First evaluation ACE-SAP project October the 19th, 2009

T ABLE O F C ONTENTSA2-wp1 - Genetic diversity and adaptive traits in the filamentous cyanobacterium A2-wp3 - The adaptive potential of natural populations of two aquatic insect species

ACE-SAP projectAlpine ecosystems in a Changing Environment:

Biodiversity Sensitivity and Adaptive Potential

First evaluation ACE-SAP project

October the 19th, 2009

Edmund Mach Foundation, Via Mach1, San Michele all’Adige, Italy

T ABLE O F C ONTENTS

A2-wp1 - Genetic diversity and adaptive traits in the filamentous cyanobacterium

A2-wp3 - The adaptive potential of natural populations of two aquatic insect species in

First evaluation ACE-SAP project

Evaluator: Chris Bowler

Place: Fondazione Edmund Mach

Date: 19.10.2009

Proposed day schedule

9:00-9:20 Welcome - Roberto Viola, Director CRI FEM.

9:20-9:40 Project overview – David Neale, UCD, Scientific Coordinator

9:40-10:00 ACE-SAP Activities and Work packages Overview – Claudio

Varotto, FEM.

10:00-10:30 Biological Conservation - Cristiano Vernesi, FEM - Patterns of

genetic diversity and structure among populations of viviparous

lizard (Zootoca vivipara).

10:30-11:00 Adaptation - Aquatic Environments - Valeria Lencioni, MTSN- Cold

and warm resistance in the stenothermal chironomid

Pseudodiamesa branickii.

11:00-11:30 Coffee break

11:30-12:00 Adaptation - Land Environments - David Neale, UCD- Adaptive

genetic diversity in alpine conifers of Trentino.

12:00-12:30 Modelling - Emanuele Eccel, FEM.

12:30-13:00 Popularisation - Claudia Beretta, MCR.

13:00-14:00 Lunch

14:00-16:00 Meeting of Prof Bowler with ACE-SAP executive committee

16:00-18:00 Visit to FEM campus Additional meetings to be scheduled on

request

Key to acronyms and details

CRI - Research and Innovation Centre

FEM - Fondazione Edmund Mach

MCR - Museo Civico di Rovereto

MTSN - Museo Tridentino di Scienze Naturali

UCD - University of California, Davis

Abstracts of each project activity and work package

A1 -Biological Conservation

A1-wp3 - Adaptive genetic variation in the highly endangered Salmo carpio L 1758

Participants: Paolo Gratton, Andrea

Gandolfi

Within the S trutta complex, Salmo carpio

exhibits very specific ecological features,

including gregarious behaviour, planktonic

diet and peculiar reproductive biology It is

therefore commonly accepted as a ‘true’

species, strictly endemic to the Garda lake

and critically endangered according to the IUCN Red List of Threatened Species

The carpione is a middle-sized trout, attaining a maximum size and weight of 35-40 cm and ca

500 g Exceptional specimens may reach 50 cm and 1 kg The body shape is characterized by relatively small head The background colour is silver grey, with small blackish dots on the back During spawning seasons there is some sexual dimorphism, with males showing a dark bronzed background and blackish fins

Eggs are laid in two spawning seasons (peaking in December-January and July-August,

respectively) in deep (50-200 m) lakebeds with plenty of oxygen

The species has gregarious attitudes, particularly during seasonal migration between

spawning and foraging areas The diet of Salmo carpio is mostly zooplanktonic.

Genetic analysis of mitochondrial and nuclear data led different authors to propose a hybrid

origin for S carpio, by secondary contact in recent times of different evolutionary lineages of the S trutta complex This hypothesis is consistent with the post-glacial origin of the Garda lake Admitting that S carpio might have a hybrid origin does not negate its recognition as a

species under the Phylogenetic Species Concept as it is an independent and diagnosable lineage Moreover, the adaptive differentiation from the two parental lineages adds interest forthe species in an evolutionary context

