Effects of sheep and horse manure and pine bark amendments on metal distribution and chemical properties of contaminated mine soils.

Mine soils usually contain large levels of heavy metals and poor fertility conditions which limit their reclamation and the application of phyto-remediation technologies. Two organic waste materials (pine bark compost and sheep and horse manure compost), with different pHs and varying degrees of humification and nutrient contents, were applied as amendments to assess their effects on copper (Cu) and zinc (Zn) bioavailability and on fertility conditions of mine soils. Soil samples collected from two abandoned mining areas near Madrid (Spain) were mixed with 0, 30 and 60 t ha?1 of the organic amendments. The concentrations of metals among the different mineral and organic fractions of soil were determined by several extraction procedures to study the metal distribution in the solid phase of the soil affected by the organic amendments. The results showed that the manure amendment increased the soil pH and the cation exchange capacity and enhanced the nutrient levels of these soils. The pine bark amendment decreased the soil pH and did not significantly change the nutrient status of soil. Soil pH, organic matter content and its degree of humification, which were altered by the amendments, were the main factors affecting Cu fractionation. Zn fractionation was mainly affected by soil pH. The addition of manure not only improved soil fertility, but also decreased metal bioavailability resulting in a reduction of metal toxicity. Conversely, pine bark amendment increased metal ioavailability. The use of sheep and horse manure could be a cost-effective practice for the restoration of contaminated mine soils.

Physical characterization of growing media using standard methods (CEN)- Limitations of Applicability for pine bark and vermiculite

CEN standards have helped to harmonize analytical methods for substrate analysis.Though, for special substrates or constituents the applicability might be Iimited. In this paper a comparative study of implementation of CEN standards to samples of pine bark and vermiculite has been carried out. For composted pine bark, an elongation of the equilibrium period up to 72 instead of 48 hours might increase the accuracy of determinations physical parameters according to EN 13041. For vermiculite, we suggest pycnometry as a feasible technique for the determination of particle density (PD), as the determination of organic matter (OM) as requested by EN 13041 for the calculation of the PD seems not to be applicable for this kind of material.

Fine spatial pattern of an epiphytic lichen species is affected by habitat conditions in two forest types in the Iberian Mediterranean region

Persistence and abundance of species is determined by habitat availability and the ability to disperse and colonize habitats at contrasting spatial scales. Favourable habitat fragments are also heterogeneous in quality, providing differing opportunities for establishment and affecting the population dynamics of a species. Based on these principles, we suggest that the presence and abundance of epiphytes may reflect their dispersal ability, which is primarily determined by the spatial structure of host trees, but also by host quality. To our knowledge there has been no explicit test of the importance of host tree spatial pattern for epiphytes in Mediterranean forests. We hypothesized that performance and host occupancy in a favourable habitat depend on the spatial pattern of host trees, because this pattern affects the dispersal ability of each epiphyte and it also determines the availability of suitable sites for establishment. We tested this hypothesis using new point pattern analysis tools and generalized linear mixed models to investigate the spatial distribution and performance of the epiphytic lichen Lobaria pulmonaria, which inhabits two types of host trees (beeches and Iberian oaks). We tested the effects on L. pulmonaria distribution of tree size, spatial configuration, and host tree identity. We built a model including tree size, stand structure, and several neighbourhood predictors to understand the effect of host tree on L. pulmonaria. We also investigated the relative importance of spatial patterning on the presence and abundance of the species, independently of the host tree configuration. L. pulmonaria distribution was highly dependent on habitat quality for successful establishment, i.e., tree species identity, tree diameter, and several forest stand structure surrogates. For beech trees, tree diameter was the main factor influencing presence and cover of the lichen, although larger lichen-colonized trees were located close to focal trees, i.e., young trees. However, oak diameter was not an important factor, suggesting that bark roughness at all diameters favoured lichen establishment. Our results indicate that L. pulmonaria dispersal is not spatially restricted, but it is dependent on habitat quality. Furthermore, new spatial analysis tools suggested that L. pulmonaria cover exhibits a distinct pattern, although the spatial pattern of tree position and size was random.

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.

Fine spatial pattern of an epiphytic lichen species is affected by habitat conditions in two forest types in the Iberian Mediterranean region

Persistence and abundance of species is determined by habitat availability and the ability to disperse and colonize habitats at contrasting spatial scales. Favourable habitat fragments are also heterogeneous in quality, providing differing opportunities for establishment and affecting the population dynamics of a species. Based on these principles, we suggest that the presence and abundance of epiphytes may reflect their dispersal ability, which is primarily determined by the spatial structure of host trees, but also by host quality. To our knowledge there has been no explicit test of the importance of host tree spatial pattern for epiphytes in Mediterranean forests. We hypothesized that performance and host occupancy in a favourable habitat depend on the spatial pattern of host trees, because this pattern affects the dispersal ability of each epiphyte and it also determines the availability of suitable sites for establishment. We tested this hypothesis using new point pattern analysis tools and generalized linear mixed models to investigate the spatial distribution and performance of the epiphytic lichen Lobaria pulmonaria, which inhabits two types of host trees (beeches and Iberian oaks). We tested the effects on L. pulmonaria distribution of tree size, spatial configuration, and host tree identity. We built a model including tree size, stand structure, and several neighbourhood predictors to understand the effect of host tree on L. pulmonaria. We also investigated the relative importance of spatial patterning on the presence and abundance of the species, independently of the host tree configuration. L. pulmonaria distribution was highly dependent on habitat quality for successful establishment, i.e., tree species identity, tree diameter, and several forest stand structure surrogates. For beech trees, tree diameter was the main factor influencing presence and cover of the lichen, although larger lichen-colonized trees were located close to focal trees, i.e., young trees. However, oak diameter was not an important factor, suggesting that bark roughness at all diameters favoured lichen establishment. Our results indicate that L. pulmonaria dispersal is not spatially restricted, but it is dependent on habitat quality. Furthermore, new spatial analysis tools suggested that L. pulmonaria cover exhibits a distinct pattern, although the spatial pattern of tree position and size was random.

Valorización de subproductos agrícolas y forestales como sustratos de cultivo en el estado de Oaxaca (México)

