Census, reproductive biology, and germination of Astragalus gines-lopezii (Fabaceae), a narrow and endangered endemic species of SW Spain

Astragalus gines-lopezii Talavera, Podlech, Devesa & F.M.Vázquez (Fabaceae) is a threatened endemic species with a distribution restricted to a very small area in Badajoz Province (Extremadura Region, SW Spain) and only 2 populations are known. This species was catalogued in the ?Endangered? category in the 2008 Red List and the 2010 Threatened Spanish Vascular Flora List. Despite its status as an endangered species, at present very little is known about the distribution, census, and reproductive biology of this species. In this study we have carried out an exhaustive census of A. gines-lopezii , and we have evaluated the production of flowers, fruits, and seeds and the existence or not of intra- and interpopulation variability in seed germination. Results have highlighted the high reproductive capacity of this species on the basis of a high production of flowers, fruits, and seeds. Mechanical scarification of seeds was effective for increasing germination. Thus, initial germination (22%?60%) was increased to 97%?99% when seeds were rubbed with sandpapers. A high intra- and interpopulation variability in seed germination was found in this species. A. gines-lopezii produces seeds with different degrees of physical dormancy, varying this grade among different individuals within a population.

Census, reproductive biology, and germination of Astragalus gines-lopezii (Fabaceae),a narrow and endangered endemic species of SW Spain.

Astragalus gines-lopezii Talavera, Podlech, Devesa & F.M.Vazquez (Fabaceae) is a threatened endemic species with a distribution restricted to a very small area in Badajoz Province (Extremadura Region, SW Spain) and only 2 populations are known.This species was catalogued in the "Endangered" category in the 2008 Red List and the 2010 Threatened Spanish Vascular Flora List. Despite its status as an endangered species, at present very little is known about the distribution, census, and reproductive biology of this species. In this study we have carried out anexhaustive census of A. gines-lopezii, and we have evaluated the production of flowers, fruits, and seeds and the existence or not of intra- and interpopulation variability in seed germination. Results have highlighted the high reproductive capacity of this species on the basis of a high production of flowers, fruits, and seeds. Mechanical scarification of seeds was effective for increasing germination. Thus, initial germination (22%-60%) was increased to 97%-99% when seeds were rubbed with sandpapers. A high intra- and interpopulation variability in seed germination was found in this species. A. gines-lopezii produces seeds with different degrees of physical dormancy, varying this grade among different individuals within a population.

Morphological and germination response variability in seeds of wild yellow gentian (Gentiana lutea L.) accessions from northwest Spain

Gentiana lutea L. (yellow gentian, Gentianaceae) is an important medicinal plant under protection as endangered species in most European countries. The aim of this work was to evaluate variation in seed mass, seed water content, and seed germination among 56 wild accessions of G. lutea. The effect of gibberellic acid (GA3), putrescine, moist chilling, and level of ripeness of seeds on subsequent germination was also investigated. Seeds of G. lutea showed physiological dormancy (final germination percentages ranged from 0% to 11%, depending on the accession) and GA3 enhanced seed germination drastically in all the accessions. The highest germination (99%) of GA3-treated seeds was reached at 15 °C. Final germination percentage and germination rate (as expressed by mean germination time), as well as seed mass and seed water content, varied significantly among accessions. In general, 1 year moist chilling did not significantly enhance G. lutea seed germination. For most accessions, no significant differences were found between fully ripe seeds and less ripe seeds for seed water content, seed mass, and seed germination. Applications of GA3 were always most effective than those of putrescine for increasing seed germination.