Some peculiar features of the S carpio life-cycle could lie within the wide range of phenotypic plasticity of the S trutta complex, and be a direct effect of the Genotype-Environment

interactions in the specific environmental conditions and factors faced in the Garda lake (e.g trophic spectrum) However, behavioural and life-history traits directly associated to

reproduction (two spawning seasons a year and spawning areas in deep waters of the lake)

that create a strong reproductive barrier isolating the carpione from the lacustris phenotype

of the S trutta complex, sympatric in the Garda Lake, are likely to represent a rapid adaptive

(genetic?) response to a strong selection imposed by the environment Divergent natural selection (selection on ecologically relevant traits that favours different alleles in different environments) could have been the main driver to the incipient ecological speciation within the Garda lake

The aim of this WP is to characterize neutral and potentially functional polymorphisms (SNPs)

in the Salmo carpio genome Putatively neutral polymorphic loci available from other salmonid

species will be assayed and compared to genetic variation in genes potentially under positive selection (candidate genes), which will be preliminarily searched for by comparison of

expression profiles (DNA microarrays, Quantitative Real-Time PCR assays)

The description of genetic variation, according to this approach, will be a valuable tool for i)

phylogenetic and population studies within the S trutta complex, and ii) to formulate new hypotheses on the S carpio rapid adaptive evolution and incipient speciation.

A1-wp2 - Conservation genetics of three threatened vertebrate species

Participants: Luca Cornetti, Barbara Crestanello, Michele

Menegon, Elena Pecchioli, Paolo Pedrini, Cristiano Vernesi

The main goal is to provide a preliminary ecological and

molecular survey of three vertebrate species, selected for their

conservation value

The species of this WP are: common lizard (Zootoca vivipara),

yellow-bellied toad (Bombina variegata) and rock ptarmigan

(Lagopus muta).

The common lizard is an Eurasian lacertid that has both viviparous and oviparous

populations The first are widely distributed from British Isles to northeastern Asia (Takenaka,1991); the latter have only been observed in Slovenia, Italian NE Alps, Prealps According to mitochondrial DNA and karyotype, oviparous populations from Slovenia and Italian Prealps

belong to a different subspecies, Z v carniolica (Surget-Groba et al 2002) In Trentino Z v

carniolica has been recently discovered (Menegon et al., 2003), being relegated to low and

middle altitude bogs Z vivipara is considered one of the vertebrate taxa facing the highest risk

of extinction within the regional boundary

B variegata, is distributed over much of central and southern Europe with an altitudinal range

from 100 to 2100 m, while it prefers low-mid altitudes The species has experienced some local extinctions and population declines In Italy it is present to the north of river Po with fragmented populations Populations of this species might be locally threatened by the loss of suitable habitat due to anthropogenic pressure (e.g transportation and discharge of pollutantsinto wetlands; from Amphibia Data, IUCN) and mycosis The species is listed on Appendix II of the Berne Convention, on Annexes II and IV of the EU Natural Habitats Directive In Trentino

there are evidences of rapid decline of some population of B variegata (Caldonazzi et al

2002)

The rock ptarmigan (Lagopus muta helvetica) is a bird inhabiting the arctic and alpine tundra

of North America and northern Eurasia Isolated populations are found on mountain chains of southern Europe (Holder & Montgomerie, 1993) This species can be considered well adapted

to high altitude and cold environment, being therefore particularly sensitive to perturbations posed by global warming and human activities In fact some range contractions with local extinctions have been reported (Flint, 1995) The isolated and fragmented Alpine populations are facing several threats: habitat fragmentation and degradation linked to climate change, tourism activities and overhunting (Ménoni & Magnani 1998, Zeitler & Glänzer 1998) The

subspecies, L m helvetica, is added in Annex I of EU Wild Birds Directive; it is considered

Vulnerable in Italian Red List and “Endangered” in Trentino

Taking advantage of previous research conducted by participants, we will first provide basic information about where natural populations occur across Trentino and about habitat

selectivity This allows carefully selecting sampling sites, which should be equally subdivided between the Eastern and the Western side of Adige valley

Molecular analyses will involve determination of nucleotide sequence variation at

mitochondrial DNA and allele frequency estimation at some (6-10) nuclear microsatellites Statistical data analysis will address estimation of genetic diversity within population and differentiation among populations by means of standard approaches (e.g nucleotide diversity,

expected heterozygosity, Fst, AMOVA, etc.) and more advanced coalescent-based

methodologies (Bayesian estimation of migration rate, effective population size and time of divergence in non equilibrium models, individual-based assignment, etc.)

