AN INTEGRATED APPROACH TO THE CONSERVATION OF PERIPHERAL ISOLATED PLANT POPULATIONS

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UNIVERSITÀ DI PISA

Dipartimento di Biologia

Dottorato in:

“Scienze Biologiche e Molecolari”

A

N INTEGRATED APPROACH TO THE CONSERVATION OF

PERIPHERAL ISOLATED PLANT POPULATIONS

Settore scientifico-disciplinare BIO/02

TESI DI DOTTORATO

Angelino Carta

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Photographs:

p 1 peat bog, Fociomboli (Italy), Apuan Alps Regional Park

May 2008 (G. Trombetti).

p 6 stream with Arenaria balearica L., Montecristo Island (Italy), Parco Nazionale Arcipelago Toscano

May 2012 (A. Carta).

p 20 shallow soft-water pools with Hypericum elodes L., Bad-Bentheim (Germany)

Sep 2012 (A. Carta).

p 73 Hypericum elodes L. flowering, Bosco del Palazzetto (Italy), Parco Regionale San Rossore

Sep 2013 (M. Calbi).

p 93 shallow pools with Hypericum elodes L., Bosco del Palazzetto (Italy), Parco Regionale San Rossore

June 2013 (A. Carta).

p 106 peat bog with Eriophorum angustifolium Honck., Fociomboli, Apuan Alps (Italy), Parco Regionale

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A

CKNOWLEDGEMENTS

After three years of research, I must say that if things have worked out

reasonably well it has been thanks to a good deal of help from many people. I

had the best of supervisors, Gianni Bedini and Lorenzo Peruzzi, and they

stroke a perfect balance between guidance and autonomy. I am truly grateful

to them for always being helpful and willing. Robin Probert gave me the

chance to join the Seed Conservation Department, Millennium Seed Bank

(MSB) at the Royal Botanic Gardens and taught me the art in designing the

science. At the MSB I felt at home and many people provided all the

logistical support I needed. Especially I received intellectual, technical and

moral assistance from Jonas Müller, Efisio Mattana, Alice Di Sacco and

Tiziana Ulian. Many people taught me about botany, especially Giuliano

Frangini and Brunello Pierini. Bruno Foggi help me in ecological

interpretations and gave me the opportunity to join numerous botanical field

work in our wonderful Archipelago. For the publications I thank the

co-authors and all the other contributors that I acknowledge at the end of each

chapter. Last but not least, I thank my parents and Marta for enduring me for

so long and giving me much joy.

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S

UPERVISORS

Dr. Gianni Bedini

Dr. Lorenzo Peruzzi

I

NVOLVED INSTITUTIONS

Università di Pisa, Department of Biology

Università di Pisa, Botanic Garden

Royal Botanic Gardens, Kew, Seed Conservation Department

Parco Nazionale Arcipelago

Parco Regionale San Rossore

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ecological and evolutionary processes that differ from central populations. Peripheral populations may have either a high evolutionary potential or be prone to extinction and therefore, they are considered an important source of biodiversity and included in conservation actions.

This work addresses the identification of PIPPs in Tuscany and the analysis of the main patterns of biological and ecological strategies which allow PIPPs to survive. I assessed the state of knowledge about the PIPPs in Tuscany, by means of a combination of different approaches ranging from quantitative floristic analysis, seed ecology experiments, mating system investigations and species distribution modelling.

Based on the results, many disjunct species of different geographical affinities occur in Tuscany. Few relationship between germination niche width and distribution range were found. Indeed, plastic responses, such as seed dormancy variation modulated by local climate and mixed mating system, may be successful reproduction traits under mosaic and unpredictable habitats. These outcomes support the idea that peripheral populations may be able to cope with ecological and biological constraints. On the contrary, distribution modeling approach predicted low survival of peripheral populations under unbalanced climatic conditions.

Distribution modeling established a fundamental baseline for assessing the consequences of climate change on peripheral populations. Specifically, ‘core localities’, that could have the potential to withstand climate change, were identified. The populations occurring there could serve as long-term in situ stocks and others that are predicted to be under threat from climate change, presently and in the medium term, represent assessment priorities for ex situ conservation.