El presente trabajo aborda el aprovechamiento de algunos subproductos agrícolas (bagazo de maguey y fibra de coco) y forestales (corteza de pino) en el Estado de Oaxaca (Sur de México). El objetivo principal se centra en localizar, cuantificar y caracterizar estos con vistas a su aplicación como sustratos o componentes de sustratos en cultivos ornamentales, forestales y hortícolas, y a su uso como enmiendas en cultivos tipo. Así mismo se persigue reducir el uso de la turba y la tierra de monte como sustratos mayoritarios en la actualidad. Para la localización de los subproductos se utilizaron los datos de los registros parcelarios de los productores de coco para la obtención de copra (generadores de fibra de coco) de la región costa y de los productores de mezcal (generadores del residuo de bagazo de maguey) de la región valles centrales, así como las ubicaciones de los aserraderos forestales en el Estado de Oaxaca. Se emplea un Sistema de Información Geográfica (SIG) con una cartografía digitalizada de los elementos del medio (clima, geología y suelo), de los cultivos generadores (bagazo de maguey, fibra de coco y corteza de pino), de la agricultura protegida como receptora (tomate) y de la agricultura extensiva con cultivos receptores de enmienda (café, hule, limón, mango, palma de coco y maguey). La producción anual de los residuos se cartografía y cuantifica con los siguientes resultados: bagazo de maguey 624.000 t, fibra de coco 86.000 m3 y 72.000 t de corteza de pino. Mediante el estudio de las características de los suelos de los cultivos receptores y de los requerimientos de materia orgánica de cada cultivo se calcularon las necesidades totales de materia orgánica para cada suelo. Los resultados de las cantidades globales para cada cultivo en todo el Estado muestran una necesidad total de 3.112.000 t de materia orgánica como enmienda. Con los datos obtenidos y a través de un algoritmo matemático se realiza una propuesta de localización de dos plantas de compostaje (de bagazo de maguey y fibra de coco) y cuatro plantas de compostaje de corteza de pino. Con el fin de conocer los subproductos a valorizar como sustrato o componente de sustrato se caracteriza su composición física‐química, siguiendo Normas UNE‐EN, y se analizan mediante Resonancia Magnética Nuclear (RMN). Para el acondicionamiento de bagazo de maguey y la corteza de pino se realizaron ensayos de compostaje. Al final de 241 días la temperatura y la humedad de ambos procesos se encontraban en los rangos recomendados, indicando que los materiales estaban estabilizados y con calidad para ser utilizados como sustrato o componente de sustrato. Para la fibra de coco se realizó el proceso de molienda en seco de conchas de coco provenientes de la comunidad de Río Grande Oaxaca (Principal zona productora de copra en Oaxaca). Posteriormente se emplean los materiales obtenidos como componentes para sustratos de cultivo. Se estudia el compost de bagazo de maguey y siete mezclas; el compost de corteza de pino y ocho mezclas y la fibra de coco con tres mezclas. Estos sustratos alternativos permiten obtener mezclas y reducir el uso de la tierra de monte, turba, arcilla expandida y vermiculita, siendo por tanto una alternativa sostenible para la producción en invernadero. Se elaboraron mezclas especificas para el cultivo de Lilium hibrido asiático y oriental (siete mezclas), sustratos eco‐compatibles para cultivo de tomate (nueve mezclas), para la producción de planta forestal (siete mezclas) y para la producción de plántula hortícola (ocho mezclas). Como resultados más destacados del bagazo de maguey, corteza de pino y las mezclas obtenidas se resume lo siguiente: el bagazo de maguey, con volúmenes crecientes de turba (20, 30, 50 y 60 %) y la corteza de pino, con volúmenes de turba 40 y 60%, presentan valores muy recomendados de porosidad, capacidad de aireación, capacidad de retención de humedad y equilibrio agua‐aire. Para la fibra de coco, la procedente de Río Grande presenta mejor valoración que la muestra comercial de fibra de coco de Morelos. Por último se llevó a cabo la evaluación agronómica de los sustratos‐mezclas, realizando cinco experimentos por separado, estudiando el desarrollo vegetal de cultivos tipo, que se concretan en los siguientes ensayos: 1. Producción de Lilium asiático y oriental en cama para flor de corte; 2. Producción de Lilium oriental en contenedor para flor de corte; 3. Producción de plántula forestal (Pinus greggii E y Pinus oaxacana M); 4. Producción de tomate (Solanum lycopersicum L) y 5. Producción de plántula de tomate en semillero (Solanum lycopersicum L). En relación a la producción de Lilium hibrido asiático en cama los sustratos corteza de pino (CPTU 80:20 v/v), corteza de pino + sustrato comercial (CPSC 80:20 v/v) y corteza de pino+turba+arcilla expandida+vermiculita (CPTAEV2 30:40:15:15 v/v) presentan los mejores resultados. Dichos sustratos también presentan adecuados resultados para Lilium hibrido oriental con excepción de la corteza de pino + turba (CPTU 80:20 v/v). En la producción de Lilium hibrido oriental en contenedor para flor de corte, además de los sustratos de CPSC y CPTAEV2, la mezcla de corteza de pino+turba+arcilla expandida+vermiculita (CPTAEV 70:20:5:5 v/v) manifestó una respuesta favorable. En el ensayo de producción de plántulas de Pinus greggii E y Pinus oaxacana Mirov, las mezclas con corteza de pino+turba+arcilla expandida+vermiculita (CPTAEV2 30:40:15:15 v/v) y bagazo de maguey turba+arcilla expandida+vermiculita (BMTAEV2 30:60:5:5 v/v) son una alternativa que permite disminuir el empleo de turba, arcilla expandida y vermiculita, en comparación con el sustrato testigo de turba+arcilla expandida+vermiculita (TAEV 60:30:10 v/v). En la producción de tomate (Solanum lycopersicum L) frente a la utilización actual del serrín sin compostar (SSC), las mezclas alternativas de bagazo de maguey+turba (BMT 70:30 v/v), fibra de coco de Río Grande (FCRG 100v/v) y corteza de pino+turba (CPT 70:30 v/v), presentaron los mejores resultados en rendimientos. Así mismo, en la producción de plántulas de tomate las dos mezclas alternativas de bagazo de maguey+turba+ arcilla expandida+vermiculita (BMTAEV5 50:30:10:10 v/v) y (BMTAEV6 40:40:10:10 v/v) presentaron mejores resultados que los obtenidos en la mezcla comercial (Sunshine 3), mayoritariamente utilizada en México en la producción de plántula de tomate y hortícola. ABSTRACT This paper addresses the use of some agricultural products (maguey bagasse and coconut fiber) and forestry (pine bark) in the State of Oaxaca (southern Mexico). The principal purpose is to locate, quantify and characterize these with the idea of applying them as substrates or substrate components in ornamental crops, forestry, horticultural, and their use as crop amendments. On the other hand, the reduction of peat and forest soil as main substrates is pursued. For the location of the products, registry parcel data from copra producers (coconut fiber generators) of the coastal region and mescal producers (maguey bagasse residue generators) of the central valleys region, as well as the locations of forest mills in the State of Oaxaca. A Geographic Information System (GIS) with digital mapping of environmental factors (climate, geology and soil), crop generators of residues (maguey bagasse, coconut and pine bark) receptors of amendments such as protected agriculture (tomato) and extensive agriculture crops (coffee, rubber, lemon, mango, coconut and agave). The annual production of waste is mapped and quantified with the following results: 624,000t maguey bagasse, coconut fiber 72,000 m3 and 86,000 t of pine bark. Through the study of receiving crops soils properties of and organic matter requirements of each crop, total needs of organic matter for each soil were estimated. The results of the total quantities for each crop across the state show a total of 3,112,000 t of organic matter needed as amendment. Using that data and a mathematical algorithm, the location of two composting plants (agave bagasse and coconut fiber) and four composting plants pine bark was proposed. In order to know the by‐products that were going to be used as substrates or substrate components, their physical‐chemical composition was analyzed following UNE‐EN technics. Furthermore they were analyzed by Nuclear Magnetic Resonance (NMR). For conditioning of maguey bagasse and pine bark, composting essays were conducted. At the end of 241 days the temperature and humidity of both processes were at the recommended ranges, indicating that the materials were stabilized and had reached the quality to be used as a substrate or substrate component. Coconut shells from the community of Rio Grande Oaxaca (Main copra producing area in Oaxaca) were put through a process of dry milling. Subsequently, the obtained materials were used as components for growing media. We studied the maguey bagasse compost and seven mixtures; the pine bark compost and eight blends and coconut fiber with three mixtures. These alternative substrates allow obtaining mixtures and reduce the use of forest soil, peat, vermiculite and expanded clay, making it a sustainable alternative for greenhouse production. Specific mixtures were prepared for growing Lillium, Asian and eastern hybrids (seven blends), eco‐compatible substrates for tomato (nine mixtures), for producing forest plant (seven mixtures) and for the production of horticultural seedlings (eight mixtures). Results from maguey bagasse, pine bark and mixtures obtained are summarized as follows: the maguey bagasse, with increasing volumes of peat (20, 30, 50 and 60%) and pine bark mixed with 40 and 60% peat by volume, have very recommended values of porosity, aeration capacity, water retention capacity and water‐air balance. Coconut fiber from Rio Grande had better quality than commercial coconut fiber from Morelos. Finally the agronomic evaluation of substrates‐mixtures was carried out conducting five experiments separately: 1. Production of Asiatic and Eastern Lilium in bed for cut flower, 2. Production of oriental Lillium in container for cut flower, 3.Production of forest seedlings (Pinus greggii E and Pinus oaxacana M), 4. Production of tomato (Solanum lycopersicum L) and 5. Tomato seedling production in seedbed (Solanum lycopersicum L). In relation to the production of hybrid Asian Lilium in bed, pine bark substrates (CPTU 80:20 v/v), pine bark + commercial substrate (CPSC 80:20 v/v) and pine bark + peat + expanded clay + vermiculite (CPTAEV2 30:40:15:15 v/v) showed the best results. Such substrates also have adequate results for Lilium Oriental hybrid except pine bark + peat (CPTU 80:20 v / v). In the production of Lilium oriental hybrid container for cut flower, besides the CPSC and CPTAEV2 substrates, the mixture of pine bark + peat + vermiculite expanded clay (CPTAEV 70:20:5:5 v / v) showed a favorable response. In the production of Pinus greggii E and Pinus oaxacana Mirov seedlings trial, mixtures with pine bark + peat + expanded clay + vermiculite (CPTAEV2 30:40:15:15 v/v) and maguey bagasse+ peat+ expanded clay + vermiculite (BMTAEV2 30:60:5:5 v / v) are an alternative which allows reducing the use of peat, vermiculite and expanded clay in comparison with the control substrate made of peat + expanded clay+ vermiculite (60:30 TAEV: 10 v/v). In the production of tomato (Solanum lycopersicum L), alternative mixes of maguey bagasse + peat (BMT 70:30 v/v), coconut fiber from Rio Grande (FCRG 100 v / v) and pine bark + peat (CPT 70:30 v / v) showed the best results in yields versus the current use of sawdust without compost (SSC). Likewise, in the production of tomato seedlings of the two alternative mixtures maguey bagasse + peat expanded clay + vermiculite (BMTAEV5 50:30:10:10 v/v) and (BMTAEV6 40:40:10:10 v/v) had better results than those obtained in the commercial mixture (Sunshine 3), mainly used in Mexico in tomato seedling production and horticulture.