Whenever possible, the results of genetic analyses will be integrated with spatial ecological features in order to correlate the size and quality of the habitat with the level of genetic

variation and differentiation for each species

A1-wp3 - Taxonomic delimitation and conservation biology of three endemic plant taxa

Participants: Thomas Abeli, Alessio Bertolli,

Costantino Bonomi, Simone Fior, Margherita

Lega, Gilberto Parolo, Filippo Prosser, Graziano

Rossi, Claudio Varotto

Species richness is a traditional measure of

biological diversity It relies on the number of

species present in a given habitat or region The

proper taxonomic definition of endemic taxa,

potentially more threatened with extinction

than widespread relatives, is therefore

particularly relevant to set conservation

priorities aimed at maintaining biodiversity The identification of the closest relative (sister group) of a given taxon is moreover relevant for comparative studies, e.g to compare the inter and intra-population genetic variation of the two taxa

In the Alps several endemic taxa have a dubious taxonomic position Two interesting case

studies are taxa belonging to Brassicaceae, provisionally called Brassica repanda subsp

baldensis Bertolli & Prosser and Erysimum aurantiacum Leyb Both these species are narrow

endemics of the South-Eastern Alps with a very limited distribution

Brassica repanda subsp baldensis Bertolli & Prosser is a Brassica repanda subspecies

described for the first time only two years ago (Bertolli and Prosser, 2007, Willdenowia 37:

191-198) It has the closest morphological affinities to B repanda subspecies of the eastern Iberian Peninsula (subsp blancoana, subsp cadevallii and in particular subsp maritima) and

not to the three subspecies known from the Alps and NE Italy

Erysimum aurantiacum Leyb differs form the closely related species E rhaeticum and E sylvestre by a few traits The most relevant difference, however, is the orange pigmentation of

its petals, a trait which inspired the name given to this taxon

Another endemic taxon present in this region is Aquilegia thalictrifolia Schott & Kotschy This

taxon is recognized as a true species, but its sister species is not known The species shows signs of decline associated to habitat change expected as a consequence of the ongoing climatechange which may seriously threaten this species Moreover, little is known about the

conservation status of this plant (population ecology and dynamics)

Both in the case of B repanda subsp baldensis Bertolli & Prosser and E aurantiacum Leyb a

phylogenetic reconstruction based on molecular markers will be carried out to determine the taxonomic status of this taxa An AFLP markers based exploratory study will be first

performed on these two species and related taxa in order to orientate the successive research line The phylogenetic reconstruction of A thalictrifolia Schott & Kotschy will allow the

identification of its sister group

Genetic analyses of all taxa under study will be carried out to characterize the genetic

variation within and among populations Microsatellite markers developed for Arabidopsis and close relatives will be tested in both B repanda subsp baldensis Bertolli & Prosser and E

aurantiacum Leyb and other SSR markers may be developed within the frame of this

project.AFLP markers will also be tested for these two taxa A good number of SSR markers

already available for Aquilegia species will be applied to the genetic characterization of A

thalictrifolia Schott & Kotschy populations

In parallel the collection of ecological and demographic data will provide a detailed

description of the largely unknown biology of these species In particular, for Aquilegia

thalicrifolia, measurements of fluctuating asymmetry, seed set and germination will provide

direct estimates of population fitness The genetic and ecological data collected will be used toassess the treat status (IUCN 2001 criteria), propose management strategies and conservationguidelines for the taxa under study

A1-wp4 - Flora of Mt Baldo

Participants: Alessio Bertolli, Filippo Prosser

Objectives: to find priority conservation actions for a wide but circumscribed mountain

territory, using as indicator all vascular flora The collected information is extended to a wide territory and to all vascular flora, even if not detailed