Estudio de la ecología de Ulmus laevis Pallas en la Península Ibérica

Esta tesis doctoral pretende profundizar en el conocimiento de la ecología de Ulmus laevis Pallas, especie autóctona en peligro de extinción en la Península Ibérica, con el fin de proponer medidas adecuadas para su conservación. Se ha estudiado la distribución natural de la especie atendiendo a aspectos edáficos. Los resultados muestran que U. laevis presenta menor capacidad de acidificación de la rizosfera, menor actividad de la reductasa férrica y menor homeostasis que U. minor Mill. cuando crecen en sustratos con una disponibilidad de hierro limitada. Estas diferencias ayudan a comprender la distribución de ambas especies en la Península Ibérica: U. laevis se ve restringido a suelos ácidos o moderadamente ácidos, mientras que U. minor es capaz de habitar tanto suelos ácidos como básicos. Se han analizado las propiedades hidráulicas y anatómicas de U. laevis, constatando que sus características son favorables en ambientes con gran disponibilidad hídrica y que se trata del olmo ibérico más vulnerable a la cavitación por estrés hídrico, por lo que la aridificación del clima y la pérdida de los freáticos supone un riesgo para sus poblaciones. Para evaluar la capacidad de recuperación de la especie se han estudiado la diversidad y estructura genética espacial de las dos mayores poblaciones españolas. Los resultados evidencian que estas poblaciones mantienen niveles de diversidad equiparables o ligeramente superiores a los europeos, pese a haber sufrido un cuello de botella prolongado durante las glaciaciones y a las reducciones poblacionales recientes. En la actualidad la endogamia no representa un riesgo para estas poblaciones. También se ha analizado la producción, dispersión y predación de semillas en Valdelatas (Madrid). Los resultados han mostrado que el viento dispersa las sámaras a corta distancia (<30 m) y que los años no veceros las probabilidades de establecimiento de regenerado son bajas. Además, la producción de sámaras vanas puede tratarse de un carácter adaptativo que aumenta la eficiencia biológica de la especie, ya que favorece la supervivencia de las semillas embrionadas disminuyendo sus tasas de predación pre- y post-dispersión. La modificación del hábitat de esta especie como consecuencia de las actividades humanas afecta de manera negativa al establecimiento del regenerado. La conservación de esta especie a largo plazo requiere la recuperación de los niveles freáticos y de regímenes hidrológicos que permitan avenidas, ya que estas crean las condiciones adecuadas para el establecimiento de regenerado al eliminar la vegetación preexistente y depositar barro. ABSTRACT Ulmus laevis Pallas is an endangered species in the Iberian Peninsula. Therefore, in order to be able to propose adequate management guidelines for its conservation, this PhD Thesis intends to advance the knowledge on the species ecology in the region. Firstly, the species natural distribution was studied in relation to soil nature. Results show that U. minor Mill. had a higher root ferric reductase activity and proton extrusion capability than U. laevis, and maintained a better nutrient homeostasis when grown under iron limiting conditions. These differences in root Fe acquisition efficiencies proved helpful to understand the distribution of these species in the Iberian Peninsula, where U. laevis is restricted to acid or moderately acid soils, whereas U. minor can grow both in acid and basic soils. Secondly, we studied Ulmus laevis’ xylem anatomy and hydraulic traits. These proved favourable for growing under high water availability, but highly susceptible to drought-stress cavitation. Therefore, this species is vulnerable to the Iberian Peninsula’s aridification. Spatial genetic structure and diversity were evaluated in two of the biggest U. laevis populations in Spain in order to evaluate their recovery capabilities. These populations maintain similar or slightly higher diversity levels than European populations, despite having undergone an ancestral genetic bottleneck and having suffered recent population size reductions. No inbreeding problems have been detected in these populations. Seed production, dispersal and predation were assessed in Valdelatas’ elm grove (Madrid). Despite U. laevis samaras being winged nuts, wind dispersed them short distances from the mother tree (<30 m). The seed shadow models show that non-mast years provide very few chances for the stand to regenerate due to their low full seed flux. Empty samaras deceive pre- and post-dispersal predators increasing full seed survival probabilities. Therefore, empty fruit production might be an adaptive trait that increases plant fitness. Finally, human-induced changes in water-table levels and river regulation may affect U. laevis seed dispersal and regeneration establishment negatively. The long-term conservation and expansion of this species in the Iberian Peninsula requires the recovery of water-tables and of natural hydrological regimes, as flooding eliminates vegetation, creating open microhabitats and deposits mud, creating the ideal conditions for seedling establishment.

Metodología para la estimación de indicadores armonizados a partir de los inventarios forestales nacionales europeos con especial énfasis en la biodiversidad forestal