HAPTER

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BACKGROUND

The analysis of species–environment relationship has been a central issue in ecology and biogeography for a long time. The importance of species/population distribution patterns has been recognized from the beginning of the 19th_{century (Humboldt & Bonpland, 1807;}

Watson, 1835; Candolle, 1855; Wallace, 1876). Climate in combination with other biotic and abiotic factors has been much used to explain species and communities distribution around the world and recently a review of the models explaining species range limits formation was evaluated (Sexton et al., 2009). In this context, the evolutionary significance of peripheral isolated plant populations (PIPPs hereafter) has been recognized early (Darwin, 1859; McArthur & Wilson, 1967).

PIPPs grow in fragmented spots at the margin of a species’ range, they are separated from central populations by spatial distance and are often relatively small (Channell, 2004). The isolation from central and more continuous populations may be followed by a reduction of gene flow (Sexton et al., 2009). In many cases, peripheral populations are also ecologically marginal because they may experience a different ecological context with respect to the species’ optimum condition (Soulé, 1973). Thus, since PIPPs may be exposed to ecological and evolutionary processes that differ from central populations, they may have either a high evolutionary potential or be prone to extinction (Levin, 1970; Holt & Keitt, 2005). Peripheral populations have been a topic of scientific debate in Conservation Biology for about 15 years and they are still an active research subject (Sagarin et al., 2006; Thompson et al., 2010; Wiens, 2011; Sun et al., 2012; Martínez-Meyer et al., 2013; Abeli et al., 2014). The conservation value of peripheral populations depends upon their evolutionary potential (Lesica & Allendorf, 1995). International conventions, such as the European Strategy for Plants Conservation 2008/2014 (ESPC, target 8.1), address the issue of their conservation and the IUCN has suggested that isolated subpopulations should be included in the Red List categorization system at the regional level (IUCN, 2012).

Despite in the last years more attention has been paid to peripheral plant populations performance (Gargano et al., 2007; 2009; Abeli et al., 2012) and their conservation issues in Italy (Abeli et al., 2009; Gentili et al., 2011), this is insufficient in the Tuscan scenario. Hence, a study of conservation of PIPPs in Tuscany is a relevant research subject.

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- 3 - DEFINITION OF THE BIOLOGICAL CONTEXT

Defining peripheral isolated population is not a straightforward task, given the traditional use of the term “relict” in a broad sense. The term relicts is often applied to species with a former more widespread distribution range, but is sometimes used for specific populations in parts of a species distribution (thus called relict populations). Furthermore, in phytogeographic analysis, the concept of “element” is often generically attributed to a group of taxa sharing one or more characteristics (Braun-Blanquet, 1919). Thus, the generic term “relict elements” it also used. However, an exact determination of the concept of element cannot ignore the definition of the type of connection (geographical, ecological, historical, genetic) (Arrigoni, 1973) and the floristic territory (Christ, 1867). In this context, in order to study peripheral isolated populations, they should be projected in the whole ecological-geographical context of the species bearing in mind the historical-genetic factors that may explain the origin of such populations.

AIMS AND STRUCTURE OF THE WORK

This work has been conducted in the laboratories of the Department of Biology (University of Pisa) in association with the Botanical Garden of Pisa and the Seed Conservation Department (Royal Botanic Gardens, Kew). As such, it is part of a broader project on the systematics, evolution and conservation of endemic species, relict populations and habitat-specialist plants in Tuscany. More specifically, in this dissertation I will focus on PIPPs in Tuscany, concerning the identification of the biological and ecological features allowing their survival in Tuscany. I used a combination of different approaches, ranging from quantitative floristic analysis, seed ecology experiments, mating system investigations and species distribution modelling. In chapter 2, I examine the distribution pattern at the regional level, considering geographical and historic constrains. Then I focus on biological properties, such as the seed ecology and the breeding system (chapter 3 and 4) because the knowledge of reproductive strategies is crucial for gaining deeper insights into the biology, evolution, ecology, and conservation of angiosperms. In chapter 5 I model the present and future predicted distribution of H. elodes L. (one of the most remarkable PIPPs in Tuscany), and identify priorities in order to facilitate appropriate decision making for conservation, monitoring and future research. Finally, in chapter 6 I discuss the results, and summarize the main conclusion.

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REFERENCES

Abeli T., Gentili R., Mondoni A., Orsenigo S., Rossi G. 2014. Effects of marginality on plant population performance. Journal of Biogeography 41: 239–249.