Mt Baldo (66-2218 m, 411 km2) is ecologically various and well delimited by Lake Garda and Adige Valley Moreover Mt Baldo is one of the more famous floristic area of the world Calzolari

in 1566 wrote the first field flora Linnaeus in his Flora Alpina (1756) gave to Mt Baldo the same importance as Swiss or Pyrenees

MCR collected data from this territory in order to create a database At the time the database consists on 30.000 records bibliography and herbarium data (from 1554 up to now) and on 140.000 records collected on the field in the years 1991-2008 The data structure is: taxon, locality (observation site), date, altitude (min-max), observer(s) All data collected on the field are georeferenced (with points, polylines or regions), but we have not quantitative and

ecological information For each observation site we have generally recorded only part of the taxa

At the time we have finished to

check the data, we are still

uploading bibliographical data and

we have to georeference the

bibliographical data

We can give the following figures:

we have found about 2133 taxa

Among them 1792 (84%) are

native or naturalized and still

present; 94 (4,4%) are native and

not confirmed by recent

observations; 247 (11,6%) are

casual occurrences About 200 taxa

(12 %) we have found were not reported by previous authors We listed about 338 taxa

reported by previous authors as erroneous In a rough way we can answer to some interesting questions: at which altitude did the flora change?

We can also produce maps of all species, showing generally very interesting geographical patterns The database seems to be very suitable to obtain any kind of elaboration regarding geographical distribution of biodiversity at species level We also expect to infer good

information in order to plan conservation actions

A2 - Adaptation - Aquatic Environments

A2-wp1 - Genetic diversity and adaptive traits in the filamentous cyanobacterium

Planktothrix rubescens

Participants: Domenico D’Alelio, Andrea Gandolfi,

Monica Tolotti, Adriano Boscaini, Giovanna Flaim,

Graziano Guella, Nico Salmaso

Planktothrix rubescens (DeCandolle ex Gomont)

Anagnostidis et Komárek 1988 (Cyanoprokaryota;

Order: Oscillatoriales; Family: Phormidiaceae;

Subfamily: Phormidioideae) is a freshwater

microcystins-producing filamentous cyanobacterium

living in the planktonic environment, in both

meso-eutrophic and strongly thermally stratifying large lakes and stagnant waters, where it

produces red water blooms during the summer season; in winter, it often colours the water red under the ice The species occurs in several regions in northern temperate zones, where it effectively colonizes limnetic basins (lakes in Austria; SE France, Germany, Switzerland, N Italyetc.); outside of these limited areas, the species occurs occasionally over the whole temperate zone Seasonal blooms of the species were reported in sub-Alpine lakes in Northern Italy (i.e Como, Garda, Idro, Ledro, Levico, Pusiano) In this organism, cells are joined into straight or slightly curved thricomes, solitary, free-floating, which sometimes form purple-red (or, when dried, violet) irregular clusters that, in turns, accumulate in dense blooms provoking water discolorations

In the frame of the project ACE-SAP, natural populations of Planktothrix rubescens will

be collected from a set of key lakes in Trentino, distributed along morphometric (with focus

on maximum depth), altitudinal and trophic gradient, where water temperature, light and chemical dynamics will be investigated in detail Taxonomical identification of the species used in this study will be carried out with classical morphometric methods (light microscopy).Environmental samples from different habitats (different lakes and different depths from eachlake) will be used for the isolation of single filaments used as inoculum for cultures in

controlled experimental conditions Both strains corresponding to environmental samples andpure cultures will be characterized metabolically Analysis of the secondary metabolites and membrane lipid profiles will be carried out through advanced mass spectrometric techniques (MALDI/TOF and LC-ESI-ION TRAP) and, eventually, through Nuclear Magnetic Resonance

measurements if the investigated strains will produce new metabolites which require a full and ab initio structural elucidation Genetic analysis will include specifically the

characterization of genes involved in the production of gas vesicles (gvp genes) Upon

identification of temperature and light optima for different strains composing natural

populations of Planktothrix, quality and quantity of target secondary metabolites will be

investigated in cultures under optimal and stress (temperature, light) conditions The genetic characterization of strains producing different types of gas vesicles will be carried out from cultured natural strains