Phytoextraction of heavy metals from mine soils using hyperaccumulator plants.

Phytoextraction is an environmental-friendly and cost-effective technology that uses metal hyperaccumulator plants to remove heavy metals from soils. The metals are absorbed by the roots, transported and accumulated in the aerial parts of the plants, which can be harvested and eliminated. The aim of this work was to study some hyperaccumulator species that could be useful to decontaminate mine soils and also to investigate the bioavailability and uptake of these metals by plants with the addition of organic amendments. Pot experiments were performed with soil samples collected from two mining areas in the north of Madrid, where there was an intense mining activity more than 50 years ago. Three species (Thlaspi arvense, Brassica juncea and Atriplex halimus) were grown under controlled conditions in pots filled with contaminated soils mixed with 0 Mg, 30 Mg and 60 Mg per hectare of two different organic amendments: a commercial compost made of pine bark, peat and wood fiber and other made of horse and sheep manure and wood fiber. Plants were harvested at the end of their crop cycle and were digested in order to measure metal concentration (Zn, Cu and Cd) in roots and shoots. Highest plant metal concentration was observed in pots treated with pine bark amendment and with pure soil due to an increase in metal bioavailability with decreasing pH. Also in those treatments the total plant biomass was lower, even some plants could not germinate. On the contrary, there was a lower metal concentration in plant tissues of pots with manure because its higher pH whereas plant growth was significantly larger so there was an incresing amount of metals removed from soil by plants. Comparing the three species results indicate a higher total metal uptake in A. halimus than B. juncea and T. arvense. In conclusion, results show that pH affects metal bioavailability and uptake by hyperaccumulator plants. Addition of organic amendments could be a successful technique for stabilization of metals in contaminated soils.

Bioavailability and extraction of heavy metals from contaminated soil by Atriplex halimus

Pot experiments were performed to evaluate the phytoremediation capacity of plants of Atriplex halimus grown in contaminated mine soils and to investigate the effects of organic amendments on the metal bioavailability and uptake of these metals by plants. Soil samples collected from abandoned mine sites north of Madrid (Spain) were mixed with 0, 30 and 60 Mg ha?1 of two organic amendments, with different pH and nutrients content: pine-bark compost and horse- and sheep-manure compost. The increasing soil organic matter content and pH by the application of manure amendment reduced metal bioavailability in soil stabilising them. The proportion of Cu in the most bioavailable fractions (sum of the water-soluble, exchangeable, acid-soluble and Fe?Mn oxides fractions) decreased with the addition of 60 Mg ha?1 of manure from 62% to 52% in one of the soils studied and from 50% to 30% in the other. This amendment also reduced Zn proportion in water-soluble and exchangeable fractions from 17% to 13% in one of the soils. Manure decreased metal concentrations in shoots of A. halimus, from 97 to 35 mg kg?1 of Cu, from 211 to 98 mg kg?1 of Zn and from 1.4 to 0.6 mg kg?1 of Cd. In these treatments there was a higher plant growth due to the lower metal toxicity and the improvement of nutrients content in soil. This higher growth resulted in a higher total metal accumulation in plant biomass and therefore in a greater amount of metals removed from soil, so manure could be useful for phytoextraction purposes. This amendment increased metal accumulation in shoots from 37 to 138 mg pot?1 of Cu, from 299 to 445 mg pot?1 of Zn and from 1.8 to 3.7 mg pot?1 of Cd. Pine bark amendment did not significantly alter metal availability and its uptake by plants. Plants of A. halimus managed to reduce total Zn concentration in one of the soils from 146 to 130 mg kg?1, but its phytoextraction capacity was insufficient to remediate contaminated soils in the short-to-medium term. However, A. halimus could be, in combination with manure amendment, appropriate for the phytostabilization of metals in mine soils.

Phytoextraction of heavy metals from mine soils using hyperaccumulator plants

The aim of this work was to study some hyperaccumulator species that could be useful to decontaminate mine soils and also to investigate the bioavailability and uptake of these metals by plants with the addition of organic amendments. Pot experiments were performed with soil samples collected from two mining areas in the north of Madrid, where there was an intense mining activity more than 50 years ago. Three species (Thlaspi arvense, Brassica juncea and Atriplex halimus) were grown under controlled conditions in pots filled with contaminated soils mixed with 0 Mg, 30 Mg and 60 Mg per hectare of two different organic amendments: a commercial compost made of pine bark, peat and wood fiber and other made of horse and sheep manure and wood fiber. Plants were harvested at the end of their crop cycle and were digested in order to measure metal concentration (Zn, Cu and Cd) in roots and shoots. Highest plant metal concentration was observed in pots treated with pine bark amendment and with pure soil due to an increase in metal bioavailability with decreasing pH. Also in those treatments the total plant biomass was lower, even some plants could not germinate. On the contrary, there was a lower metal concentration in plant tissues of pots with manure because its higher pH whereas plant growth was significantly larger so there was an incresing amount of metals removed from soil by plants. Comparing the three species results indicate a higher total metal uptake in A. halimus than B. juncea and T. arvense. In conclusion, results show that pH affects metal bioavailability and uptake by hyperaccumulator plants. Addition of organic amendments could be a successful technique for stabilization of metals in contaminated soils.

