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.

Pollen record during the Eemian from the Fuentillejo maar-lake sequence (Ciudad Real, Spain)

The Fuentillejo maar is located in the Central Spanish Volcanic Field of Campo de Calatrava (Ciudad Real). Fuentillejo maar-Iake is a c10sed system covering over 142 m depth oflacustrine sediments; it is one ofthe best examples oflong and continuous cores at terrestrial site from the Iberian Peninsula. PalynoIogical, mineralogical and sedimentary facies analysis were performed to characterize the sedimentary record during the Last Interglacial. In core FUENT-l this period (dated in 133 ka-120 ka) is detected between 45,90-56,90 m depth. Sedimentology point of view is characterized by develop of lacustrine facies, fineIy laminated black-brown doIomicrite mud and sapropeIIayers (Sedimentary Units 16,6-17-18). The vegetation is characterised by high polIen taxa diversity (around 50 polIen taxa of terrestriaI types, 5 polIen taxa of aquatic types, spores and 9 types of non palynological microfossils-NPM) together with a high content in the Mediterranean and mesic forest components (Quercus evergreen, Oleaceae, Quercus decidous and CoryIus), tipical ofwarm and humid conditions, and a few content on Artemisia, Pinus and Juniperus taxa (typicaI of coId or warm arid phases). The scarce forest development can be interpreted from the polIen record of mesophilus and thermophilous vegetation of the FUENT-1 sequence, in which only 40-50% of total polIen come from arboreaI associations. These vaIues for arboreal pollen content are low compared with other European polIen sequences.

The late Miocene macroflora of the La Cerdaña Basin, Eastern Pirenees, Spain. Towards a Synthesis

The fossil plant-bearing beds of the Tortonian (late Miocene) intramontane basin of La Cerdanya (Eastern Pyrenees, Catalonia, Spain) have been investigated for more than a century, and 165 species from 12 outcrops have been described in previous publications. The sediments with rich plant fossil assemblages, which correspond to lacustrine diatomitic deposits, contain large numbers of plant remains, mainly leaf compressions and impressions. These assemblages are well preserved, a consequence of the rapid accumulation of plant remains in the sediments of the basin's ancient lake, and the often close proximity of its shores to wetland and upland vegetation. This paper provides a comprehensive taxonomic and nomenclatural review of the historic and new collections of late Miocene macroflora for the La Cerdanya Basin. Examination of the newer materials allowed emendments to be made to the diagnoses ofAbies saportana, Acer pyrenakum,Alnus occidentalis, Quercus hispanka and Tilia vidali provided by REROLLE for the basin at the end of the 19th century. In addition, 24 species of vascular plants are identified for the basin for the first time, including one horsetail, three conifers, 19 arboreal or bushy dicotyledonous angiosperms, and one monocotyledonous angiosperm. Indeed, this is the first time that Cedrela helkonia (UNGER) KNOBLOCH, Decodon sp„ Hedera cf multinervis KOLAKOVSKII, Mahonia cf pseudosimplex KVACEK & WALTHER, Smilax cf. aspera L. vm.fossilis and Ulmus cf. plurinervia UNGER have been recorded anywhere in the Iberian Peninsula. The La Cerdanya Basin plant assemblages of the late Miocene mainly consisted of conifers and deciduous broadleaved taxa of Arctotertiary origin; evergreen Palaeotropical elements were less well represented. This flora is similar to those recorded at coeval sites in northern Greece, northern Italy and central and eastern France. Within the Iberian Peninsula, the late Miocene macroflora reported for the nearby Seu d'Urgell Basin is the most similar.

Nuevos mecanismos moleculares de tolerancia a sequía y otros tipos de estrés abiótico en especies arbóreas de interés económico

