Emerging wildlife diseases represent a serious threat to conservation efforts. Impacts of emerging multi-host pathogens can vary greatly among species as well as geographically, and understanding which populations will be at greatest risk is essential for conserving biodiversity. Snake fungal disease (SFD), caused by the fungal pathogen Ophidiomyces ophidiicola, is responsible for lethal infections in snakes and has contributed to the decline of multiple North American snake populations. However, which species are most affected by this disease and how infections vary regionally remains unknown. Here we sampled 44 different species across 14 sites throughout the Southeastern and Mid-Atlantic United States. We found a strong effect of latitude on both pathogen prevalence and severity, with more severe infections at more northern latitudes. We also found high variability in pathogen prevalence and infection severity among species. There was a strong positive relationship between pathogen prevalence and disease severity, suggesting that SFD is not just highly prevalent in some populations but also highly virulent. More broadly, our results support that SFD likely has continued impacts on snake populations with some species experiencing greater disease than others attributed to spatial and host variation. / Master of Science / Conserving biodiversity is a significant challenge. Wildlife species are under multiple threats including habitat loss, changing climate, species introductions, pollution, and infectious diseases. Emerging wildlife diseases can pose a major problem for wildlife as they often go undetected until they cause substantial declines for the affected species, sometimes leading to population extirpations and extinction events. Snake fungal disease (SFD) is an emerging disease caused by the fungal pathogen Ophidiomyces ophidiicola, which has contributed to the decline of some North American snake populations. However, little is known about differences in infection, transmission, and host responses to SFD in a broader community context. To investigate the dynamics of this pathogen, we collected swab samples from 44 species from a total of 14 sites in New Jersey, Virginia, North Carolina, South Carolina, Georgia, Florida, and Louisiana. We sampled individual snakes to examine variation over a geographic gradient and among species. We found high variability among sites with more severe disease at northern sites. There was also high variability among species and some populations experienced both high pathogen prevalence and disease severity. Our results show that SFD is highly variable within snake communities and may still be causing population level effects.

Chytridiomycosis is a fungal skin disease in amphibians that is primarily caused by Batrachochytrium dendrobatidis (Bd). We analyzed whole genome sequences of 40 bacterial isolates that had been previously cultured from the skin of four amphibian species from Virginia, USA, and tested for their ability to inhibit Bd growth via an in vitro challenge assay. These 40 isolates spanned 11 families and 13 genera. The aim of this study was to identify genomic differences among the amphibian skin bacterial isolates and generate hypotheses about possible differences that could contribute to variation in their ability to inhibit the growth of Bd. We identified sixty-five gene families that were present in all 40 isolates. We also looked for the presence of biosynthetic gene clusters. While this set of isolates contained a wide variety of biosynthetic gene clusters, the two most abundant clusters with potential anti-fungal activity were siderophores (N=17) and Type III polyketide synthases (N=20). We then analyzed the isolates belonging to the phylum Proteobacteria in more detail. We identified 197 gene families that were present in all 22 Proteobacteria. We examined various subsets of the Proteobacteria for genes for specific compounds with known activity against fungi, including chitinase and violacein. We identified a difference in the number, as well as amino acid sequences, of predicted chitinases found in two isolates belonging to the genus Agrobacterium that varied in their inhibition of Bd. After examining the annotated genomes, we identified a predicted chitinase in a Sphingomonas isolate that inhibited the growth of Bd that was absent from the five Sphingomonas isolates that did not inhibit Bd growth. The genes vioA, vioB, vioC, vioD and vioE are necessary to produce violacein, a compound which inhibits the growth of Bd. Differences in these genes were identified in three out of the four Janthinobacterium isolates. Of these three isolates, two showed strong inhibition of Bd growth, while the third inhibited Bd growth to a lesser extent. Using comparative genomics, we generated several testable hypotheses about differences among bacterial isolates that could contribute to variation in ability to inhibit Bd growth. Further work is necessary to test the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd. / Master of Science / Many amphibian population declines around the world have been caused by chytridiomycosis, a skin disease. This disease is caused by the fungus Batrachochytrium dendrobatidis (Bd). The skin of amphibians is also home to many bacteria that can provide important functions for the amphibian host, like preventing infection by Bd. To understand how these bacteria might provide protection, we examined the entire genomes of 40 bacterial isolates that reside on the skin of four amphibian species from Virginia, USA. These bacteria were previously tested for their ability to prevent Bd growth and 40 of them were chosen for sequencing based on selecting closely related isolates that varied in their ability to inhibit Bd growth. This allowed us to compare their genomes and generate hypotheses about possible genomic differences that could contribute to the variation in Bd growth inhibition. We identified sixty-five gene families that were present in all 40 bacteria. We also looked for sets of genes (biosynthetic gene clusters) that are known to produce secondary metabolites, which are compounds that can include antifungals. The two most abundant clusters we identified that had the potential to produce compounds that inhibit fungal growth were siderophores and Type III polyketide synthases. We then looked for genes that were not part of biosynthetic gene clusters that could produce specific compounds that can inhibit Bd growth, such as chitinase and violacein. We found variation in chitinase genes in several isolates that seemed to be associated with the ability to inhibit Bd growth. In addition, there were some differences in violacein genes that should be examined more in future studies. Overall, we suggest that using comparative genomic approaches can be valuable for identifying key bacterial functions in the microbiome.