Abeli T., Gentili R., Rossi G., Bedini G., Foggi B. 2009. Can the IUCN criteria be effectively applied to peripheral isolated plant populations? Biodiversity and Conservation 18: 3877–3890.

Abeli T., Rossi G., Gentili R., Mondoni A., Cristofanelli, P. 2012. Response of alpine plant flower production to temperature and snow cover fluctuation at the species range boundary. Plant Ecology 213: 1– 13.

Arrigoni P.V. 1973. Le categorie corologiche in botanica. Lavori Società Italiana Biogeografia n.s. 4: 101– 110.

Braun-Blanquet J. 1919. Essai sur les notions d' "élément" et de "territoire" phytogögraphiques. Archives des

Sciences Physiques et Naturelles, Genève, ser. 5, 1: 497–512.

Channell R. 2004. The conservation value of peripheral populations: the supporting science. Proceedings of the Species at Risk 2004 Pathways to Recovery Conference (ed. By T.D. Hooper), pp. 1–17. Species at Risk 2004 Pathways to Recovery Conference Organizing Committee, Victoria, BC.

Christ H. 1867. Ueber die Verbreitung der Pflanzen der alpinen Region der europäischen Alpenkette (Vol. 22, No. 7). Schweizerische Gesellschaft für die gesammten Naturwissenschaften.

Darwin C.R. 1859. On the origin of species by means of natural selection, or the preservation of favoured races in the struggle for life, 1st edn. John Murray, London.

De Candolle A.P. 1855. Essai élémentaire de géographie botanique. Levrault.

Gargano D., Bellusci F., Pellegrino G., Palermo A.M., Bernardo L., Musacchio A. 2009. The conservation perspectives and value of small and isolated plant populations: preliminary clues for Gentianella crispata (Gentianaceae) at the western boundary of its range. Annales Botanici Fennici 46: 115–124).

Gargano D., Fenu G., Medagli P., Sciandrello S., Bernardo L. 2007. The status of Sarcopoterium spinosum (Rosaceae) at the western periphery of its range: Ecological constraints lead to conservation concerns. Israel

Journal of Plant Sciences 55: 1–13.

Gentili R., Rossi G., Abeli T., Bedini G., Foggi, B. 2011. Assessing extinction risk across borders: integration of a biogeographical approach into regional IUCN assessment? Journal for Nature Conservation 19: 69–71.

Holt R.D., Keitt T.H. 2005. Species’ borders: an unifying theme in ecology. Oikos 108: 3–6.

IUCN. 2012. Guidelines for Application of IUCN Red List Criteria at Regional and National Levels: Version 4.0. Gland, Switzerland and Cambridge, UK: IUCN.

Lesica P., Allendorf F.W. 1995. When are peripheral populations valuable for conservation? Conservation

Biology 9: 753–760.

Levin D.A. 1970. Developmental instability and evolution in peripheral isolates. American Naturalist 104: 343–353.

MacArthur R.H., Wilson E.O. 1967. The theory of island biogeography. Princeton University Press, Princeton, NJ.

Martínez-Meyer E., Díaz-Porras D., Peterson A.T., Yáñez-Arenas C. 2013. Ecological niche structure and rangewide abundance patterns of species. Biology letters 9: 20120637.

Sagarin R.D., Gaines S.D., Gaylord B. 2006. Moving beyond assumptions to understand abundance distributions across the ranges of species. Trends in Ecology and Evolution 21: 524–530.

Sexton J.P., McIntyre P.J., Angert A.L., Rice K.J. 2009. Evolution and ecology of species range limits.

Annual Reviews of Ecology, Evolution, and Systematics 40: 415–436.

Soulé M. 1973. The epistasis cycle: a theory of marginal populations. Annual Review of Ecology, Evolution,

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Thompson J.D., Gaudeul M., Debussche M. 2010. Conservation value of sites of hybridization in peripheral populations of rare plant species. Conservation Biology 24: 236–245.

von Humboldt A., Bonpland A. 1807. Ideen zu einer Geographie der Pflanzen nebst einem Naturgemälde der Tropenländer von A.I. von Humboldt und A. Bonpland. Bearbeitet u. herausgegeben von dem erstem. Tübingen u. Paris.

Wallace A.R. 1876. The geographical distribution of animals. Harper & Brothers, New York.

