importance, supporting high environmental and amenity values, and relatively rich biological diversity while producing important ecosystem services. They have been witnesses of different and fast changes developed in the last century. Most of the research developed in this dissertation has conducted within dehesas. This thesis provides: i) the global change evidence of the tree layer and grazing management experienced in the land-use range of a Mediterranean scattered oak woodland (dehesa) over the last 60 years; ii) the important role of scattered trees and adequate management grazing in the improvement of grassland yield, quality and diversity - which it is important, in turn, for the system profitability - under different climate scenarios and site quality; and iii) the lack of oak regeneration evidence under some given representative management regimes and how is the growth development of these plants to assure the viability and persistence of Mediterranean scattered oak woodlands. Tree layer experienced a significant reduction in dehesas during 1950-1980 period where the highest human impacts took place. Sheep herd decreased drastically during the 1970s and, in contrast, cattle have been increasing gradually since then. On the other hand, same livestock grazing management (especially cattle) during long time (minimum 30 years) within Mediterranean scattered oak woodlands reduced strongly the density of young oak plants and showed high probability of herbivory occurrence and intensity. Young plant growth pattern was greatly modified by livestock. Cattle grazing generated stunted plants and sheep grazing generated slender plants favoring the height growth. Microsites created by large trees modified the herbaceous yield according the water availability of the year and generated high plant diversity within herbaceous communities. Especially, ecotone microsite supported high values of herbaceous diversity. The presence of livestock species increased the herbaceous yield and maintained a more diverse community under continuous grazing at both moderate and high intensities; especially cattle. Thus, around the influence of scattered trees there is a high amount of different interactions among livestock, trees and grasslands maintaining and enhancing the quality of whole dehesa system. The results of this thesis highlight how important is balancing management and preservation of Mediterranean scattered oak woodlands to obtain the optimum ecosystem services while the system conservation is assured for a long-term. It is crucial to design management plans with conservation goals that include appropriate silvopastoral practices in Mediterranean scattered oak woodlands.

Development of somatic embryogenesis for cloning and conservation of mature holm oak trees (Quercus ilex L.)

La encina (Quercus ilex L.) es una de las especies forestales mediterráneas más importantes. Constituye gran parte del estrato arbóreo de dehesas o montados, produce bellota como alimento del ganado y establece simbiosis con hongos micorrizógenos de gran valor económico. La encina está considerada como una especie recalcitrante en términos de conservación de semillas y capacidad morfogénica, lo que dificulta los programas de conservación de recursos genéticos y la mejora de la especie. La propagación vegetativa es una potente herramienta de los programas de mejora, por lo que es preciso desarrollar protocolos de regeneración somática en encina. La embriogénesis somática está considerada como la modalidad más adecuada de regeneración basada en técnicas de cultivo de tejidos vegetales utilizada en biotecnología forestal. Este trabajo se centra en el estudio de determinados aspectos de la embriogénesis somática para la regeneración clonal de encinas adultas. La memoria de esta tesis se ha dividido en capítulos que se corresponden con diferentes aspectos del sistema embriogénico. La embriogénesis somática se indujo en tegumentos maternos de óvulos en desarrollo procedentes de bellotas inmaduras de encinas adultas. A pesar de las bajas frecuencias de inducción, las líneas embriogénicas generadas se amplificaron mediante embriogénesis secundaria observándose cierta pérdida de la capacidad de diferenciación con el tiempo. Tanto el genotipo como la formulación del medio de cultivo influyeron en la respuesta embriogénica, concluyendo que la formulación de macronutrientes de Schenk y Hildebrant del medio sin reguladores de crecimiento fue la combinación más efectiva en la inducción. Los resultados sugirieron la existencia de una ventana en el desarrollo del óvulo más sensible a la inducción. El genotipo in[luyó en la capacidad proliferativa de los cultivos y en la conversión de los embriones somáticos, que se incrementó suplementando el medio con ácido indol-3-butírico y 6-benciladenina. El cultivo en medio líquido de líneas embriogénicas en condiciones de inmersión transitoria incrementó el crecimiento, dependiendo del genotipo, con respecto al cultivo en medio semisólido. Sin embargo, no mejoró la capacidad de diferenciar embriones cotiledonares aislados. Se estableció un protocolo de inicio y mantenimiento de cultivos en suspensión para varias líneas embriogénicas mediante inoculación en alta densidad de agregados embrionarios procedentes del medio semisólido. Para evitar la pérdida de vigor y la capacidad morfogénica debida al cultivo