Las masas forestales tienen una importancia colosal para nuestra sociedad y el conjunto de la biosfera. Estudios recientes a escala mundial indican que la sequía es el factor abiótico que más afecta a su crecimiento y supervivencia, seguida por las temperaturas extremas y la salinidad. Aunque comprender los mecanismos con que las especies arbóreas toleran estas formas de estrés tiene un interés aplicado evidente, dichos mecanismos se han estudiado mucho más en especies herbáceas modelo o de interés agronómico. Existen sin embargo diferencias notables entre ellas, como se demuestra en esta tesis y en otros trabajos recientes. Nuestro estudio se centra concretamente en la respuesta molecular del chopo –el sistema modelo forestal más desarrollado– al estrés abiótico, con particular énfasis en la sequía. Utilizando una estrategia proteómica y tratamientos controlados, hemos identificado componentes mayoritarios de dicha respuesta. Su participación en la misma se ha validado mediante análisis transcripcionales detallados utilizando tecnología qRT-PCR (PCR cuantitativa en tiempo real). Hemos identificado proteínas cuyo nexo funcional con mecanismos de tolerancia ya era conocido, como chaperonas moleculares sHSP o enzimas que atenúan el estrés oxidativo, pero también proteínas cuya relación funcional con el estrés es menos clara o incluso novedosa, como polifenol oxidasas (PPO), deshidrogenasas/reductasas de cadena corta (SDR), o bicupinas (BIC), entre otras. El cuerpo central de la tesis consiste en la caracterización detallada de una PPO inusual, cuya inducción por estrés hídrico se describe por vez primera. Estas enzimas están ampliamente distribuidas en plantas, si bien su número es muy variable de unas especies a otras. Algunas, como nogal, tienen un único gen, mientras que Arabidopsis no tiene ninguno. En la última versión del genoma de chopo hemos identificado un total de 12 miembros bona fide, corrigiendo trabajos previos, y hemos caracterizado su expresión individual ante diferentes situaciones de estrés controlado y tratamientos hormonales. La isoforma antedicha es el único miembro de la familia que responde claramente a la deshidratación. También responde a salinidad y a la mayor parte de tratamientos hormonales ensayados, pero no a daño mecánico o tratamientos con metil jasmonato. Esto la diferencia de enzimas homólogas presentes en otras especies de plantas, que se han relacionado experimentalmente con estrés biótico. Los patrones de acumulación de transcritos en árboles adultos son compatibles con un papel protector frente a la sequía. La integración de nuestros estudios funcionales y filogenéticos sugiere que la familia ha sufrido un proceso reciente de diversificación y neofuncionalización, siendo la protección frente a deshidratación su papel primigenio. Aunque se conoce la actividad bioquímica in vitro de este tipo de enzimas, sus sustratos naturales son esencialmente una incógnita. Mediante expresión heteróloga en Escherichia coli BL21(DE3) hemos detectado que la enzima de chopo es capaz de oxidar L-DOPA a dopaquinona, siendo menos activa frente a otros sustratos. Por otra parte, hemos demostrado su localización cloroplástica mediante transformación transitoria de protoplastos con fusiones a la proteína fluorescente YFP. Mediante la obtención de plantas transgénicas de A. thaliana hemos demostrado que la enzima de chopo aumenta considerablemente la tolerancia in vivo frente a la deshidratación y al estrés salino. El análisis fenotípico detallado de las líneas transgénicas, combinando múltiples metodologías, nos ha permitido sustanciar que la tolerancia tiene una base compleja. Esta incluye una mayor protección del sistema fotosintético, una capacidad antioxidante muy incrementada y la acumulación de solutos osmoprotectores como la prolina. Los análisis metabolómicos nos han permitido asociar la expresión de la proteína a la síntesis de un flavano no descrito previamente en A. thaliana, vinculando la enzima de chopo con la síntesis de fenilpropanoides. También hemos observado alteraciones en los niveles hormonales que podrían subyacer a efectos pleiotrópicos con interés aplicado, como un aumento consistente del tamaño de la planta o el acortamiento del ciclo de crecimiento. Además de aportar datos novedosos sobre la funcionalidad in vivo de esta familia de oxidasas, los resultados de esta tesis demuestran que los árboles son sistemas de estudio interesantes para caracterizar nuevas estrategias de tolerancia al estrés abiótico con potencial aplicado. ABSTRACT Forests masses have an extraordinary importance for our society and the biosphere. Recent worldwide studies indicate that drought is the abiotic factor that affects more their growing and survival, followed by extreme temperatures and salinity. The understanding of how the arboreal species tolerate the stress has an evident practical interest, but their mechanisms have been studied much more in herbaceous species or with agronomic interest. However, considerable differences exist between them, as this thesis and recent studies show. Our study is focused on the molecular response of the poplar –the more developed forestry model system- to abiotic stress, specifically focused in the drought. Using a proteomic strategy and controlled treatments, we have identified main components in such response. Its participation has been validated through transcriptional analysis using qRT-PCR technology. We have identified proteins whose functional connection with tolerance mechanisms were already known, as molecular chaperones sHSP or enzymes that attenuate the oxidative stress, but also some proteins whose functional relationship with the stress is less clear or even novel, as polifenol oxidases (PPO), short chain deshidrogenases/reductases (SDR), or bicupines (BIC), among others. The central body of the thesis consists of the detailed characterization of an unsual PPO, whose induction due to drought stress is first described. These enzymes are thoroughly distributed in plants, but their number of members is very variable among species. Some of them, as the walnut tree, have a single gene, while Arabidopsis has none. We have identified a total of 12 members in the last version of the poplar genome, correcting previous works, and have characterized their individual expression against different situations of controlled stress and hormone treatments. The aforementioned isoform is the only member of the family that responds clearly to the drought. It also reacts to salinity and the majority of hormonal treatments tested, but it does not respond to mechanical damage or treatments with methyl jasmonate. This is the difference with homologue enzymes present in other plant species, which have been related experimentally with abiotic stress. The accumulation patterns of transcripts in adult trees are compatible with a protector role against drought. The integration of our functional and phylogenetic studies suggests that the family has suffered a recent process of diversification and neofunctionalization, being the protection against drought their original role. Although the in vitro biochemistry activity of this kind of enzymes is already known, their natural substracts are essentially a mystery. By means of heterologous expression of Escherichia coli BL21(DE3) we have detected that the enzyme of poplar is able to oxidize L-DOPA to dopaquinone, being less active against other substrates. Additionally, we have proven its chloroplastic location with transitory transformation of protoplasts with YFP protein fusion. By means of getting transgenic plants of A. thaliana, we have demonstrated that the poplar enzyme increases notably the in vivo tolerance against the drought and salinity stresses. The phenotypic analysis of the transgenic lines, and the use of multiple methodologies, allowed us to test the complexity of the tolerance. This includes a major protection of the photosynthetic system, a very increased antioxidant capacity and the accumulation of osmoprotectant solutes as the proline. The metabolic analysis has allowed to associate the protein expression with the synthesis of a Flavan non described previously in A. thalaiana, linking the enzyme of poplar with the synthesis of phenylpropanoids. We have observed alterations in the hormonal levels that could underlie pleiotropic effects with applied interest, as a consistent increase of the size of the plant and the reduction of the growth cycle. The results of this thesis, in addition to provide novel data about the in vivo functionality of the oxidase family, demonstrate that the trees are interesting systems of study to characterize new strategies of tolerance against abiotic stress with applied potential.