Phytostabilization of metals in mine soils using Brassica juncea in combination with organic amendments

Background and aims The high metal bioavailability and the poor conditions of mine soils yield a low plant biomass, limiting the application of phytoremediation techniques. A greenhouse experiment was performed to evaluate the effects of organic amendments on metal stabilization and the potential of Brassica juncea L. for phytostabilization in mine soils. Methods Plants were grown in pots filled with soils collected from two mine sites located in Central Spain mixed with 0, 30 and 60 tha?1 of pine bark compost and horse- and sheep-manure compost. Plant biomass and metal concentrations in roots and shoots were measured. Metal bioavailability was assessed using a rhizosphere-based method (rhizo), which consists of a mixture of low-molecular-weight organic acids to simulate root exudates. Results Manure reduced metal concentrations in shoots (10?50 % reduction of Cu and 40?80 % of Zn in comparison with non-amended soils), bioconcentration factor (10?50 % of Cu and 40?80 % of Zn) and metal bioavailability in soil (40?50 % of Cu and 10?30 % of Zn) due to the high pH and the contribution of organic matter. Manure improved soil fertility and was also able to increase plant biomass (5?20 times in shoots and 3?30 times in roots), which resulted in a greater amount of metals removed from soil and accumulated in roots (increase of 2?7 times of Cu and Zn). Plants grown in pine bark treatments and in non-amended soils showed a limited biomass and high metal concentrations in shoots. Conclusions The addition of manure could be effective for the stabilization of metals and for enhancing the phytostabilization ability of B. juncea in mine soils. In this study, this species resulted to be a potential candidate for phytostabilization in combination with manure, differing from previous results, in which B. juncea had been recognized as a phytoextraction plant.

Variation throughout the tree stem in the physical-mechanical properties of the wood of Abies alba Mill. from the Spanish Pyrenees

This study analyses the variation of main physical-mechanical properties of wood along the longitudinal and radial directions of the tree for Abies alba Mill. growing in the Spanish Pyrenees. Small clear specimens were used to study the properties of volumetric shrinkage (VS), density (?), hardness (H), bending strength (MOR), modulus of elasticity (MOE), maximum compressive strength parallel to the grain (MCS) and impact strength (K). Several models of properties variation in the longitudinal and radial directions were analyzed. Main trends of variation of properties throughout the tree stem were identified although none of them could be fitted to predictive statistical models. Along the longitudinal direction, the properties studied followed a downward trend from the base to the crown, which was not significant in all cases, indicating that no differences in quality existed. Throughout the radial direction the trend is upward for the first 40-50 growth rings, after which it slopes downwards, more gently at first until rings 70-75 and then more steeply. This behaviour is related to variation in wood structure from the pith to the bark, depending on whether the wood is juvenile, sapwood or heartwood, and to wood maturity and microfibril angle. Authors encourage carrying further studies on other populations of A. alba in the Spanish Pyrenees to check if the trends found in this study apply to other provenances.

Coleópteros Saproxílicos de los Bosques de Montaña en el Norte de la Comunidad de Madrid

La fauna saproxílica ha cobrado mucha relevancia en los últimos años. Por una parte, debido a los múltiples papeles que juega en la ecología de los bosques y por otra, por encontrarse muchas especies de ese grupo amenazadas como consecuencia de la intensificación de las actividades forestales. Se supone que los bosques de Europa meridional albergan una fauna saproxílica rica y variada. Sin embargo apenas se han realizado estudios que permitan conocer la composición de las biocenosis saproxílicas, así como el estatus y grado de amenaza a que está sometida cada especie. En esta tesis se han muestreado de forma sistemática las comunidades de coleópteros saproxílicos de cuatro montes del norte de la Comunidad de Madrid, muy diferentes a pesar de su cercanía: Dehesa Bonita de Somosierra, Hayedo de Montejo, Dehesa de Madarcos y Pinar de La Maleza. Para llevar a cabo la recogida de muestras se definió una estación de muestreo tipo, compuesta por cuatro trampas aéreas con cebo oloroso, dos trampas de ventana y una trampa de embudos. En los dos primeros montes mencionados se desplegaron seis estaciones de muestreo, por sólo tres en los otros dos. El primer objetivo de esta tesis fue conocer las especies de coleópteros que constituyen la fauna de los cuatro montes estudiados. Los muestreos sistemáticos reportaron la presencia de un total de 357 especies de coleópteros saproxílicos, siendo el Hayedo de Montejo el bosque con la diversidad más alta, 220 especies; le siguen la Dehesa de Madarcos con 116; el pinar de La Maleza con 115; y la Dehesa de Somosierra con 109, si bien la fauna de este ultimo bosque podría ser mucho más variada dado que la interferencia del ganado con algunos dispositivos de captura hizo que se perdiera parte del material allí recolectado. Se han encontrado nueve especies nuevas para la fauna de la Península Ibérica, y otras muchas desconocidas previamente en el centro peninsular. Un total de 50 especies se encuentran incluidas en la Lista Roja Europea de coleópteros saproxílicos. El segundo objetivo fue estimar la riqueza de fauna de coleópteros saproxílicos en cada bosque. Partiendo de los datos de los respectivo muestreos se calcularon diferentes estimadores, paramétricos y no paramétricos, y se elaboraron las curvas de rarefacción para cada bosque y para el conjunto. El bosque con más biodiversidad ha resultado ser el Hayedo de Montejo, que albergaría entre 254 y 332 especies. En el Pinar de la Maleza se encontrarían de 132 a 223; de 128 a 205 en la Dehesa de Somosierra; y entre 134 y 188 en la Dehesa de Madarcos. Para el conjunto del área se estimó la presencia de entre 411 y 512 especies. El tercer objetivo fue evaluar la influencia de algunos factores como la especie arbórea dominante y la cantidad de madera muerta en la riqueza y diversidad de coleópteros saproxílicos. El estudio se realizó en el Hayedo de Montejo, encontrando una alta correlación positiva entre cantidad y calidad de madera muerta, y diversidad y riqueza de especies de coleópteros saproxílicos. El cuarto objetivo fue evaluar la eficacia y complementariedad de los diferentes tipos de dispositivos de captura empleados en los muestreos. El más eficaz resultó ser la trampa de ventana, seguido por la trampa aérea con cebo oloroso, y finalmente la trampa de embudos. La mayor complementariedad se encontró entre trampas de ventana y aéreas con cebo oloroso. No obstante, si se quiere optimizar la exhaustividad del inventario no se debe prescindir de ninguno de los sistemas. En cualquier caso, puede afirmarse que la efectividad de los tres tipos de dispositivos de captura utilizados en los muestreos fue baja, pues para la gran mayoría de especies presentes se capturó un número de ejemplares realmente bajo. El bajo rendimiento de captura implica un bajo impacto sobre las poblaciones de las especies muestreadas, y esto supone una importante ventaja desde el punto de vista de la conservación. Finalmente, se dejan algunas recomendaciones de manejo a aplicar en cada uno de los montes con el fin de preservar o mejorar los hábitats utilizables por la fauna saproxílica que garanticen el mantenimiento y mejora de dichas comunidades. ABSTRACT The saproxylic fauna has become increasingly important in recent years. It has been due, on the one hand, to the multiple roles they play in the forest ecosystems and, on the other, because of the large proportion of endangered saproxylic species as a result of the intensification of forestry. It is generally assumed that southern Europe forests are home to a rich and diverse saproxylic fauna. However, there are hardly any studies leading to reveal the composition of saproxylic biocenosis, or the stage and extent of the threat each species is suffering. For the purpose of this thesis the communities of saproxylic beetles of four mountain forests in northern Comunidad de Madrid have been systematically sampled: Dehesa Bonita de Somosierra, Hayedo de Montejo, Dehesa de Madarcos and Pinar de La Maleza. They are very different from each other in spite of not being too far apart. In order to carry out sample collection, a standard sampling station was defined as follows: four smelly bait aerial traps, two window traps and one funnel trap. Six sampling stations were deployed in each of the first two forests mentioned above; put only three in each of the other two. The first aim of this thesis was to determine the composition of saproxylic beetles fauna inhabiting each of the four forests studied. Systematic sampling reported the presence of a total of 357 species of saproxylic beetles. Hayedo de Montejo, with 220 species, is the forest with the highest diversity, followed by Dehesa de Madarcos, 116; Pinar de La Maleza, 115, and Dehesa de Somosierra, 109. The fauna of the latter forest, however, could be much more varied, since cattle interference with some capture devices caused the loss of part of the material collected there. Nine new species in the fauna of the Iberian Peninsula were found, and many others previously unknown in the center of the Peninsula. A total of 41 of those species are included in the European Red List of saproxylic beetles. The second aim was to estimate the richness of saproxylic (beetle) fauna in each forest. From the data of the respective sampling, different parametric and nonparametric estimators were calculated, and rarefaction curves for each forest, as well as for the four of them together, were drawn. The most biodiverse forest turned out to be Hayedo de Montejo, which houses between 254 and 332 species. In Pinar de La Maleza, between 132 and 223 species were found; between 128 and 205 in Dehesa de Somosierra, and between 134 and 188 in Dehesa de Madarcos. The estimated diversity of species for the whole area ranges from 411 to 512. The third aim was to evaluate the influence of such factors as the dominant tree species and the amount of dead wood in the richness and diversity of saproxylic beetles. The study was conducted at Hayedo de Montejo, finding a high positive correlation between quantity and quality of coarse woody debris and diversity and richness of saproxylic beetle species. The fourth aim was to evaluate the effectiveness and complementarity of the different sampling methods used in this research work. The most effective proved to be the window trap, followed by the smelly bait aerial trap and the funnel trap, in that order. The greater complementarity was found between window and aerial traps. However, in order to optimize the completeness of the inventory, neither of the systems should be discarded. Nevertheless, the effectiveness of the three types of capture devices used in this piece of research was on the whole rather low, since for the vast majority of species, a significant low number of specimens were captured. Poor trapping performance implies a low impact on the populations of the sampled species, and this is an important advantage in terms of conservation. Finally, this thesis gives some recommendations with regard to the management of each of those four forests, leading to preserve and improve the habitats of the saproxylic wildlife and so ensure the maintenance and growth of their communities.