Durante las últimas décadas el objetivo principal de la silvicultura y la gestión forestal en Europa ha pasado de ser la producción de madera a ser la gestión sostenible de los ecosistemas, por lo que se deben considerar todos los bienes y servicios que proporcionan los bosques. En consecuencia, es necesario contar con información forestal periódica de diversos indicadores forestales a nivel europeo para apoyar el desarrollo y la implementación de políticas medioambientales y que se realice una gestión adecuada. Para ello, se requiere un seguimiento intensivo sobre el estado de los bosques, por lo que los Inventarios Forestales Nacionales (IFN), (principal fuente de información forestal a gran escala), han aumentado el número de variables muestreadas para cumplir con los crecientes requerimientos de información. Sin embargo, las estimaciones proporcionadas por los diferentes países no son fácilmente comparables debido a las diferencias en las definiciones, los diseños de muestreo, las variables medidas y los protocolos de medición. Por esto, la armonización de los datos que proporcionan los diferentes países es fundamental para la contar con una información forestal sólida y fiable en la Unión europea (UE). La presente tesis tiene dos objetivos principales: (i) establecer el diseño de una metodología para evaluar la biodiversidad forestal en el marco del Inventario forestal nacional de España teniendo en cuenta las diferentes iniciativas nacionales e internacionales, con el objetivo de producir estimaciones comparables con las de otros países de la UE y (ii) armonizar los indicadores más relevantes para satisfacer los requerimientos nacionales e internacionales. Como consecuencia del estudio realizado para alcanzar el primer objetivo, la metodología diseñada para estimar la biodiversidad fue adoptada por el Tercer Inventario forestal nacional. Ésta se componía de indicadores agrupados en: cobertura del suelo, composición de árboles y especies de arbustos, riqueza de especies herbáceas y helechos, especies amenazadas, estructura, madera muerta, y líquenes epífitos. Tras el análisis del diseño metodológico y de los datos proporcionados, se observó la conveniencia de modificarla con el fin de optimizar los costes, viabilidad, calidad y cantidad de los datos registrados. En consecuencia, en el Cuarto Inventario Forestal Nacional se aplica una metodología modificada, puesto que se eliminó el muestreo de especies herbáceas y helechos, de líquenes epífitos y de especies amenazadas, se modificaron los protocolos de la toma de datos de estructura y madera muerta y se añadió el muestreo de especies invasoras, edad, ramoneo y grado de naturalidad de la masa. En lo que se refiere al segundo objetivo, se ha avanzado en la armonización de tres grupos de variables considerados como relevantes en el marco de los IFN: los indicadores de vegetación no arbórea (que juegan un papel relevante en los ecosistemas, es donde existe la mayor diversidad de plantas y hasta ahora no se conocían los datos muestreados en los IFN), la determinación de los árboles añosos (que tienen un importante papel como nicho ecológico y su identificación es especialmente relevante para la evaluación de la biodiversidad forestal) y el bosque disponible para el suministro de madera (indicador básico de los requerimientos internacionales de información forestal). Se llevó a cabo un estudio completo de la posible armonización de los indicadores de la vegetación no arbórea en los IFN. Para ello, se identificaron y analizaron las diferentes definiciones y diseños de muestreo empleados por los IFN, se establecieron definiciones de referencia y se propusieron y analizaron dos indicadores que pudiesen ser armonizados: MSC (mean species cover) que corresponde a la media de la fracción de cabida cubierta de cada especie por tipo de bosque y MTC (mean total cover). Se estableció una nueva metodología que permite identificar los árboles añosos con los datos proporcionados por los inventarios forestales nacionales con el objetivo de proporcionar una herramienta eficaz para facilitar la gestión forestal considerando la diversidad de los sistemas forestales. Se analizó el concepto de "bosque disponible para el suministro de madera" (FAWS) estudiando la consistencia de la información internacional disponible con el fin de armonizar su estimación y de proporcionar recomendaciones para satisfacer los requerimientos europeos. Como resultado, se elaboró una nueva definición de referencia de FAWS (que será adoptada por el proceso paneuropeo) y se analiza el impacto de la adopción de esta nueva definición en siete países europeos. El trabajo realizado en esta tesis, puede facilitar el suministrar y/o armonizar parcial o totalmente casi la mitad de los indicadores de información forestal solicitados por los requerimientos internacionales (47%). De éstos, prácticamente un 85% tienen relación con los datos inventariados empleando la metodología propuesta para la estimación de la biodiversidad forestal, y el resto, con el