Crecimiento forestal en el bosque tropical de montaña: efectos de la diversidad florística y de la manipulación de nutrientes

El bosque tropical de montaña, es considerado zona de mega diversidad y de alto grado de endemismo, por las diferentes zonas ecológicas que presenta. Durante las últimas décadas estos bosques han recibido mayor atención por parte de investigadores, sin embargo, aún existe poca investigación en determinar cuáles son las respuestas de los bosques a los cambios ambientales a los que son sometidos. Estos bosques están sufriendo serias amenazas como pérdida de cobertura vegetal y cambios en los ciclos de nutrientes. El trabajo se dividió en cuatro objetivos específicos. i) Caracterización y análisis de patrones altitudinales de la riqueza de especies en el bosque tropical de montaña en el sur del Ecuador; con el fin de conocer cómo varía la diversidad de especies riqueza lo largo de un gradiente altitudinal. ii) Conocer los patrones espaciales del crecimiento en tres remanentes boscosos de un bosque tropical de montaña para determinar cómo la vecindad y la semejanza funcional de ésta influyen en el crecimiento forestal. iii) Conocer los efectos de la fertilización en el crecimiento diamétrico de especies arbóreas, en el bosque tropical de montaña; se analizó cómo reaccionan los árboles a la adición de nutrientes N y P en tres tipos de bosque. iv) Saber la respuesta de la comunidad de árboles a la adición de nutrientes en el bosque montano andino; este objetivo se basó con el supuesto de la deficiencia de tres tipos de nutrientes N, P y Ca, en esta formación boscosa y cómo reaccionan los árboles a la adición de nutrientes. El presente trabajo se llevó a cabo, en el bosque tropical de montaña que se encuentra localizada en la parte adyacente del Parque Nacional Podocarpus (PNP) en la cordillera del Consuelo, forma parte de la cadena oriental de los Andes del sur del Ecuador El trabajo de desarrollo entre los años 2008 y 2014. Para abordar el primer objetivo se establecieron 54 parcelas ubicadas aleatoriamente a lo largo de un gradiente altitudinal (3 niveles de altitud) y se e midieron e identificaron todos los individuos mayores a 5cm de DAP. Se construyó una filogenia con Phylocom y se calcularon diferentes componentes de diversidad para cada parcela ( riqueza taxonómica, diversidad filogenética y edad media de las especies). Ajustando modelos lineares se contrastó el efecto de la altitud sobre dichos componentes y se vio que la riqueza taxonómica y la edad media de las especies aumentaron con la altitud, en sentido contrario a las predicciones de la "hipótesis del conservadurismo tropical" (Tropical Conservatism Hypothesis). Para abordar el segundo objetivo se realizó una remedición de todos los árboles cartografiados en tres parcelas permanentes de alrededor de 5000 m2 cada una, representativas de tres estados diferentes de la sucesión del bosque montano. A partir de las coordenadas y de los datos de registrados, y empleando diferentes funciones de correlación de marca se analizó la distribución espacial del tamaño y del crecimiento relativo y del tamaño. Se constató que mientras que el tamaño de los árboles presentó una correlación espacial negativa, el crecimiento presentó correlación espacial positiva, en ambos casos a distancias cortas. El rango y la magnitud de ambas correlaciones aumentaron al avanzar la sucesión. La distribución espacial del crecimiento mostró una correlación negativa con la distribución espacial de tamaños. Por otro lado, la distribución espacial del crecimiento mostró una correlación negativa para árboles semejantes funcionalmente y positiva cuando se calculó entre árboles con diferente estrategia funcional. En conjunto, los resultados obtenidos señalan un aumento de la importancia de procesos competitivos y una mayor estructuración espacial del crecimiento y de la distribución de tamaños al avanzar la sucesión. Para el tercer y cuarto objetivo se instalaron 52 parcelas distribuidas en bloques donde se fertilizaron dos veces al año durante 6,4 años, se identificaron todos los individuos mayores a 10 cm de DAP, y se midió el crecimiento diamétrico durante estos años Con la adición de nutrientes realizada a los diferentes tipos de bosque en la gradiente altitudinal, encontramos que el efecto sobre el crecimiento diamétrico en la comunidad varia con el rango