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Activity 3: Adaptation - Land Environments

A3-wp 1 - Adaptation in Alpine Conifers

Participants: Elena Mosca, Nicola La Porta, Giovanni G. Vendramin, David B. Neale

Coniferous trees form the dominant plant species in many alpine landscapes, which are subject to climate change and other anthropogenic factors. Alpine coniferous tree species in the Italian Alps may be responding to climate change by adapting to the modified environmental conditions or by migrating to more suitable habitats. Our goal is quantify and understand the standing adaptive genetic diversity in alpine conifers so that we may obtain a baseline reference to monitor genetic change in populations. Moreover, this study will provide useful information for forestry management, such as diagnostic tools for assisted migration according to the species potential distribution. We focus on five species: Pinus cembra L., Larix decidua Mill., Pinus mugo Turra, Abies alba Mill and Picea abies (L.) H. Karst.

To begin, we have studied the level of DNA sequence polymorphism in four species (Picea abies was investigated in a prior study). For each species, 12 individuals were sampled from several diverse mountain locations, such as Pyrenees, Massif Central, Alps, Apennines and Carpathian. Nearly 800 candidate genes were resequenced to identify single nucleotide polymorphisms (SNP) in the four species. Because Sanger sequencing primers were derived from loblolly pine (Pinus taeda L.) a higher percentage of amplicons could be resequenced in the pines than fir or larch. In Pinus mugo and Pinus cembra 4500 and 2500 SNPs were discovered, respectively. In Larix decidua and Abies alba 1800 and 1300 SNPs were discovered, respectively. We are currently estimating measures of nucleotide diversity and performing tests of neutrality with these sequence data.

The second aim of the research is to determine the complex patterns of the adaptation to changing environment by performing population genetic analyses in the four species. Genotyping chips for the four species will be designed and genotyping will be conduced for 1000 trees per species in Italian Alps. The sampling sites have been identified to capture the full spectrum of adaptive genetic diversity and to study the possible interaction among species that share the same environment.

A3-wp2 - Cold Regulated (COR) genes and cold tolerance in Brassicaceae

Participants: Lino Ometto, Claudio Varotto

The response to adverse abiotic factors is a key adaptive response in plants, sessile organisms that are forced by their immobility to cope with environmental stress. The exposure to low temperature is an important stimulus promoting flowering in winter annual and perennial plant species. Sudden temperature drops are however a common source of stress, especially in mountain regions. To cope with low temperatures, plants have developed a series of physiological adaptations which are normally triggered by cold stress.

The CBF transcription factors are key molecular switches which, upon perception of cold stress, activate a signalling cascade involving the up-regulation of about 100 genes (called Cold Responsive or briefly COR genes) taking part into the physiological changes that accompany the onset of cold resistance (Miura et al, 2007). Despite the pretty detailed understanding of the activation mechanism of CBF genes in the model species Arabidopsis thaliana, little is known about the relative relevance of CBF and COR genes in the increased cold resistance commonly observed in high altitude plants (orophytes). The comparison among congeneric species adapted to different altitudes may provide a good model system to address this point.

To this purpose, we selected two Arabidopsis relatives which are adapted to disjoint altitudinal ranges: Cardamine resedifolia, a species growing above 1800 m of altitude and Cardamine impatiens, a species normally growing below 1200 m asl.

The identification of the genes with higher adaptive value with regard to cold adaptation is going to be carried out with different approaches. The isolation and sequencing of CBF and COR genes from these two species will provide a first assessment of the relative evolutionary rates in comparison to Arabidopsis orthologs. The determination of genetic variation among species will be complemented by intra-specific measurements of nucleotide variation in selected populations from both species.

Comparison of expression levels of COR genes in C. resedifolia and C. impatiens will be used as a further screening method for the identification of COR genes which are candidates for the differential cold resistance of these two species. The most promising candidates identified may be functionally tested in Arabidopsis thaliana for their ability to modulate the adaptive response to cold stress through genetic transformation.

A3-wp3 - Common frog landscape genetics

Participants: Luca Cornetti, Barbara Crestanello, Michele Menegon, Elena Pecchioli, Paolo Pedrini, Cristiano Vernesi

The common frog (Rana temporaria) is an anuran amphibian widespread across Europe. In the Alps it shows altitudinal and anthropogenic gradients that provide a suitable model to study adaptive responses to global change. As most amphibians, the common frog is sensitive to changes in levels of abiotic stresses. Beside, fungal diseases affect many amphibians whose diffusion has been linked to global warming (Pounds et al, 2006). Due to all these threats the amphibians are facing a major global decline becoming more threatened than birds or mammals. Evidence of coding genes directly associated to adaptive traits is still lacking, but some studies found a significant positive relationship between fitness related traits and the amount of variability at neutral markers as microsatellites (Lesbarrères et al, 2005; Johansson et al, 2007).