To date, sampling sessions have been carried out in the Levico Lake, where the

planktonic community was collected from the water column by using a phytoplankton net (mesh = 40 µm) Vertical net sampling – i.e from the bottom of the lake (ca 40 m) to the surface – allowed the collection of the actual planktonic community, which was successively sorted out in the lab Though the species was not producing a dense bloom in the lake waters

at the time of the sampling, several strains of P rubescens were identified and isolated in light

microscopy and were eventually brought in culture These cultures are now incubated under costant conditions (temperature = 13°C, photoperiod = 12:12h L:D, irradiance < 40 µmol photon · m-2 · s-1) in order to get material enough to begin preliminary biomolecular analysis, which are aimed at i) veryfing the usefulness of existing protocols and ii) standardising

methods to be used during the species’ bloom season, for project’s purposes

A2-wp2 - Target species: Bangia atropurpurea (Roth) C Agardh

Participants: Marco Cantonati, Daniel Spitale,

Alessia Scalfi, Nicola Angeli, Graziano Guella, Rita

Frassanito, Carlo Andreoli, Nicoletta La Rocca,

Isabella Moro, Katia Sciuto

Bangia atropurpurea is widely distributed in marine

intertidal areas as well as in some freshwater

habitats (rivers and lakes in North America, Europe

and Asia) Concerning the distribution in Lake

Garda, B atropurpurea occurs mainly on rocky

substrates in the central-northern Garda and

sporadically in the southern part B atropurpurea typically occurs in the upper and mid

eulittoral zones Here it experiences, both diurnally and seasonally, extreme environmental fluctuations, resulting in desiccation, freezing, osmotic and radiation stress (including UV)

Since L Garda experiences wide seasonal water-level fluctuations, Bangia is supposed to be

adapted to a very stressful habitat Understanding the mechanisms of resistance to UV

exposition and desiccation of B atropurpurea is the main aim of our study within the ACE_SAP

Project

When growing outside the water, B atropurpurea exhibits a set of ecological,

phenological, and bioorganic adaptations Preliminary results suggest that populations of B

atropurpurea grow desynchronized along a small-scale spatial gradient outside the water As a

consequence, near the water mainly wider filaments (monosporangia) occur while the upper

zone is filled mainly by vegetative filaments The patterns observed suggest that B

atropurpurea might possess different levels of tolerance according to the phenological state In

addition, along the same gradient, B atropurpurea shows several shifts of pigments

concentration, probably related to the different duration of air exposure These preliminary

results suggest that the combination of adaptive traits in B atropurpurea changes along the

depth gradient

Since the beginning of 2008, the seasonal cycle of B atropurpurea has been studied by

means of frequent (fortnightly, monthly) sampling surveys Detailed seasonal studies are being carried out in two sites (eastern shore, northern part of the lake) Moreover, the

distribution of the target species in the whole lake has been investigated considering six localities, and, within each one, an impacted and a non-impacted site + replicates Diatoms

epiphytic on B atropurpurea were studied in the two main sampling localities while epilithic

diatom communities (used also for an ecological characterization of the shores) were

investigated in the six localities Results have already been (3rd Central European Diatom Meeting, Utrecht, The Netherlands; 57th North American Benthological Society Meeting, Grand Rapids, MI, USA) and will be (7th International Symposium “Use of Algae for MonitoringRivers”, Luxembourg) presented at international congresses

We are planning a field experiment in which artificial substrata (limestone tiles)

previously colonized by B atropurpurea are translocated to obtain two main treatments: (i)

moisture level (hydrated and exposed to air) and (ii) radiation (ambient, ambient minus UVA, and ambient minus UVA and UVB) Within these main treatments, we will study the response

of B atropurpurea with ecological, morphological, target metabolites analysis, and genetic

approaches Candidate genes will be selected on the basis of literature and preliminary

ecological, morphological, ultrastructural, and bioorganic analyses The expression of the selected genes at the different experimental conditions will be assessed by RT-PCR and/or real time PCR Stress induced by desiccation and by different radiation types will be evaluated

by morphological and ultrastructural analysis, paying special attention to cellular membranes organization and to the stress-protection compounds In addition, photosynthetic apparatus will be investigated concerning a set of specific target proteins (i.e photosystem II reaction centre protein D1)