establecimiento de la definición de bosque disponible para el suministro de madera. No obstante, y pese a que esta tesis supone un avance importante, queda patente que las necesidades de información forestal son cambiantes y es imprescindible continuar el proceso de armonización de los IFN europeos. ABSTRACT Over the last few decades, the objectives on forestry and forest management in Europe have shifted from being primarily focused on wood production to sustainable ecosystem management, which should consider all the goods and services provided by the forest. Therefore, there is a continued need for forest indicators and assessments at EU level to support the development and implementation of a number of European environmental policies and to conduct a proper forest management. To address these questions, intensive monitoring on the status of forests is required. Therefore, the scope of National Forest Inventories (NFIs), (primary source of data for national and large-area assessments), has been broadened to include new variables to meet these increasing information requirements. However, estimates produced by different countries are not easily comparable because of differences in NFI definitions, plot configurations, measured variables, and measurement protocols. As consequence, harmonizing data produced at national level is essential for the production of sound EU forest information. The present thesis has two main aims: (i) to establish a methodology design to assess forest biodiversity in the frame of the Spanish National Forest Inventory taking into account the different national and international initiatives with the intention to produce comparable estimates with other EU countries and (ii) to harmonize relevant indicators for national and international requirements. In consequence of the work done related to the first objective, the established methodology to estimate forest biodiversity was adopted and launched under the Third National Forest Inventory. It was composed of indicators grouped into: cover, woody species composition, richness of herbaceous species and ferns, endangered species, stand structure, dead wood, and epiphytic lichens. This methodology was analyzed considering the provided data, time costs, feasibility, and requirements. Consequently, in the ongoing Fourth National Forest Inventory a modified methodology is applied: sampling of herbaceous species and ferns, epiphytic lichens and endangered species were removed, protocols regarding structure and deadwood were modified, and sampling of invasive species, age, browsing impact and naturalness were added. As regards the second objective, progress has been made in harmonizing three groups of variables considered relevant in the context of IFN: Indicators of non-tree vegetation (which play an important role in forest ecosystems, it is where the highest diversity of plants occur and so far the related sampled data in NFIs were not known), the identification of old-growth trees (which have an important role as ecological niche and its identification is especially relevant for the assessment of forest biodiversity) and the available forest for wood supply (basic indicator of international forestry information requirements). A complete analysis of ground vegetation harmonization possibilities within NFIs frame was carried on by identifying and analyzing the different definitions and sampling techniques used by NFIs, providing reference definitions related to ground vegetation and proposing and analyzing two ground vegetation harmonized indicators: “Mean species cover” (MSC) and “Mean total cover” (MTC) for shrubs by European forest categories. A new methodology based on NFI data was established with the aim to provide an efficient tool for policy makers to estimate the number of old-growth trees and thus to be able to perform the analysis of the effect of forest management on the diversity associated to forest systems. The concept of “forest available for wood supply” (FAWS) was discussed and clarified, analyzing the consistency of the available international information on FAWS in order to provide recommendations for data harmonization at European level regarding National Forest Inventories (NFIs). As a result, a new reference definition of FAWS was provided (which will be adopted in the pan-European process) and the consequences of the use of this new definition in seven European countries are analyzed. The studies carried on in this thesis, can facilitate the supply and/or harmonization partially or fully of almost half of the forest indicators (47%) needed for international requirements. Of these, nearly 85% are related to inventoried data using the proposed methodology for the estimation of forest biodiversity, and the rest, with the establishment of the definition of forest available for wood supply. However, despite this thesis imply an important development, forest information needs are changing and it is imperative to continue the process of harmonization of European NFIs.