With this study we aim at understanding which are the most relevant environmental variables (e.g. altitude, mean annual temperature, precipitation, etc.) responsible for the observed patterns of intra-population variability and inter-population genetic differentiation when comparing natural populations adapted to different ecological features.

This objective will be accomplished adopting a landscape genetics approach: genetic data will be correlated to a set of pre-defined environmental features within a GIS-based framework. The flow chart of this activity can be summarized as follows: careful selection of populations displaying supposedly different adaptation regimes; sampling of a reasonable amount of individuals from each population (at least 15-20) recording, whenever possible, the GPS coordinates for every single specimen; typing the samples at 15-20 microsatellites loci; retrieving from available databases the information about the environmental features of the sampling areas; analysing the correlation between genetic and environmental data in a Bayesian framework by adopting a Reversible Jump Markov Chain algorithm which allows to simultaneously take into account several different models.

We strongly believe that, taking advantage of similar approaches pursued in taxa studied in other WPs and Activities and sampled at the same sites as common frog, this activity will permit distinguishing species-specific factors from factors being relevant in all species and therefore likely to be relevant for the whole ecosystem.

A3-wp4 - Molecular basis of host-parasite interaction

Participants: Barbara Crestanello, Annapaola Rizzoli, Fausta Rosso, Valentina Tagliapietra, Cristiano Vernesi

This activity is mainly aimed at trying to better identify the molecular basis of the adaptive response to host-parasite interaction, focusing on a wide array of infective agents, including viruses, bacteria and helminths. The two species selected are Apodemus flavicollis and Tetrao tetrix.

Apodemus flavicollis, common name yellow-necked field mouse, is widespread throughout Europe showing large temporal variation in abundance, with periodical population peaks mostly related to seed production (Angelstam et al., 1987). Climate warming and land use changes are currently considered the main causes of demographic increase in some rodent species including Apodemus sp. These species are now recognised among the most important reservoirs of emerging human diseases in Europe (http://www.eden-fp6project.net).

Immunogenetics provides insights into the relative influence of genetic variation and environmental factors on host-pathogen interactions. An association between MHC class II genes and Puumala hantavirus infection in vole has been recently recorded (Deter et al, 2008). Specific rodent-borne viruses and tick borne diseases will be selected for the yellow-necked field mouse species on the basis of previous investigation.

The black grouse Tetrao tetrix, is among the threatened galliform species in Europe (Storch & Segelbacher, 2001). Habitat fragmentation, hunting pressure, predation, climate change and disease are considered among the major threats. In Trentino, the species is undergoing a progressive reduction both in spatial distribution and density, with temporal fluctuation in abundance. Previous epidemiological studies performed in different areas of Trentino showed that this species harbours several helminth species; some of them, as Ascaridia compar, may be relevant in processes of parasite mediated competition. Insofar few immunogenetic studies have carried out on this species.

The general objective of this activity is twofold: 1) identification of the relationship between specific alleles of candidate genes and resistance to pathogens. 2) assessment of the specific alleles frequency in the different populations.

While for black grouse it seems now affordable only the analysis of genes of class II of the Major Histocompatibility Complex (MHC), in the yellow-necked field mouse we will take advantage of the large genomic resources of the closely phylogenetically related domestic mouse for analysing not only MHC genes but also other candidate loci. Since pathogens have different pathways, in terms of intracellular or extracellular activity, this differentiation is crucial for the selection of specific immune-related genes. We will consider some loci of the Interferon (IFN) and Tumor necrosis factor (TNF) gene families which are involved in the response against intracellular pathogens.

In the same specimens typed at neutral markers, we will therefore analyse molecular variation in MHC genes and in IFN and TNF genes by means of resequencing. Conducting veterinary analyses on the same specimens, by means of serological, parasitological and molecular assays, depending on the pathogens investigated, will allow testing the hypothesis that specific alleles can confer resistance to pathogens, thus unveiling the basis of adaptation to disease resistance. The evaluation, by means of maximum likelihood and Bayesian methods, of the ratio of synonymous versus non-synonymous substitutions will allow understanding which is the most likely selective regime acting on the loci under examination. The frequency of these supposed specific alleles in the different populations will be correlated with the most relevant environmental features of each population so to try to understand the ecological drivers of adaptation to pathogens.