altitudinal, y el tipo de nutriente, analizando a nivel de las especies, en la mayoría de los casos las especies comunes no tuvieron cambios significativos a la adición de nutrientes. Los resultados de este estudio aportan nuevas evidencias para el entendimiento de la diversidad, estructura y dinámica de los bosques tropicales de montaña. ABSTRACT The montane tropical forest is considered a megadiverse habitat that harbor an enormous degree of endemism. This is mainly due to the high degree of environmental heterogeneity found and the presence of different well defined ecological areas. These forests have received more attention during the last decades, however, the information regarding the responses of these forests to environmental change, is still scarce. These forests are seriously endangered and are suffering serious threats, such as loss of vegetative cover, changes in the nutrient cycles. The work was divided in four specific objectives: i) Characterization and analysis of the species richness altitudinal patterns in the montane tropical forest of south Ecuador. Specifically, how species diversity changes along altitudinal gradients. ii) Exploring the spatial patterns of tree growth in three remnants of a montane tropical forest, and analyze how tree neighborhood and functional similarity among trees influence tree growth. Tropical Conservatism Hypothesis iii) Understanding the effects of fertilization in arboreal species growth (increase in diameter) of the montane tropical forest. Specifically we studied the effects of P and N addition on three different forests across an altitudinal gradient. iv) Know the response of the community of trees to the addition of nutrients in the Andean montane forest; this objective was based on the supposition of deficiency of three types of nutrients: P, N and Ca in this forest all formation and how the trees react to the addition of these nutrients. The present work was carried out in the montane tropical forest located in Bombuscaro, San Francisco and Cajanuma close to Podocarpus National Park (PNP) on Consuelo mountain range (Andean oriental range) at South of Ecuador. Field work was carried out during 2008 and 2014. To address the first objective, we randomly placed 54 plots along an altitudinal gradient. In these plots, every individual larger than 5 cm of DBH was measured and identified. A phylogeny was build with Phylocom and different diversity components (taxonomic richness, phylogenetic diversity and average species age) were computed for each plot. Linear models were used to test the effects of altitude on the diversity components. Our results showed that, contrary to the Tropical Conservatism Hypothesis, both taxonomic richness and average species age increased with altitude. To address our second objective, all mapped trees in three successional permanent plots (around ~5000 m2 each) were re-measured. Using different mark correlation functions, we analyzed the spatial distribution of tree-size and tree relative growth rate. Whereas tree size showed negative spatial correlation at fine spatial scales, relative growth rate showed positive correlation at the same scales. The range and magnitude of those correlations increased along successional stage. The spatial distribution of the relative growth rate was negatively correlated with the spatial distribution of tree sizes. Additionally, we found that the spatial correlation of the relative growth rate was negative for functionally similar trees and positive when computed for functionally different trees. In synthesis, our results point to an increase of competitive processes and strong spatial structure of relative growth rate and tree size along succession. For the third and fourth objectives, 52 plots were placed in a block design and were fertilized twice a year for 6,4 years. In these plots all the individuals with DBH > 10 cm were identified, and the diametrical growth was measured during these years. The nutrient addition at the three different altitude forests, revealed that the effect on the diametrical growth in the community varied with the altitudinal range. When analyzed at species level, the addition of nutrients was no significant in most cases. These results represent new evidences that will improved our understanding of diversity patterns and structure, and the dynamics of tropical montane forests.