A2-wp3 - The adaptive potential of natural populations

of two aquatic insect species in relation to

temperature variations and pollutants

Participants: Paola Bernabò, Luigi Caputi, Alessandra

Franceschini, Rita Frassanito, Graziano Guella, Olivier

Jousson, Valeria Lencioni

Abstract:

Knowledge as to how aquatic insects will potentially react

and adapt in face of increasing human impacts is one of the

major challenge in prediction of future freshwater

biodiversity trends

Two main drivers of biodiversity change have been

individuated, global warming and land use In relation to

these drivers, two adaptive traits were selected, resistance

to adverse temperature conditions and resistance to

pollutants, in two target insect species (Diptera:

Chironomidae): the cold stenothermal and stenotope

Pseudodiamesa branickii (Nowicki, 1873) and the euriecious

Chironomus riparius Meigen, 1804 The former is frequent in

Pseudodiamesa branickii

Chironomus riparius

cold mountain springs and streams (<7-8 °C), the latter colonizes warm and eutrophic lakes and rivers and agricultural/industrial waste waters The general aim of this WP is to give new insights to understand: 1) how could aquatic insects react to the oncoming temperature variation related to global climate change; 2) how aquatic insects from polluted freshwaters develop toxic resistance and detoxification capacity This by the i) characterisation of genetic determinants of resistance to abiotic stresses in natural populations of the two selected target species and ii) definition of the role of metabolites such as proteins and sugars and of the membrane lipid composition in development of such resistances

Five candidate genes have been indicated as determinants for resistance to cold and warm

temperature (hsc70, hsp70, hsp90, afps) and to pollutants (cytP450) Heat shock proteins are

known to be involved in temperature variation resistance in many organisms, as well as antifreeze proteins in cold resistance and cytochrome P450 monooxygenase in detoxification processes

Experiment will be carried out on IV instar larvae of P branickii collected in the Noce Bianco stream (Trentino) and of C riparius collected in the Lambro River (Lombardy) and reared in

the laboratory Survival curves will be performed on larvae exposed to different temperatures and toxic compounds The expression levels of all genes will be analysed in stressed and control larvae via quantitative real-timeRT-PCR The main membrane lipid components (fatty acids, glycerolipids and phospholipids) will be characterized with mass spectrometry

techniques Particular attention will be paid to lysophospholipids and phospholipids,

membrane constituents that are expected to change in response to freezing, both in the acyl chains and/or in their polar heads The identification and quantification of cryoprotectans such as polyols and sugars involved in cold hardiness will be also carried out by different spectroscopic techniques

Overall, the experimental approach outlined will lead to a better understanding of the abiotic factors that represent major selective constrains to various aquatic taxa

A3 - 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 speciesthat 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 abioticstresses 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 studieshave carried out on this species

The general objective of this activity is twofold: 1) identification of the relationship betweenspecific 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 theMajor 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, ofthe 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.

A4 - Modelling

A4-wp1 - Climatic Modelling

Participants: Piero Cau, Emanuele Eccel

The production of a high-resolution climate characterization is

the focus of the investigation on the existing time series of

temperature, precipitation and derived climatic indicators

Particularly for the past 20 – 30 years, possible links will be

sought between the assessed climatic shifts and the observable

changes in Trentino’s biota In order to define in a high spatial

detail the state of the physical environment and its evolution,

first of all the present conditions will be assessed A4-WP2 will

collaborate to produce a spatial interpolation on the main

climatic quantities: temperature, precipitation, direct indices

(such as absolute maximum, mean, extreme values) and derived

indices (e.g., length of vegetative season, monthly rain days,

etc ) According to the different use of processed data, the

integration time will range from day (for derivation of indices)