<p>Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease worldwide. Even though this fungus has not evolved specifically to cause human disease, it has a remarkable ability to adapt to many different environments within its infected host. C. neoformans adapts by utilizing conserved eukaryotic and fungal-specific signaling pathways to sense and respond to stresses within the host. Upon infection, two of the most significant environmental changes this organism experiences are elevated temperature and high pH. </p><p>Conserved Rho and Ras family GTPases are central regulators of thermotolerance in C. neoformans. Many GTPases require prenylation to associate with cellular membranes and function properly. Using molecular genetic techniques, microscopy, and infection models, I demonstrated that the prenyltransferase, geranylgeranyl transferase I (GGTase I) is required for thermotolerance and pathogenesis. Using fluorescence microscopy, I found that only a subset of conserved GGTase I substrates requires this enzyme for membrane localization. Therefore, the C. neoformans GGTase I may recognize its substrate in a slightly different manner than other eukaryotic organisms. </p><p>The alkaline response transcription factor, Rim101, is a central regulator of stress-response genes important for adapting to the host environment. In particular, Rim101 regulates cell surface alterations involved in immune avoidance. In other fungi, Rim101 is activated by alkaline pH through a conserved signaling pathway, but this pathway had yet been characterized in C. neoformans. Using molecular genetic techniques, I identified and analyzed the conserved members of the Rim pathway. I found that it was only partially conserved in C. neoformans, missing the components that sense pH and initiate pathway activation. Using a genetic screen, I identified a novel Rim pathway component named Rra1. Structural prediction and genetic epistasis experiments suggest that Rra1 may serve as the Rim pathway pH sensor in C. neoformans and other related basidiomycete fungi. </p><p> To explore the relevance of Rim pathway signaling in the interaction of C neoformans with its host, I characterized the Rim101-regulated cell wall changes that prevent immune detection. Using HPLC, enzymatic degradation, and cell wall stains, I found that the rim101Δ mutation resulted in increased cell wall chitin exposure. In vitro co-culture assays demonstrated that increased chitin exposure is associated with enhanced activation of macrophages and dendritic cells. To further test this association, I demonstrated that other mutant strains with increased chitin exposure induce macrophage and dendritic cell responses similar to rim101Δ. We used primary macrophages from mutant mouse lines to demonstrate that members of both the Toll-like receptor and C-type lectin receptor families are involved in detecting strains with increased chitin exposure. Finally, in vivo immunological experiments demonstrated that the rim101Δ strain induced a global inflammatory immune response in infected mouse lungs, expanding upon our previous in vivo rim101Δ studies. These results demonstrate that cell wall organization largely determines how fungal cells are detected by the immune system.</p> / Dissertation

Cattlemen in the southeastern United States widely plant annual ryegrass [Lolium perenne L. spp. multiflorum (Lam.) Husnot] (ARG). It is often planted earlier than the recommended window. This exposes juvenile stands to increased pressure from gray leaf spot (Pyricularia oryzae Cavara) (GLS), causing stand loss. This study was designed to screen for resistance to P. oryzae in two ARG genotypes, Vertyl and a heat-tolerant germplasm (HTARG5) with the objective of developing a crossbred population to be improved using restricted, recurrent phenotypic selection. Experiments were conducted from 2021 – 2022. Disease severity (DS) was assessed 3wk after inoculation with P. oryzae. Vertyl had a significantly lower DS in the preliminary screenings compared to the other genotypes. In experiments 3 and 4, mean DS of Vertyl, HTARG5, and Marshall was not significantly different. These results confirm Vertyl has greater resistance to GLS than HTARG5 and can be a candidate for disease resistance breeding.