Conservación de plantas regeneradas in vitro y análisis de la variación somaclonal de Cinchona officinalis, Linneo

Cinchona officinalis (Rubiaceae), especie endémica del Valle de Loja, ubicado en la región sur del Ecuador, es un recurso forestal de importancia medicinal y ecológica, además la especie ha sido catalogada como planta nacional y es un ícono de la región sur por su aporte a la farmacopea mundial. Esta especie, entre los siglos XVII-XIX sufrió una gran presión en sus poblaciones debido a la extracción masiva de la corteza para la cura del paludismo. Aunque la actividad extractiva generó grandes ingresos a la Corona Española y a la región Sur del Ecuador, ésta fue poco o nada sustentable ecológicamente, provocando la desaparición de la especie en muchos sitios de la provincia, pues, en su momento, no se consideraron alternativas de recuperación de las poblaciones naturales. Actualmente la extracción y consumo de la corteza en la zona de origen es baja o nula, sin embargo esta zona enfrenta nuevas amenazas. La deforestación a causa de proyectos de desarrollo en infraestructuras, la práctica de actividades agrícolas y de ganadería, y los efectos del cambio climático han ocasionado, en estos últimos años, la fragmentación de los ecosistemas. La mayoría de los bosques del sur del Ecuador se han convertido en parches aislados (los bosques en los que se distribuye C. officinalis no son la excepción) siendo esta la principal causa para que la especie se encuentre en estado de amenaza. Los individuos de la especie tienen una alta capacidad de rebrote y producen semillas durante todo el año; sin embargo la capacidad germinativa y la tasa de sobrevivencia son bajas, además de estas dificultades la especie requiere de la asociación con otras especies vegetales para su desarrollo, lo cual ha limitado su distribución en pequeños parches aislados. Con esta problemática, la recuperación natural de las poblaciones es una necesidad evidente. Varios trabajos y esfuerzos previos se han realizado a nivel local: i. Identificación de la distribución actual y potencial; ii. Determinación de la fenología y fructificación iii. Programas de educación ambiental, iv. Análisis moleculares para determinar la diversidad genética. v. Ensayos de propagación vegetativa; y otras acciones de tipo cultural. No obstante, el estado de conservación y manejo de las poblaciones naturales no ha mejorado significativamente, siendo necesaria la aplicación de estrategias integradas de conservación in situ y ex situ, que permitan la recuperación y permanencia de las poblaciones naturales a largo plazo. El presente trabajo tiene como fin dar alternativas para el cultivo de tejidos in vitro de Cinchona officinalis centrados en la propagación masiva a partir de semillas, análisis de la fidelidad genética y alternativas de conservación de tejidos. Los objetivos específicos que se plantean son: i. Analizar el proceso de germinación y proliferación in vitro. ii. Evaluar la estabilidad genética en explantes cultivados in vitro, mediante marcadores ISSR. iii. Establecer protocolos de conservación in vitro mediante limitación del crecimiento y criopreservación de segmentos nodales y yemas. Los resultados más significativos de esta investigación fueron: i. El desarrollo de protocolos eficientes para mejorar los porcentajes de germinación y la proliferación de brotes en explantos cultivados in vitro. Para evaluar el efecto de los fenoles sobre la germinación, se determinó el contenido total de fenoles y el porcentaje de germinación en semillas de C. officinalis comparados con una especie de control, C. pubescens. Para inducir a proliferación, se utilizaron segmentos nodales de plántulas germinadas in vitro en medio Gamborg (1968) suplementado con diferentes combinaciones de reguladores de crecimiento (auxinas y citoquininas). Los resultados obtenidos sugieren que el contenido de compuestos fenólicos es alto en las semillas de C. officinalis en comparación con las semillas de C. pubescens. Estos fenoles pueden eliminarse con peróxido de hidrógeno o con lavados de agua para estimular la germinación. La formación de nuevos brotes y callos en la mayoría de las combinaciones de reguladores de crecimiento se observó en un período de 45 días. El mayor porcentaje de proliferación de brotes, formación de callos y presencia de brotes adventicios se obtuvo en medio Gamborg (B5) suplementado con 5.0 mg/l 6-bencil-aminopurina y 3.0 mg/l de ácido indol-3-butírico. ii. La evaluación de la fidelidad genética de los explantes obtenidos con distintas combinaciones de reguladores de crecimiento vegetal y diversos subcultivos. Se realizó el seguimiento a los explantes obtenidos de la fase anterior, determinando el índice de multiplicación y analizando la fidelidad genética de los tejidos obtenidos por las dos vías regenerativas: brotación directa y regeneración de brotes a partir de callos. Este análisis se realizó por amplificación mediante PCR de las secuencias ubicadas entre microsatélites-ISSR (Inter simple sequence repeat). El medio Gamborg (B5) con 3.0 mg/l de AIB y 5.0 mg/l de BAP usado como medio de inducción en la primera etapa de cultivo generó el mayor índice de proliferación (11.5). Un total de 13 marcadores ISSR fueron analizados, 6 de éstos fueron polimórficos. El mayor porcentaje de variación somaclonal fue inducido en presencia de 1.0 mg/l 2,4-D combinado con 0.2 mg/l Kin con un 1.8% en el segundo sub-cultivo de regeneración, la cual incrementó a 3.6% en el tercer sub-cultivo. Todas las combinaciones con presencia de 2,4-D produjeron la formación de callos y presentaron variación genética. Por su parte la fidelidad genética se mantuvo en los sistemas de propagación directa a través de la formación de brotes a partir de meristemos preformados. iii. El establecimiento de protocolos de conservación in vitro y crioconservación de segmentos nodales y yemas. Para la conservación limitando el crecimiento, se cultivaron segmentos nodales en los medios MS y B5 en tres concentraciones de sus componentes (25, 50 y 100%); y en medio B5 más agentes osmóticos como el manitol, sorbitol y sacarosa en diferentes concentraciones (2, 4 y 8%); los cultivos se mantuvieron por 12 meses sin subcultivos. Para el establecimiento de protocolos para la crioconservación (paralización del metabolismo) se usaron yemas axilares y apicales a las cuales se les aplicaron los métodos de encapsulación-deshidratación y vitrificación. La efectividad de los protocolos usados se determinó en función de la sobrevivencia, reducción del crecimiento y regeneración. Los resultados obtenidos en este apartado reflejan que un crecimiento limitado puede mantener tejidos durante 12 meses de almacenamiento, usando medio B5 más manitol entre 2 y 8%. En los