prolongado se desarrolló un protocolo de crioconservación de líneas embriogénicas mediante vitrificación. Al determinar la influencia de los agentes crioprotectores antes y después de su inmersión en nitrógeno líquido se concluyó que las respuestas de capacidad de crecimiento y de diferenciación del material embriogénico son independientes, además de estar bajo influencia del genotipo y el tipo de material crioconservado. La combinación de sacarosa y PVS2 previa a la inmersión en nitrógeno líquido proporcionó la mayor tasa de recuperación. Cuando las líneas fueron crioconservadas 30 días la capacidad de diferenciación se perdió en todas ellas. El análisis de SSR detectó variación somaclonal en el material crioconservado a corto plazo. SSR y RAPD mostraron importantes diferencias genéticas entre los árboles donantes y el material embriogénico que dependieron del genotipo. El grado de detección dependió del marcador empleado. Ambos marcadores revelaron baja inestabilidad intraclonal. Los RAPD revelaron variación genética intra-individuo en las encinas donantes. Se discuten la variación genética pre-existente en encina, su aparición durante las primeras fases de la inducción de embriogénesis, y la presencia de tejidos provenientes de la fertilización en el explanto materno. Esto hace preciso definir la identidad genética del material donante y acometer ensayos de detección precoz de variación somaclonal. ABSTRACT Holm oak (Quercus ilex L.) is one of the most important Mediterranean forest species. It conforms the tree layer of dehesas or montados, it produces acorns to feed the livestock and it establishes symbiosis with profitable mycorrhizal fungi. Holm oak is considered as recalcitrant species in terms of seed conservation and morphogenic capacities, which complicates the development of genetic conservation and improvement programs. Vegetative propagation is one of the mightiest tools for breeding programs therefore; developing protocols for clonal regeneration of holm oak is essential. Somatic embryogenesis is considered the best tissue culture-based way of plant regeneration in forest biotechnology. The present study is focused on the study of certain aspects of somatic embryogenesis for clonal regeneration of mature holm oak. This thesis manuscript is divided into several chapters that match with different aspects of the embryogenic system. Somatic embryogenesis induction was achieved on maternal teguments of developing ovules from immature acorns of adult holm oak trees. Despite the low induction frequencies, the generated embryogenic lines were amplified by secondary embryogenesis. A decline in the differentiation capacity over time was also observed. It was concluded that both genotype and culture media formulation influenced the embryogenic response, being the Schenk and Hildebrandt´s macronutrients formulation from culture medium and the lack of plant growth regulators the most effective combination for the induction of the embryogenic response. It has been suggested the existence of a developmental window in which ovules are prone to induction. Genotype influenced the proliferation capacity and the plant conversion of somatic embryos, which was also favoured by the presence of indol-3-butyric acid and 6-bencyladenine. The use of temporary immersion systems as proliferation in liquid culture of the embryogenic lines increased the growth depending on genotype, when compared to semisolid cultures. However, it did not improve the differentiation of single cotyledonary embryos. A protocol for the initiation and maintenance of embryogenic suspension cultures was established for several embryogenic lines with highly dense inoculi of embryogenic clusters from proliferating semisolid cultures. In order to avoid the loss of vigour and morphogenic ability of embryogenic lines due to prolonged cultures, a cryopreservation protocol for embryogenic lines of holm oak has been developed. During the determination of the influence of cryoprotective agents on the growth and differentiation capacities before and after liquid nitrogen immersion, it was concluded that both responses were independent from each other and also under the influence of genotype and the type of cryopreserved material. The combination of sucrose and PVS2 prior liquid nitrogen immersion provided higher recovery rates. When the same embryogenic lines were cryopreserved for 30 days, none was able to differentiate. The SSRs analysis of the short-term cryopreserved material detected somaclonal variation. Both SSR and RAPD markers showed high sensitivity to detect genetic differences between the donor trees and the generated embryogenic material. Nevertheless, the degree of instability detection depended on the marker. The SSR analysis indicated a relationship between genotype, the studied loci and the located polymorphisms. Also, both markers revealed low intraclonal genetic variation. The RAPD detected genetic variation within the donor trees. The presence of pre-existent genetic variation within mature trees, in addition to its occurrence during the early stages of the embryogenic induction, and the presence of tissues of fertilisation origin within the maternal explants are all discussed. Nonetheless, the determination of the genetic identity of donor material is required, in addition to early detection methods of somaclonal variation.

