The European Native Seed conservation Network



ACE-SAP project

Alpine 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

Table Of Contents

First evaluation ACE-SAP project 4

Abstracts of each project activity and work package 6

A1 -Biological Conservation 6

A1-wp3 - Adaptive genetic variation in the highly endangered Salmo carpio L. 1758 6

A1-wp2 - Conservation genetics of three threatened vertebrate species 7

A1-wp3 - Taxonomic delimitation and conservation of three endemic plant taxa 8

A1-wp4 - Flora of Mt. Baldo 9

A2 - Adaptation - Aquatic Environments 10

A2-wp1 - Genetic diversity and adaptive traits in the filamentous cyanobacterium Planktothrix rubescens 11

A2-wp2 - Target species: Bangia atropurpurea (Roth) C. Agardh 12

A2-wp3 - The adaptive potential of natural populations of two aquatic insect species in relation to temperature variations and pollutants 13

A3 - Adaptation - Land Environments 14

A3-wp 1 - Adaptation in Alpine Conifers 14

A3-wp2 - Cold Regulated (COR) genes and cold tolerance in Brassicaceae 15

A3-wp3 - Common frog landscape genetics 16

A3-wp4 - Molecular basis of host-parasite interaction 17

A4 - Modelling 18

A4-wp1 - Climatic Modelling 18

A4-wp2 - GIS modelling - landscape genetics 20

A4-wp3 - Ecological Modelling 21

A5 - Popularisation 22

A5-wp1 - Dissemination 22

NOTES 22

ACE-SAP People 22

Housekeeping details 22

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 for the 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 pollutants into 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 climate change 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 to assess the treat status (IUCN 2001 criteria), propose management strategies and conservation guidelines 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.

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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 Italy etc.); 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 each lake) will be used for the isolation of single filaments used as inoculum for cultures in controlled experimental conditions. Both strains corresponding to environmental samples and pure 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 Monitoring Rivers”, 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 cold mountain springs and streams ( ................
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