protocolos de crioconservación, se obtuvo el mayor porcentaje de recuperación tras la congelación en NL en el tratamiento control seguido por el método crioprotector de encapsulación-deshidratación. Este trabajo brinda alternativas para la propagación de C. officinalis bajo condiciones in vitro, partiendo de material vegetal con alta diversidad genética. El material propagado puede ser fuente de germoplasma para la recuperación y reforzamiento de las poblaciones naturales así como una alternativa de producción para las comunidades locales debido a la demanda actual de corteza de la zona de origen para la elaboración de agua tónica. ABSTRACT Cinchona officinalis (Rubiaceae) is endemic to the Loja Valley, located in the southern area of Ecuador. The importance of this plant as medical and ecological resource is so great that it has been designated as the national flower and is an icon of the southern region for its contribution to the world pharmacopoeia. Between XVII-XIX centuries its population suffered great reduction due to massive harvesting of the bark to cure malaria. Although extraction activity generated large revenues to the Spanish Crown and the southern region of Ecuador, this was not ecologically sustainable, causing the disappearance of the species in many areas of the province, because during that time alternatives to prevent extinction and recover natural populations were not taken in account. Currently the extraction and consumption of bark in the area of origin is almost absent, but this species faces new threats. Deforestation due to infrastructure development, the practice of farming and ranching, and the effects of climate change had led to the fragmentation of ecosystems during the recent years. Most of the forests of southern Ecuador have become isolated patches, including those where C. officinalis is diffused. The lack of suitable habitat is today the main threat for the species. The species has a high capacity for regeneration and produces seeds throughout the year, but the germination rate is low and the growth is slow. In addition, the species requires the association with other plant species to develop. All these factors had limited its distribution to small isolated patches. The natural recovery of populations is essential to face this problem. Several studies and previous efforts had been made at local level: i. Identification of current and potential distribution; ii. Phenology determination. iii. Environmental education programs, iv. Molecular analisis to determine the genetic diversity. v. Testing of vegetative propagation; and other actions of cultural nature. Despite these efforts, the state of conservation and management of natural populations has not improved significantly. Implementation of integrated in situ and ex situ conservation strategies for the recovery and permanence of long-term natural populations is still needed. This work aims to provide alternatives for in vitro culture of tissue of Cinchona officinalis focused on mass propagation from seeds, genetic fidelity analysis and tissue conservation alternatives. The specific aims are: i. Analyze the process of germination and proliferation in vitro. ii. To evaluate the genetic stability of the explants cultured in vitro by ISSR markers. iii. Establish protocols for in vitro conservation by limiting growth and cryopreservation of nodal segments and buds. The most significant results of this research were: i. The development of efficient protocols to improve germination rates and proliferation of buds in explants cultured in vitro. To study the effect of phenols on germination, the total phenolic content and percentage germination was measured in C. officinalis and in a control species, C. pubescens, for comparison. The content of phenolic compounds in C. officinalis seeds is higher than in C. pubescens. These phenols can be removed with hydrogen peroxide or water washes to stimulate germination. To analyze the regeneration, we used nodal explants from seedlings germinated in vitro on Gamborg medium (1968) supplemented with different combinations of growth regulators (auxins and cytokinins) to induce proliferation. The formation of new shoots and calluses was observed within a period of 45 days in most combinations of growth regulators. The highest percentage of shoot proliferation, callus formation and adventitious buds were obtained in B5 medium supplemented with 5.0 mg/l 6-benzyl-aminopurine and 3.0 mg/l indole-3-butyric acid. ii. Evaluating genetic fidelity explants obtained with various combinations of plant growth regulators and different subcultures. The genetic fidelity was analyzed in tissues obtained by the two regenerative pathways: direct sprouting and shoot regeneration from callus. This analysis was performed by PCR amplification of the sequences located between microsatellite-ISSR (Inter Simple Sequence Repeat). Among a total of 13 ISSR markers analyzed, 6 were polymorphic. The highest percentage of somaclonal variation was induced in the presence of 1.0 mg/l 2,4-D combined with 0.2 mg/l Kin with 1.8% in the second round of regeneration, and increased to 3.6% in the third round. The presence of 2,4-D induced genetic variation in all the combinations of growth regulators. Meanwhile genetic fidelity remained systems propagation through direct shoot formation from meristems preformed. iii. Establishing conservation protocols in vitro and cryoconservation of nodal segments and buds. For medium-term conservation (limited growth) nodal segments were cultured in MS and B5 media at three concentrations (25, 50 and 100%); we tested B5 medium with different concentrations of osmotic agents such as mannitol, sorbitol and sucrose (2, 4 and 8%); cultures were maintained for 12 months with regular subculturing. To establish protocols for cryoconservation (cessation of metabolism) different methods of encapsulation-dehydration and vitrification were applied to axillary and apical buds. The effectiveness of the used protocols is determined based on the survival, growth and regeneration success. The results show that these tissues can be maintained in storage for 12 months, using B5 medium plus mannitol between 2 and 8%. The cryoconservation protocol with highest percentage of recovery was obtained by contral treatment, followed by freezing in NL with encapsulation-dehydration method. This work provides alternatives for the propagation in vitro of C. officinalis, starting from plant material with high genetic diversity. The obtained material represents a source of germplasm to support the recovery and strengthening of natural populations as well as a creation of alternative sources for local communities due to the current demand of bark for the preparation of tonic water.