Balancing management and preservation of mediterranean scattered oak woodlands (Dehesas) in human-dominated landscapes

Los sistemas agroforestales mediterráneos tienen una gran importancia ecológica y socioeconómica, y mantienen altos valores medioambientales y de diversidad biológica a la vez que producen importantes servicios ecosistémicos. Estos sistemas han sido testigos de diversos cambios rápidos y drásticos en su gestión y aprovechamiento durante el último siglo. La mayor parte de la investigación desarrollada en esta tesis doctoral ha sido llevada a cabo en las dehesas españolas. Esta tesis nos muestra: i) la evidencia de la existencia de un cambio global del estrato arbóreo y del manejo del pastoreo en el todo el área de distribución de la dehesa durante los últimos 60 años; ii) la importancia del papel que juega el arbolado disperso y el adecuado manejo del ganado en la mejora de la producción, calidad y diversidad de las comunidades herbáceas, que a su vez, un pasto herbáceo bien desarrollado es importante para la rentabilidad del sistema, evaluando estos efectos bajo distintos escenarios de clima y calidad de estación; y iii) la evidencia de la falta de regeneración en sistemas agroforestales mediterráneos bajo distintos tipos de manejo del pastoreo, y además se evalúa el crecimiento y desarrollo de las pocas plántulas existentes que serán las que aseguren la viabilidad y persistencia y de estos sistemas. El arbolado disperso de estos sistemas ha experimentado una reducción importante en su densidad arbórea y fracción de cabida cubierta durante el periodo entre 1950-1980 donde tuvieron lugar importantes transformaciones en la actividad agropecuaria. La cabaña ganadera de ovino disminuyó drásticamente en los años 70 en comparación a la de bovino que desde entonces ha aumentado progresivamente hasta la actualidad. Por otro lado, el mismo tipo de manejo del ganado doméstico (especialmente bovino) durante bastante tiempo (mínimo 30 años) provocó una reducción significativa de la densidad de las plántulas. Además la probabilidad de ocurrencia y la intensidad de daños por herbivoría fue mayor bajo pastoreo bovino (con daños más intensos y consistentes) que bajo pastoreo ovino o sin pastoreo doméstico (presencia de ciervos). También el patrón de crecimiento de las plantas jóvenes estuvo afectado por el tipo de manejo, generando plántulas achaparradas en el caso del bovino y plántulas esbeltas favoreciendo el crecimiento en altura en el caso del ovino. La presencia de un arbolado disperso generó una mayor diversidad y variación en la producción de las comunidades herbáceas según las condiciones de disponibilidad de agua. Especialmente, el ecotono como microhábitat sostuvo altos valores de diversidad herbácea. La presencia del ganado bajo pastoreo continuo de intensidad moderada a alta, especialmente el bovino, incrementó los rendimientos de producción y diversidad del estrato herbáceo. Los resultados de esta tesis nos muestran la importancia que tiene la existencia de un equilibrio entre la producción y la conservación de los sistemas agroforestales mediterráneos para obtener una producción sostenible de servicios ecosistémicos mientras se asegura la perpetuación del sistema a largo plazo. Es crucial diseñar planes de gestión incorporando objetivos de conservación que integren técnicas silvopastorales apropiadas para poder aplicar en los sistemas agroforestales mediterráneos. ABSTRACT Mediterranean scattered oak woodlands have great ecological and socio-economic importance, supporting high environmental and amenity values, and relatively rich biological diversity while producing important ecosystem services. They have been witnesses of different and fast changes developed in the last century. Most of the research developed in this dissertation has conducted within dehesas. This thesis provides: i) the global change evidence of the tree layer and grazing management experienced in the land-use range of a Mediterranean scattered oak woodland (dehesa) over the last 60 years; ii) the important role of scattered trees and adequate management grazing in the improvement of grassland yield, quality and diversity - which it is important, in turn, for the system profitability - under different climate scenarios and site quality; and iii) the lack of oak regeneration evidence under some given representative management regimes and how is the growth development of these plants to assure the viability and persistence of Mediterranean scattered oak woodlands. Tree layer experienced a significant reduction in dehesas during 1950-1980 period where the highest human impacts took place. Sheep herd decreased drastically during the 1970s and, in contrast, cattle have been increasing gradually since