No description available.

No description available.

Fungal infections of animals have become an increasingly important global issue. White-Nose Syndrome is an ongoing fungal epizootic of North American hibernating bats, caused by epidermal infections of the fungus, Pseudogymnoascus destructans. Infections emerged early in 2006 in New York State and have since spread to 35 US States and seven Canadian Provinces, with rates of mortality exceeding 90% in some bat colonies. As an emerging outbreak in North America, the transmission of P. destructans is assumed to occur in a radial fashion outwards from the point of origin. In addition, the factors that may influence P. destructans transmission have been postulated, but not tested before. Lastly, as reproduction is assumed to be strictly clonal in North America, invasive populations should have low genetic diversity, and may even accumulate deleterious mutations over time. The aim of my PhD research is to test these assumptions regarding the spread, evolution, and adaptation of P. destructans using combination of genotyping methods. My results showed how P. destructans isolates have shifted in terms of phenotypes and physiological capabilities since being introduced. In addition, I describe patterns of connectivity across the landscape, which are more consist with the level of anthropogenic activity than variation in climate. The mutations common to all invasive strains of P. destructans are associated with adaptations that have occurred since being introduced from Europe, some with relevant metabolic functions that fit their pathogenic lifestyle. Together, my results revealed significant phenotypic and genotypic changes during the spread of P. destructans in North America. The factors identified here that influence the phenotypic and genotypic changes should help developing better management strategies against the White-Nose Syndrome pathogen. / Thesis / Doctor of Philosophy (PhD)

L’étude de la structure génétique des populations d’agents pathogènes à grandes échelles reste très important dans la contexte de nouvelles invasions. Puccinia striiformis f.sp. tritici (PST), responsable de la rouille jaune du blé, constitue un modèle fongique d’intérêt pour les études d’invasion étant donné sa capacité de migration et l’apparition récurrente de nouvelles souches localement. Nous avons analysé la structure des populations de PST à l’échelle mondiale, à l’aide de marqueurs microsatellites sur un échantillon de 409 isolats issus des six continents. Les génotypes ont été répartis en six groupes génétiques correspondant à leur origine géographique. Les analyses indiquent une forte hétérogénéité géographique de diversité génotypique, avec des signatures de recombinaison dans les régions de l'Himalaya (Népal et Pakistan) et à proximité en Chine. La structure reste clonale pour les populations des autres régions. L’assignation des isolats aux différents groupes génétiques a permis de déterminer l’origine des invasions (récentes ou anciennes). Ainsi, les souches agressives adaptées à de hautes températures, répandues de par le monde depuis 2000, sont originaires de Mer rouge-Moyen Orient ; les isolats d'Amérique du Nord et du Sud et d’Australie proviennent d’Europe du Nord-Ouest. Par ailleurs, les isolats d'Afrique du Sud appartiennent au groupe génétique de la zone méditerranéenne. La subdivision marquée entre les différentes zones géographiques indique qu’elles ne sont pas fortement marquées par les migrations récentes. De plus, les voies de migration identifiées attestent de l'importance des activités humaines dans la dispersion de PST à longue distance. La biologie des populations des zones les plus diverses (Chine et Pakistan) a été finement étudiée à l’aide d‘échantillonnages réalisés deux années consécutives. Une population échantillonnée en 2004 et 2005 dans la vallée de Tianshui, (province de Gansu, Chine), s’est révélée très diverse, fortement recombinante et non structurée spatialement et temporellement. L’observation de clones identiques entre les deux échantillons temporels a permis de développer un estimateur du taux de sexualité, i.e. du rôle relatif de la reproduction sexuée par rapport à celui de la reproduction asexuée dans le maintien de la population. Ce taux de reproduction sexuée est estimé à 74 %, alors que la taille efficace de la population est de 1735, ce qui donne les premières indications du rôle du cycle sexué. L’échantillonnage réalisé au Nord du Pakistan a permis de décrire quatre groupes génétiques ayant tous une grande diversité génotypique et une structure recombinante. Le très faible taux de ré-échantillonnage de génotypes identiques au cours de deux années suggère le rôle prédominant de la reproduction sexuée dans le maintien temporel des populations locales. La forte diversité génétique et génotypique, la signature de recombinaison et la capacité à