Estudio de los factores que condicionan, a escala local, la presencia de Epidendrum rhopalostele (Orchidaceae): Implicaciones para su conservación

El análisis de los factores que determinan el establecimiento y supervivencia de orquídeas epífitas, incluyen: a) las condiciones microambientales de los bosques que las mantienen, b) preferencias por las características de los hospederos donde crecen, c) limitación en la dispersión de semillas, d) interacciones planta-planta, y e) asociaciones micorrízicas para la germinación y resultan esenciales para el desarrollo de estrategias para la conservación y manejo de este grupo de plantas. Este trabajo ha evaluado la importancia de estos factores en Epidendrum rhopalostele, orquídea epífita del bosque de niebla montano, a través de los análisis de los patrones espaciales de los árboles que la portan y de la propia orquídea, a escala de población, estudios de asociación y métodos moleculares. Estos últimos han consistido en el uso de marcadores AFLP para el análisis de la estructura genética de la orquídea y en la secuenciación-clonación de la región ITS para la identificación de los hongos micorrízicos asociados. El objetivo de esta tesis es, por tanto, una mejor comprensión de los factores que condicionan la presencia de orquídeas epífitas en los remanentes de bosque de niebla montano y una evaluación de las implicaciones para la conservación y mantenimiento de sus hábitats y la permanencia de sus poblaciones. El estudio fue realizado en un fragmento de bosque de niebla montano de sucesión secundaria situado al este de la Cordillera Real, en los Andes del sur de Ecuador, a 2250 m.s.n.m y caracterizado por una pendiente marcada, temperatura media anual de 20.8°C y precipitación anual de 2193 mm. En este fragmento se mapearon, identificaron y caracterizaron todos los árboles presentes con DBH > 1 cm y todos los individuos de Epidendrum rhopalostele. Así mismo se tomaron muestras de hoja para obtener ADN de todas las orquídeas registradas y muestras de raíces de individuos con flor de E. rhopalostele, uno por cada forófito, para el análisis filogenético de micorrizas. Análisis espaciales de patrones de puntos basados en la K de Ripley y la distancia al vecino más cercano fueron usados para los árboles, forófitos y la población de E. rhopalostele. Se observó que la distribución espacial de árboles y forófitos de E. rhopalostele no es aleatoria, ya que se ajusta a un proceso agregado de Poisson. De ahí se infiere una limitación en la dispersión de las semillas en el fragmento estudiado y en el establecimiento de la orquídea. El patrón de distribución de la población de E. rhopalostele en el fragmento muestra un agrupamiento a pequeña escala sugiriendo una preferencia por micro-sitios para el establecimiento de la orquídea con un kernel de dispersión de las semillas estimado de 0.4 m. Las características preferentes del micro-sitio como tipos de árboles (Clusia alata y árboles muertos), tolerancia a la sombra, corteza rugosa, distribución en los dos primeros metros sugieren una tendencia a distribuirse en el sotobosque. La existencia de una segregación espacial entre adultos y juveniles sugiere una competencia por recursos limitados condicionada por la preferencia de micro-sitio. La estructura genética de la población de E. rhopalostele analizada a través de Structure y PCoA evidencia la presencia de dos grupos genéticos coexistiendo en el fragmento y en los mismos forófitos, posiblemente por eventos de hibridización entre especies de Epidendrum simpátricas. Los resultados del análisis de autocorrelación espacial efectuados en GenAlex confirman una estructura genético-espacial a pequeña escala que es compatible con un mecanismo de dispersión de semillas a corta distancia ocasionada por gravedad o pequeñas escorrentías, frente a la dispersión a larga distancia promovida por el viento generalmente atribuida a las orquídeas. Para la identificación de los micobiontes se amplificó la región ITS1-5.8S-ITS2, y 47 secuencias fueron usadas para el análisis filogenético basado en neighborjoining, análisis bayesiano y máximum-likelihood que determinó que Epidendrum rhopalostele establece asociaciones micorrízicas con al menos dos especies diferentes de Tulasnella. Se registraron plantas que estaban asociadas con los dos clados de hongos encontrados, sugiriendo ausencia de limitación en la distribución del hongo. Con relación a las implicaciones para la conservación in situ resultado de este trabajo se recomienda la preservación de todo el fragmento de bosque así como de las interacciones existentes (polinizadores, micorrizas) a fin de conservar la diversidad genética de esta orquídea epífita. Si fuere necesaria una reintroducción se deben contemplar distancias entre los individuos en cada forófito dentro de un rango de 0.4 m. Para promover el reclutamiento y regeneración de E. rhopalostele, se recomienda que los forófitos correspondan preferentemente a árboles muertos o caídos y a especies, como Clusia alata, que posean además corteza rugosa, sean tolerantes a la sombra, y en el área del sotobosque con menor luminosidad. Además es conveniente que las orquídeas en su distribución vertical estén ubicadas en los primeros metros. En conclusión, la limitación en la dispersión, las características del micro-sitio, las interacciones intraespecíficas y con especies congenéricas simpátricas y las preferencias micorrízicas condicionan la presencia de esta orquídea epífita en este tipo de bosque. ABSTRACT The analysis of factors that determine the establishment and survival of epiphytic depends on factors such as a) microenvironmental conditions of forest, b) preference for host characteristics where orchids grow, c) seed dispersal limitation, d) plant-plant interaction, e) priority mycorrhizal associations for germination, are essential for the development of strategies for management and conservation. This work evaluated the importance of these factors in Epidendrum rhopalostele, an epiphytic orchid of montane cloud forest through the analysis of spatial patterns of host trees and the orchid, in a more specific scale, with association studies and molecular methods, including AFLPs for orchid population genetic structure and the sequencing of the ITS region for associated mycorrhizal fungi. The aim of this thesis is to understand the factors that condition the presence of epiphytic orchids in the remnants of montane cloud forest and to assess the implications for the conservation and preservation of their habitats and the persistence of the orchid populations. The study was carried out in a fragment of montane cloud forest of secondary succession on the eastern slope of Cordillera Real in the Andes of southern Ecuador, located at 2250 m a.s.l. characterized by a steep slope, mean annual temperature of 20.8°C and annual precipitation of 2193 mm. All trees with DBH > 1 cm were mapped, characterized and identified. All E. rhopalostele individuals present were counted, marked, characterized and mapped. Leaf samples of all orchid individuals were collected for DNA analysis. Root samples of flowering E. rhopalostele individuals were collected for phylogenetic analysis of mycorrhizae, one per phorophyte. Spatial point pattern analysis based on Ripley`s K function and nearest neighbor function was used for trees, phorophytes and orchid population. We observed that spatial distribution of trees and phorophytes is not random, as it adjusts to a Poisson cluster process. This suggests a limitation for seed dispersal in the study fragment that is affecting orchid establishment. Furthermore, the small-scale spatial pattern of E. rhopalostele evidences a clustering that suggests a microsite preference for orchid establishment with a dispersal kernel of 0.4 m. Microsite features such as types of trees (dead trees or Clusia alata), shade tolerance trees, rough bark, distribution in the first meters suggest a tendency to prefer the understory for their establishment. Regarding plant-plant interaction a spatial segregation between adults and juveniles was present suggesting competition for limited resources conditioned for a microsite preference. Analysis of genetic structure of E. rhopalostele population through Structure and PCoA shows two genetic groups coexisting in this fragment and in the same phorophyte, possibly as a result of hybridization between sympatric species of Epidendrum. Our results of spatial autocorrelation analysis develop in GenAlex confirm a small-scale spatial-genetic structure within the genetic groups that is compatible with a short-distance dispersal mechanism caused by gravity or water run-off, instead of the long-distance seed dispersal promoted by wind generally attributed to orchids. For mycobionts identification ITS1-5.8S-ITS2 rDNA region was amplified. Phylogenetic analysis was performed with neighborjoining, Bayesian likelihood and maximum-likelihood for 47 sequences yielded two Tulasnella clades. This orchid establishes mycorrhizal associations with at least two different Tulasnella species. In some cases both fungi clades were present in same root, suggesting no limitation in fungal distribution. Concerning the implications for in situ conservation resulting from this work, the preservation of all forest fragment and their interactions (pollinators, mycorrhiza) is recommended to conserve the genetic diversity of this species. If a reintroduction were necessary, distances between individuals in each phorophyte within a range of 0.4 m, are recommended. To promote recruitment and regeneration of E. rhopalostele it is recommended that phorophytes correspond to dead or fallen trees or species, such as Clusia alata. Trees that have rough bark and are shade tolerant are also recommended. Furthermore, regarding vertical distribution, it is also convenient that orchids are located in the first meter (in understory, area with less light). In conclusion, limitation on seed dispersal, microsite characteristics, plant-plant interactions or interaction with cogeneric sympatric species and mycorrhizal preferences conditioned the presence of this epiphytic orchid in this fragment forest.