then. On the other hand, same livestock grazing management (especially cattle) during long time (minimum 30 years) within Mediterranean scattered oak woodlands reduced strongly the density of young oak plants and showed high probability of herbivory occurrence and intensity. Young plant growth pattern was greatly modified by livestock. Cattle grazing generated stunted plants and sheep grazing generated slender plants favoring the height growth. Microsites created by large trees modified the herbaceous yield according the water availability of the year and generated high plant diversity within herbaceous communities. Especially, ecotone microsite supported high values of herbaceous diversity. The presence of livestock species increased the herbaceous yield and maintained a more diverse community under continuous grazing at both moderate and high intensities; especially cattle. Thus, around the influence of scattered trees there is a high amount of different interactions among livestock, trees and grasslands maintaining and enhancing the quality of whole dehesa system. The results of this thesis highlight how important is balancing management and preservation of Mediterranean scattered oak woodlands to obtain the optimum ecosystem services while the system conservation is assured for a long-term. It is crucial to design management plans with conservation goals that include appropriate silvopastoral practices in Mediterranean scattered oak woodlands.

Development of somatic embryogenesis for cloning and conservation of mature holm oak trees (Quercus ilex L.)

La encina (Quercus ilex L.) es una de las especies forestales mediterráneas más importantes. Constituye gran parte del estrato arbóreo de dehesas o montados, produce bellota como alimento del ganado y establece simbiosis con hongos micorrizógenos de gran valor económico. La encina está considerada como una especie recalcitrante en términos de conservación de semillas y capacidad morfogénica, lo que dificulta los programas de conservación de recursos genéticos y la mejora de la especie. La propagación vegetativa es una potente herramienta de los programas de mejora, por lo que es preciso desarrollar protocolos de regeneración somática en encina. La embriogénesis somática está considerada como la modalidad más adecuada de regeneración basada en técnicas de cultivo de tejidos vegetales utilizada en biotecnología forestal. Este trabajo se centra en el estudio de determinados aspectos de la embriogénesis somática para la regeneración clonal de encinas adultas. La memoria de esta tesis se ha dividido en capítulos que se corresponden con diferentes aspectos del sistema embriogénico. La embriogénesis somática se indujo en tegumentos maternos de óvulos en desarrollo procedentes de bellotas inmaduras de encinas adultas. A pesar de las bajas frecuencias de inducción, las líneas embriogénicas generadas se amplificaron mediante embriogénesis secundaria observándose cierta pérdida de la capacidad de diferenciación con el tiempo. Tanto el genotipo como la formulación del medio de cultivo influyeron en la respuesta embriogénica, concluyendo que la formulación de macronutrientes de Schenk y Hildebrant del medio sin reguladores de crecimiento fue la combinación más efectiva en la inducción. Los resultados sugirieron la existencia de una ventana en el desarrollo del óvulo más sensible a la inducción. El genotipo in[luyó en la capacidad proliferativa de los cultivos y en la conversión de los embriones somáticos, que se incrementó suplementando el medio con ácido indol-3-butírico y 6-benciladenina. El cultivo en medio líquido de líneas embriogénicas en condiciones de inmersión transitoria incrementó el crecimiento, dependiendo del genotipo, con respecto al cultivo en medio semisólido. Sin embargo, no mejoró la capacidad de diferenciar embriones cotiledonares aislados. Se estableció un protocolo de inicio y mantenimiento de cultivos en suspensión para varias líneas embriogénicas mediante inoculación en alta densidad de agregados embrionarios procedentes del medio semisólido. Para evitar la pérdida de vigor y la capacidad morfogénica debida al cultivo prolongado se desarrolló un protocolo de crioconservación de líneas embriogénicas mediante vitrificación. Al determinar la influencia de los agentes crioprotectores antes y después de su inmersión en nitrógeno líquido se concluyó que las respuestas de capacidad de crecimiento y de diferenciación del material embriogénico son independientes, además de estar bajo influencia del genotipo y el tipo de material crioconservado. La combinación de sacarosa y PVS2 previa a la inmersión en nitrógeno líquido proporcionó la mayor tasa de recuperación. Cuando las líneas fueron crioconservadas 30 días la capacidad de diferenciación