la reproduction sexuée de PST dans la région himalayenne suggèrent que cette zone est le centre d'origine potentielle de PST. Les analyses d’approximations bayésiennes confirment la thèse d’une dispersion à partir de l’Himalaya vers les autres régions du monde. La variabilité pour la capacité à produire des téleutosores, spores indispensables à l’initiation de la phase sexuée, a été analysée (56 isolats mondiaux), et s’avère liée à la variabilité génotypique et au taux de recombinaison. Ce résultat conforte la thèse de l'apparition de la sexualité dans la zone himalayenne et à proximité de cette zone et de la perte de sexualité lors de migrations dans les zones où l’hôte alternant est absent et où le cycle épidémique est essentiellement asexué. La description de l'origine, des voies mondiale de migration de PST ainsi que de son centre de diversité contribue à la compréhension du potentiel évolutif de PST et à la construction de stratégies de gestion de lutte contre l’agent pathogène. / Analyses of the large-scale population structure of pathogens enable the identification of migration patterns, diversity reservoirs or longevity of populations, the understanding of current evolutionary trajectories and the anticipation of future ones. A detailed analysis of populations in centre of diversity should enable to infer the adaptive capacity of the pathogen and identify potential sources for new invasions. Puccinia striiformis f.sp. tritici (PST) is the causal agent of wheat yellow/stripe rust, and despite a worldwide distribution, this fungus remains a model species for invasion studies, due to its long-distance migration capacity and recurrent local emergence of new strains. Little is known about the ancestral relationship of the worldwide PST population with unknown center of origin. We used multilocus microsatellite genotyping to infer the worldwide population structure of PST and the origin of new invasions, analysing a set of isolates representative of sampling performed over six continents. Bayesian and multivariate clustering methods partitioned the isolates into six distinct genetic groups, corresponding to distinct geographic areas. The assignment analysis confirmed the Middle East-Red Sea Area as the most likely source of newly spreading, high-temperature-adapted strains; Europe as the source of South American, North American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. The existence of strong population subdivision at worldwide level shows that major genetic groups are not markedly affected by recent dispersal events. However, the sources for recent invasions and the migration routes identified emphasize the importance of human activities on the recent long-distance spread of the disease. The analyses of linkage disequilibrium and genotypic diversity indicated a strong regional heterogeneity in levels of recombination, with clear signatures of recombination in the Himalayan (Nepal and Pakistan) and near-Himalayan (China) regions and a predominant clonal population structure in other regions. To explain the variability in diversity and recombination of worldwide PST populations, we assessed their sex ability in terms of telial production, the sex-specific structures that are obligatory for PST sexual cycle, in a set of 56 isolates representative of these worldwide geographical origins. We confirmed that the variability in genotypic diversity/ recombination was linked with the sex ability, pinpointing the Himalayan region as the possible center of origin of PST, from where it then spread worldwide. The reduced sex ability in clonal populations certainly reflects a loss of sexual function, associated to migration in areas where sexual alternate host is lacking, or not necessary for the completion of epidemic cycle. Approximate Bayesian computation analyses confirmed an out of Himalaya spread of PST, with Pakistan and China being the most ancestral population. A detailed analysis of Pakistani population at regional level revealed the existence of a strong population subdivision, a high genotypic diversity and the existence of recombination signature at each location reflecting the role of sexual recombination in the temporal maintenance at local level. A time spaced sampling of PST in the valley of Tianshui (China) inspired the development of a new estimator, allowing to quantify the relative contribution of sexual reproduction and effective population size on the basis of clonal resampling within and between years. A sexual reproduction rate of 74% (95% confidence interval [CI]: 38-95%) and effective population size of 1735 (95% CI: 675-2800) was quantified in Chinese PST population. The description of the origin and migration routes of PST populations worldwide and at its centre of diversity contributes to our understanding of PST evolutionary potential, and is helpful to build disease management strategies.