Evolución de la vegetación en el sector biogeográfico Castellano Cantábrico durante el Cuaternario Final, a través del registro tobáceo

En esta memoria de Tesis Doctoral se aborda el estudio paleobotánico de seis yacimientos tobáceos situados en las localidades burgalesas de Tubilla del Agua, Sedano, Herrán, Tobera y Frías, y en la alavesa de Ocio. El registro fósil encontrado en estos afloramientos se analiza de forma conjunta con el objetivo de conocer la evolución de la vegetación en el sector biogeográfico Castellano Cantábrico. Este sector se considera el territorio para el cual los hallazgos paleobotánicos son representativos y extrapolables, en tanto que constituye una región homogénea desde el punto de vista florístico, que abarca todos los yacimientos prospectados. El contexto temporal en el que se enmarca este estudio es el final del Cuaternario, desde el Pleistoceno Medio hasta la actualidad. Este intervalo se ha establecido a partir de la edad de los depósitos estudiados, la cual ha sido determinada —para los yacimientos de los que no se disponía de edades fiables— mediante la datación de muestras extraídas de las diferentes unidades litológicas identificadas. Para ello han sido empleadas las técnicas de carbono-14, desequilibrio de las series del uranio y racemización de aminoácidos. Los resultados geocronológicos obtenidos junto con el análisis geomorfológico de los yacimientos han permitido vincular la génesis de las 13 unidades litológicas identificadas con diferentes estadíos climáticos. Estos abarcan un amplio rango de condiciones ambientales, desde las más extremas del Último Máximo Glacial, hasta las más benignas de los Estadíos Isotópicos Marinos interglaciares 1 y 5. Como resultado de la prospección de los depósitos de toba fueron recuperados 1.820 fósiles, la mayoría impresiones foliares, pero también moldes de estróbilos femeninos, ramas y corteza, así como 42 carbones y restos subfósiles de Pinus sp. La identificación taxonómica de estos restos se ha realizado fundamentalmente a partir del análisis de caracteres diagnósticos morfológicos. Como resultado de ello, han sido descritos 28 taxones pertenecientes a las subclases Bryidae, Polypodiidae, Pinidae y Magnoliidae. La flora de los yacimientos estudiados se puede clasificar en tres grupos en función de sus requerimientos ecológicos: (i) uno formado por dos especies de alta tolerancia a la continentalidad —Pinus nigra y Quercus faginea—, las cuales aparecen bien representadas en la mayoría de los depósitos; (ii) otro constituido fundamentalmente por un conjunto de árboles y arbustos que habitualmente tienen el papel de especies acompañantes en los bosques ibéricos submediterráneos y eurosiberianos; y (iii) un tercer grupo compuesto por taxones hidrófitos o edafohigrófilos asociados al ecosistema del fitohermo activo y la vegetación de ribera. En el capítulo de Discusión se propone y analiza la hipótesis de que P. nigra y Q. faginea habrían sido las especies protagonistas de la vegetación zonal del sector Castellano Cantábrico durante el Cuaternario Final. Estas podrían haber persistido como tal incluso durante las épocas más frías, debido a su amplia valencia ecológica y a la capacidad de reproducirse vegetativamente en el caso del quejigo. Por el contrario, los taxones mesofíticos y eurosiberianos pudieron haber sufrido la expansión y retracción de sus poblaciones al ritmo de las oscilaciones climáticas. Sin embargo, la orografía diversa del sector Castellano Cantábrico proporciona emplazamientos en los que se combinan las diversas variables fisiográficas, de tal forma que pudieron haber existido microrrefugios en los que encontraron cobijo algunos taxones mesotérmicos y eurosiberianos durante los periodos glaciales. Por último, la historia evolutiva reciente de la vegetación de este territorio ha estado marcada por la acción antrópica, la cual empezó a ser manifiesta a partir del Neolítico. Esta se tradujo en la degradación y reducción de la cubierta forestal, así como en la extinción del pino laricio del Sector Castellano Cantábrico en los dos últimos milenios. ABSTRACT This PhD Dissertation focuses in the study of six tufa formations located nearby the villages of Tubilla del Agua, Sedano, Herrán, Tobera y Frías, all of them in the province of Burgos, and Ocio, which belongs to the province of Álava. We analyze the palaeobotanical archives of these sites with the purpose of unveiling and understanding the evolution of the vegetation of the Castilian Cantabrian biogeographical sector. This area is considered to be the territory that is represented in the palaeobotanical sample of the studied tufa archives. It is the homogeneous phitogeographical area with the lowest rank that include all the sites. The time frame of this study is the last part of the Quaternary, since the Middle Pleistocene to the present time. This interval is defined by the age of the tufa deposits, which were dated —for the ones that there were not available datings— throughout the analysis of 20 tufa samples taken from the 13 identified lithostratigraphic units. The age of the samples has been determined by using the methods of radiocarbon, U-Th dating and amino acid racemization. Chronological results, along with the chronostratigraphic study of the sites has allowed us to relate the build-up of the 13 identified lithostratigraphic units with different climatic stages. These structures were deposited in a wide range of climatic conditions, from the most extreme ones of the Last Glacial Maximum, to the warmer ones of the Marine Isotopic Stages 1 and 5. A total of 1,820 fossils were recovered from the tufa deposits, most of them were leaf impressions, but also pine cones, branches and bark moulds, along with charcoal and Pinus nigra macro remains. The taxonomical identification of these remains has been done mainly through the analysis of morphological traits. As a result of this process, 28 taxa belonging to the subclass of Bryidae, Polypodiidae, Pinidae and Magnoliidae were identified. The persistency of some taxa can be traced along different climatic stages in this fossil record. This fossil flora can be classified in three different groups: (i) the first one would be composed of two species with high continental climate tolerance —Pinus nigra y Quercus faginea—, which can be found in most of the deposits, (ii) the second group would be mostly formed by trees and shrubs that usually grow in the Iberian forests as an accessory species and (iii) the third one is composed of hydrophytes or hydrophilic taxa associate to the streams, riparian zones or the active tufa ecosystem. In the Discussion chapter we propose and analyse the hypothesis that P.nigra and Q. faginea were the main species of the zonal vegetation of the Castilian Cantabrian biogeographical sector during the last part of the Quaternary. This species could have persisted due to their wide ecological amplitude and also due to the capacity of asexual reproduction in the cases of the oak. On the other hand, mesophitic taxa could have suffered the retraction and expansion of their population following the climate oscillations. However, the diverse orography of the Castilian Cantabrian biogeographical sector provides a variety of combinations of physiographic variables, which could have been suitable refuges for some of the mesophitic taxa. The recent evolutionary history of the vegetation in this territory has been affected by human activities, which started to be relevant since the Neolithic. This led to a reduction of the forests and eventually, to the extinction of P. nigra in the Castilian Cantabrian biogeographical sector in the last two thousands of years.