se perdió en todas ellas. El análisis de SSR detectó variación somaclonal en el material crioconservado a corto plazo. SSR y RAPD mostraron importantes diferencias genéticas entre los árboles donantes y el material embriogénico que dependieron del genotipo. El grado de detección dependió del marcador empleado. Ambos marcadores revelaron baja inestabilidad intraclonal. Los RAPD revelaron variación genética intra-individuo en las encinas donantes. Se discuten la variación genética pre-existente en encina, su aparición durante las primeras fases de la inducción de embriogénesis, y la presencia de tejidos provenientes de la fertilización en el explanto materno. Esto hace preciso definir la identidad genética del material donante y acometer ensayos de detección precoz de variación somaclonal. ABSTRACT Holm oak (Quercus ilex L.) is one of the most important Mediterranean forest species. It conforms the tree layer of dehesas or montados, it produces acorns to feed the livestock and it establishes symbiosis with profitable mycorrhizal fungi. Holm oak is considered as recalcitrant species in terms of seed conservation and morphogenic capacities, which complicates the development of genetic conservation and improvement programs. Vegetative propagation is one of the mightiest tools for breeding programs therefore; developing protocols for clonal regeneration of holm oak is essential. Somatic embryogenesis is considered the best tissue culture-based way of plant regeneration in forest biotechnology. The present study is focused on the study of certain aspects of somatic embryogenesis for clonal regeneration of mature holm oak. This thesis manuscript is divided into several chapters that match with different aspects of the embryogenic system. Somatic embryogenesis induction was achieved on maternal teguments of developing ovules from immature acorns of adult holm oak trees. Despite the low induction frequencies, the generated embryogenic lines were amplified by secondary embryogenesis. A decline in the differentiation capacity over time was also observed. It was concluded that both genotype and culture media formulation influenced the embryogenic response, being the Schenk and Hildebrandt´s macronutrients formulation from culture medium and the lack of plant growth regulators the most effective combination for the induction of the embryogenic response. It has been suggested the existence of a developmental window in which ovules are prone to induction. Genotype influenced the proliferation capacity and the plant conversion of somatic embryos, which was also favoured by the presence of indol-3-butyric acid and 6-bencyladenine. The use of temporary immersion systems as proliferation in liquid culture of the embryogenic lines increased the growth depending on genotype, when compared to semisolid cultures. However, it did not improve the differentiation of single cotyledonary embryos. A protocol for the initiation and maintenance of embryogenic suspension cultures was established for several embryogenic lines with highly dense inoculi of embryogenic clusters from proliferating semisolid cultures. In order to avoid the loss of vigour and morphogenic ability of embryogenic lines due to prolonged cultures, a cryopreservation protocol for embryogenic lines of holm oak has been developed. During the determination of the influence of cryoprotective agents on the growth and differentiation capacities before and after liquid nitrogen immersion, it was concluded that both responses were independent from each other and also under the influence of genotype and the type of cryopreserved material. The combination of sucrose and PVS2 prior liquid nitrogen immersion provided higher recovery rates. When the same embryogenic lines were cryopreserved for 30 days, none was able to differentiate. The SSRs analysis of the short-term cryopreserved material detected somaclonal variation. Both SSR and RAPD markers showed high sensitivity to detect genetic differences between the donor trees and the generated embryogenic material. Nevertheless, the degree of instability detection depended on the marker. The SSR analysis indicated a relationship between genotype, the studied loci and the located polymorphisms. Also, both markers revealed low intraclonal genetic variation. The RAPD detected genetic variation within the donor trees. The presence of pre-existent genetic variation within mature trees, in addition to its occurrence during the early stages of the embryogenic induction, and the presence of tissues of fertilisation origin within the maternal explants are all discussed. Nonetheless, the determination of the genetic identity of donor material is required, in addition to early detection methods of somaclonal variation.