Estudio de la diversidad genética de Botrytis sp en orquídeas y la resistencia al ataque del hongo

En la actualidad la mayoría de plantas sufren pérdidas debido a las enfermedades que les provocan los hongos. Uno de estos grupos amenazado por el ataque de los hongos son las especies de la familia Orchidaceae, especies que se encuentran amenazadas y con numerosas especies en peligro de extinción. Uno de los problemas sanitarios más destacados es Botrytis cinerea, hongo patógeno cosmopolita, causante de enfermedades importantes en muchas plantas tales como frutas, verduras, accesiones de viveros, plantas ornamentales y huertos cultivos (Jarvis 1977; Elad et al., 2007). Este género es uno de los grupos de hongos más ampliamente conocido y distribuido. Contiene 22 especies (Hennebert 1973; Yohalem et al., 2003) y un híbrido (B. allii) (Yohalem & Alabama, 2003) vinculado a las etapas sexuales y un amplio número de huéspedes específicos (Beever y Weds, 2000); infecta más de 200 especies vegetales distintas (Williamson et al., 2007). Dada la importancia de este patógeno se realiza un estudio de caracterización morfológica y molecular del hongo, aislado de plantas de orquídeas cultivadas en condiciones de invernadero, de hortalizas y plantas frutales, con síntomas de necrosis, atizonamientos y pudriciones. El análisis de las características morfológicas (presencia de esclerocios, tamaño de conidios, presencia de estructuras sexuales in vitro) y fenotípicas (crecimiento micelial a diferentes temperaturas, germinación de esporas), nos permitió determinar características importantes del comportamiento del hongo y establecer cuáles son las mejores condiciones para su patogenicidad. Se afianzo este trabajo con estudios moleculares a través del análisis de la región ribosomal ITS1-ITS4. Entre los aislados estudiados se identificaron dos especies diferentes, Botrytis cinerea y B. fabiopsis, esta última conocida como especifica de Vicia faba, se lo aisló de una planta de Pelargonium sp. Se hizo un análisis filogenético para comparar estas dos especies, encontrándose que B. fabiopsis está estrechamente relacionada con B. cinerea y B. elliptica, pero lejanamente relacionado con B. fabae. Además, se analizó las poblaciones de los aislados de Botrytis, para ello se seleccionaron tres parejas de cebadores microsatelites con altos porcentajes de polimorfismo. Al analizar la similaridad entre los aislados se determinaron tres grupos de poblaciones de B. cinerea entre los cuales Botrytis fabiopsis comparte un grupo grande con B. cinerea. La diferenciación genética no fue significativa entre la población de aislados de orquídeas y hortalizas, la diferencia génica que fue muy baja, lo que sugiere que la especificidad de Botrytis no está dada por los hospederos, aunque la posibilidad de la especificidad con algún cultivo no puede descartarse. ABSTRACT Most plants suffer diseases caused by fungi. Orchidaceae is one of the threatened groups with many endangered species. Included into the most important problems in plant health is Botrytis cinerea, a cosmopolitan pathogen which causes major diseases in many plants of agronomic interest such as fruits, vegetables, planthouses accessions and ornamental plants (Jarvis, 1977; Elad et al, 2007). The genus Botrytis is one of the most widely and disseminated fungi. The genus contains 22 species (Hennebert 1973; Yohalem et al, 2003) and a hybrid (B. allii) (Yohalem & Alabama, 2003) linked to the sexual stages of a large number of specific hosts (Beever & Weds, 2000); infects over 200 different plant species (Williamson et al., 2007). Due to the importance of this pathogen, a study of morphological and molecular characterization of the fungus was carried out. Fungi samples were isolated from orchid plants grown in greenhouse conditions, vegetables and fruits with signs of necrosis, blight and rottening. To establish the best conditions for pathogenicity, behavioral characteristics of the fungus were studied through the analysis of morphological characteristics (presence of sclerotia, conidia size, sexual structures in vitro) and mycelial growth at different temperatures. To complete the characterization of the fungi, a molecular study was performed via the analysis of ribosomal ITS1-ITS4 region. Two different species were identified: Botrytis cinerea and Botrytis fabiopsis (known by specificity to Vicia faba). B. fabiopsis was isolated from a plant of the genus Pelargonium. A phylogenetic analysis was carried out to compare these two species leading to the conclusion that B. fabiopsis is closely related to B. cinerea and B. elliptica, but distantly related to B. fabae. The populations of Botrytis isolates were also analyzed. Three pairs of microsatellite primers with high percentages of polymorphism were selected. A similarity analysis showed three groups of populations of B. cinerea, including Botrytis fabiopsis. The genetic differentiation was not significant among the populations of isolates from orchids and vegetables; genetic differences were very low, suggesting that the specificity of Botrytis species is not given by the hosts.

Descripción de los parámetros seminales del conejo ibicenco

The ibicenco rabbit is an endangered species and it has to be consolidated as a breed. Therefore, male reproductive characteristics must be standardized, which was the objetive of this study. We also determined the effect of the sexual maturity stage (puberal <10 months; adults > 10 months) and the effect of the ejaculate order in seminal characteristics.

Is it appropriate protective figure "Plant Micro-Reserves" to protect tree species? The example of Betula pendula subsp. fontqueri in "La Garganta de los Caballeros" (Ávila)

The figure of protection "micro-reserves" was created in the Region of Valencia (ANONYMOUS, 1994) with the aim of protecting endangered plant species. This is one of the areas of greatest floristic richness and uniqueness of the western Mediterranean. In this area rare, endemic or threatened vascular flora has a peculiar distribution: they appear to form small fragments spread over the entire region (LAGUNA, 1994; LAGUNA, 2001) The protection of every these small populations of great scientific value has significant challenges. It doesn´t try to protect every species that set out in Annex IV of the by then existing Law 4 / 1989 (repealed in 2007), or to protect to the most ecological level with the creation of Natural Protected Area but an intermediate level: the plant community of small size. According to the decree: “as Micro-Reserve will be declared the natural parcels of land under 20 hectares that contain a high concentration of rare plants, endemic, threatened or of high scientific interest” (ANONYMOUS, 1994) . Of course, the statement of an area as micro-reserve carries certain prohibitions that are harmful to the vegetal community

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.