to ten-day or monthly periods (for average values) Aiming to a

snapshot of the present conditions, it would be advisable to relate the investigation to the most recent data, rather than to the standard canonical climatic period 1961-1990, at least for some species (namely animals) Therefore the majority of the available historical series datingback to about 30 years will be considered - several dozens in Trentino - having care to sample the little-anthropised territory as well as possible More detail will be sought in “target areas”, significant for sampling, as Mt Baldo Also series from neighbouring regions (South Tyrol, Lombardy, Veneto) will be considered, in order to improve spatial coverage at border areas For areas not contiguous with the province of Trento, corresponding climatic series will be provided Particular requirements, such as the climatic characterization of radiation indices, air humidity, soil temperature, snow cover, microclimatic features, will be taken into account where possible The selected instrumental series will undergo validation, aiming to excluding clearly or probably erroneous data from the analysis, data gap filling, and homogenisation, to tackle major measure shifts due to station displacements, instrument changes, data collection protocols, or systematic erroneous periods

By using the same climatic quantities, attention will be drawn on the most recent change, happened in the last 20 - 30 years and whose consequences may be already observable Secondly, climatic evolution scenarios will be considered, suitably downscaling data to reach a sound quantitative expression of the climatic signal for the future (about 50 years); the above-mentioned instrumental series will be used for this purpose Output from general circulation, coupled atmosphere - ocean models (AO-GCM) will be the base for climatic projections

An increase in time resolution, useful for the calculation of some important climatic indices, will be carried out by producing simulated series with weather generating algorithms,

yielding daily resolution from the monthly model output These algorithms will be calibrated with homogenised station daily series Climatic evolution will be represented by expressing

the relevant climatic features in "standard" time-slices: 1961-90, 2021-50, and, if useful, 99.

2071-A4-wp2 - GIS modelling - landscape genetics

Participants: Duccio Rocchini, Markus Neteler

Genetic diversity is important for the maintenance

of the viability and the evolutionary and adaptive

potential of populations and species

Two previously separate research areas, genetics

and landscape ecology have been integrated into the

new discipline “landscape genetics” (see

Holderegger and Wagner, Bioscience, 2008) This

combined approach merges population genetics,

landscape ecology and spatial statistics, typically

performed in a GIS (Geographical Information

System) environment The combination of genetic

markers with related spatio-environmental data is used to examine population demographics and evolutionary processes For this purpose, genetic characteristics of a species are mapped across a landscape or differences between population are modeled by using landscape

compositional and structural metrics Neutral markers such as mitochondrial and Y

chromosome haplotypes, microsatellite frequencies, single-nucleotide polymorphisms as well

as genetic markers like the Major Histocompatibility Complex (MHC) allele frequencies are used

Genetic diversity can be adaptive or neutral Selectively neutral genetic variation is generally believed to not affect the fitness of the species From patterns of neutral genetic diversity and differentiation demographic and evolutionary events like bottleneck, expansion, isolation, gene flow, divergence can be inferred Adaptive markers, in contrast, are subject to selection constraints and are, therefore, better suited for studying the response to environmental

changes The opportunity to analyse neutral and adaptive variation in a GIS framework makes

it possible to start evaluating i) the putative role of biotic and abiotic factors in ishaping

genetic diversity and ii) the differentiation in a determined area

For instance, the genetic and landscape ecological data can be analysed for identifying

barriers, gradients or transitions thus obtaining crucial information about connectivity amongnatural populations By separating historic and recent gene flow, global and local changes may

be identified which lead to changes, sometimes resulting in a loss of biodiversity As an

advantage, landscape genetics does not usually require to distinguish discrete populations in advance Analyses are performed at population as well as individual levels

We will collect and integrate field data with GIS and climatic data, e.g to find current and potential faunal corridors in Trentino For key species, hypothesis will be discussed about population dimensions, measured as effective population size by means of molecular markers,with regards to expected temperature increase, precipitation decrease and increasing human impact on the territory As outcome, predictive maps for the next 50 years will be created which display the expected population changes from today to future As a result of our

landscape genetics studies, the role of landscape variables in shaping genetic diversity and population structure will be better understood The outcome is relevant for managing

properly the genetic diversity of threatened and endangered populations The study of genetic differences would permit to locate biodiversity hotspots or, in a time series, to investigate whether levels of biodiversity has changed Finally, using Partial Mantel tests the effect of