Analyses of the large-scale population structure of pathogens enable the identification of migration patterns, diversity reservoirs or longevity of populations, the understanding of current evolutionary trajectories and the anticipation of future ones. A detailed analysis of populations in centre of diversity should enable to infer the adaptive capacity of the pathogen and identify potential sources for new invasions. Puccinia striiformis f.sp. tritici (PST) is the causal agent of wheat yellow/stripe rust, and despite a worldwide distribution, this fungus remains a model species for invasion studies, due to its long-distance migration capacity and recurrent local emergence of new strains. Little is known about the ancestral relationship of the worldwide PST population with unknown center of origin. We used multilocus microsatellite genotyping to infer the worldwide population structure of PST and the origin of new invasions, analysing a set of isolates representative of sampling performed over six continents. Bayesian and multivariate clustering methods partitioned the isolates into six distinct genetic groups, corresponding to distinct geographic areas. The assignment analysis confirmed the Middle East-Red Sea Area as the most likely source of newly spreading, high-temperature-adapted strains; Europe as the source of South American, North American and Australian populations; and Mediterranean-Central Asian populations as the origin of South African populations. The existence of strong population subdivision at worldwide level shows that major genetic groups are not markedly affected by recent dispersal events. However, the sources for recent invasions and the migration routes identified emphasize the importance of human activities on the recent long-distance spread of the disease. The analyses of linkage disequilibrium and genotypic diversity indicated a strong regional heterogeneity in levels of recombination, with clear signatures of recombination in the Himalayan (Nepal and Pakistan) and near-Himalayan (China) regions and a predominant clonal population structure in other regions. To explain the variability in diversity and recombination of worldwide PST populations, we assessed their sex ability in terms of telial production, the sex-specific structures that are obligatory for PST sexual cycle, in a set of 56 isolates representative of these worldwide geographical origins. We confirmed that the variability in genotypic diversity/ recombination was linked with the sex ability, pinpointing the Himalayan region as the possible center of origin of PST, from where it then spread worldwide. The reduced sex ability in clonal populations certainly reflects a loss of sexual function, associated to migration in areas where sexual alternate host is lacking, or not necessary for the completion of epidemic cycle. Approximate Bayesian computation analyses confirmed an out of Himalaya spread of PST, with Pakistan and China being the most ancestral population. A detailed analysis of Pakistani population at regional level revealed the existence of a strong population subdivision, a high genotypic diversity and the existence of recombination signature at each location reflecting the role of sexual recombination in the temporal maintenance at local level. A time spaced sampling of PST in the valley of Tianshui (China) inspired the development of a new estimator, allowing to quantify the relative contribution of sexual reproduction and effective population size on the basis of clonal resampling within and between years. A sexual reproduction rate of 74% (95% confidence interval [CI]: 38-95%) and effective population size of 1735 (95% CI: 675-2800) was quantified in Chinese PST population. The description of the origin and migration routes of PST populations worldwide and at its centre of diversity contributes to our understanding of PST evolutionary potential, and is helpful to build disease management strategies.

La température est un des principaux facteurs climatiques pilotant le développement des champignons pathogènes foliaires pendant les différentes étapes de leur cycle parasitaire. Puisque ces microorganismes se développent à la surface, puis à l’intérieur des feuilles, c’est la température de feuille (« body temperature » en écologie) qui pilote leur développement. En épidémiologie végétale, c’est toutefois la température d’air qui est utilisée pour caractériser l’effet de la température sur le développement des agents pathogènes foliaires. Or, la température de feuille peut différer significativement de la température d’air en fonction des conditions climatiques. La prise en compte de la température d’air pour étudier la dynamique des maladies foliaires ne peut donc s’affranchir de deux biais : la température mesurée n’est pas celle qui est réellement perçue par l’agent pathogène et l’hétérogénéité spatiale des températures au sein du peuplement n’est pas prise en compte. De plus, la relation entre la température et le développement des agents pathogènes est non linéaire, ce qui limite la gamme de validité autorisant l’utilisation des sommes de températures, pourtant largement employées en protestion des cultures. L’objectif général de cette thèse est de reconsidérer la prise en compte de la température pour l’étude du développement des champignons pathogènes foliaires.Le pathosystème blé-Mycosphaerella graminicola a été choisi en tant qu’objet d’étude. La stratégie adoptée pour atteindre les objectifs de la thèse combine deux approches complémentaires, l’expérimentation et la modélisation. Pour la première fois, la loi de réponse d’un agent pathogène foliaire à la température de feuille a été établie. Un dispositif expérimental innovant a permis d’établir la loi de réponse pour trois isolats sur une large gamme de températures foliaires, via la mesure en continu de la température de 191 feuilles (F et F) inoculées et l’utilisation d’un système de forçage thermique par lampe infrarouge. La loi de réponse de la période de latence de la septoriose à la température de feuille s’apparente au concept de courbe de performance thermique développé en écologie. Celle-ci étant non linéaire sur l’ensemble de la gamme de température étudiée, l’impact de l’amplitude de fluctuations de température de feuille a été caractérisé. Une amplitude élévée a conduit à plusieurs effets négatifs pour le développement de M. graminicola : l’augmentation de la durée du cycle de l’agent pathogène, la diminution de la surface sporulante des lésions et de la densité de pycnides. Les différences de cinétique de développement en fonction de l’amplitude des fluctuations ne sont que partiellement expliquées par l’effet Kaufmann (purement mathématiques), suggérant que M. graminicola atténue les conséquences négatives d’amplitudes de fluctuation plus élevées. Enfin, les simulations du développement de la septoriose réalisées à partir de données de températures foliaires diffèrent signicativement de celles réalisées à partir de températures d’air mesurées de façon standard par une station météorologique. Ces simulations ont également souligné le caractère déterminant du pas de temps considéré.Par le transfert de concepts d’écologie vers l’épidémiologie, cette thèse ouvre des pistes pour améliorer la prise en compte de la température dans les modèles épidémiologiques. Elle contribue au développement d’une meilleure compréhension des mécanismes par lesquels l’environnement affecte les microorganismes, point clé pour le développement de modèles mécanistes de réponses possibles au changement climatique / Temperature is a major force for the development of foliar fungal pathogens. Such organisms develop onto and into leaves during their growth cycle. Thus, leaf temperature is the temperature they actually perceive (“body temperature”). However, air temperature has always been used by plant pathologists to study the effect of temperature on the development of foliar fungal pathogens. Leaf temperature may significantly differ from the air temperature according to weather conditions. Therefore, considering the air temperature to study foliar pathogens can potentially cause two biases: the measured temperature is not the temperature such pathogens actually perceive and the spatial heterogeneity of leaf temperatures within the plant canopy is ignored. In addition, the relationship between temperature and the development of foliar pathogens is nonlinear. This challenges the immoderate use of degree-day sums in plant disease epidemiology. The main objective of this thesis is to reconsider the use of temperature for the study of the development of foliar fungal pathogens.The wheat-Mycosphaerella graminicola pathosystem was chosen as the model of study. The strategy to achieve the objectives of the thesis combines two complementary approaches: experimentation and modelling. For the first time, the impact of leaf temperature on the development of a leaf pathogen was characterized. An original experimental device allowed determining the response law for three isolates over a wide range of leaf temperature, using thermal infrared lamps and measuring continuously the temperature of 191 inoculated leaves (F1 and F2). The response law of M. graminicola latent period to leaf temperature is similar to the concept of thermal performance curve (TPC) developed in ecology. As this TPC is non-linear over the entire leaf temperature range investigated, the impact of the amplitude of leaf temperature fluctuations has been characterized. A high amplitude led to several negative effects on M. graminicola development: an increase in the duration of the pathogen cycle, a decrease in the final sporulating area in the pycnidium density. Differences in kinetics of development depending on the amplitude of the fluctuations were only partially explained by the Kaufmann effect (purely mathematical), suggesting that M. graminicola mitigates the negative consequences of higher amplitudes of temperatures fluctuation. Finally, simulations of the development of M. graminicola performed using leaf temperature data differed significantly from those performed using air temperatures measured in a standard way, by a weather station. Simulations also underlined the importance of the time step considered. By transferring concepts from ecology to epidemiology, this thesis provided guidelines to better take into account temperature in epidemiological models. It helped to develop a better understanding of the mechanisms by which the environment affects micoorganisms, the cornerstone for the development of mechanistic models of possible responses to climate change.