Watson H.C. 1835. Remarks on the Geographical Distribution of British Plants: Chiefly in Connection with Latitude, Elevation, and Climate. Longman, Rees, Orme, Brown, Green, and Longman, Paternoster-Row. Wiens J.J. 2011. The niche, biogeography and species interactions. Philosophical Transactions of the Royal

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C

HAPTER

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ETTING THE SCENE

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DETERMINANTS OF PRESENT

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SETTING THE SCENE:

DETERMINANTS OF PRESENT-DAY BIODIVERSITY AND GAPS OF KNOWLEDGE

ABSTRACT

By means of quantitative floristic analysis, the peripheral isolated plant populations (PIPPs) within the flora of Tuscany were identified and consist of 116 taxa. Some areas resulted particularly rich in PIPPs, such as the Tuscan Archipelago (31 taxa), the Apuan Alps (23) and the Apennines (23).

From the taxonomic point of view, 43 families and 93 genera are represented (species/genus ratio of 1.2), indicating that the species are approximately equally represented in each genus.

The analysis of biological traits (Life forms, Chorotypes and Ellenberg indices) does not help to explain the tendency to generate a peripheral population, but clearly demonstrates that a given environment could host species to which are already adapted.

Further attempts are required to fill the gaps of knowledge on the distribution, demography and functional traits to assess conservation priorities of peripheral populations that are likely to generate future evolutionary diversity.

INTRODUCTION

Biodiversity is unequally distributed in the world (Myers et al., 2000), so that “megadiverse countries” face huge challenges in gathering information about species distribution and abundance. However, also at regional levels, basic knowledge of plant diversity may show large gaps (Kier et al., 2005).

The flora of Tuscany and neighboring areas is known to be one of the richest in the Mediterranean taking into account both the absolute number of taxa (Conti et al., 2005) and their evolutionary and conservation value (Médail & Diadema, 2009; Peruzzi et al., 2012). Indeed, the Tuscan flora prompted the interest of many botanists since the XVI century (Cesalpino, 1583; Micheli, unpublished; Savi, 1808; Caruel, 1860). Despite this long tradition of study, the knowledge is far from complete and even such a basic tool as an annotated flora of this area is still lacking. This is largely due to knowledge scattered among several researchers, fragmented publications, and to the difficulty of integrating and managing large datasets established independently from one another. An effort to realize a Flora of Tuscany was launched in 2013 through an open and collaborative project named Wikiplantbase #Toscana (Peruzzi & Bedini, 2013).

Besides the endemics, which assume great importance in the interpretation of speciation events (Favarger, 1974; Garbari, 1990; Siljak-Yakovlev & Peruzzi, 2012), the most

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representative units of a flora are those species which may represent important testimonies of the evolutionary history in a defined geographical region (Holt & Keitt, 2005). In this context, Peripheral and Isolated Plant Populations (PIPPs hereafter) certainly deserve special attention at the regional level because of their biogeographical values, and additionally at the global level, being representative of potential independent evolutionary processes (Lesica & Allendorf, 1995).

The aims of this work were (1) to assess which plants may display evidences to be considered as PIPPs in Tuscany; and (2) to review the knowledge of this valuable component of the flora, through a summary of their distribution, ecology, and conservation data.

MATERIALS AND METHODS

Source of data

The distribution and ecological information were collected through the analysis of the scientific literature reported in journals (Atti della Società Toscana di Scienze Naturali, Memorie, serie B; Informatore Botanico Italiano; Webbia; Flora Mediterranea etc.), in institutional publication regarding the biodiversity in Tuscany (Sposimo & Castelli, 2005)

and in open-access online data-bases (http://www.actaplantarum.org/;

http://www.biologia.unipi.it/ortobotanico/FloraToscana/flotos_start.html). The distribution of a taxon in Tuscany was and compared with its global distribution (Meusel et al., 1978;

http://www2.biologie.uni-halle.de/bot/ag_chorologie/choro/).

The karyological information was obtained from the free online database "Chrobase.it" (Bedini et al., 2010 onwards).

The data regarding conservation status refert to the IUCN categories published in Scoppola & Spampinato (2005) and subsequent updates (Rossi et al., 2005 onwards).

Regional overview

Species with peripheral isolated populations in Tuscany were identified following a stepwise selection starting from a list of species of conservation interest in Tuscany (Re.Na.To.: 416 taxa) slightly modified and updatedfor the purpose of the study using the same criteria proposed by Sposimo & Castelli (2005) and considering recent floristical and taxonomical updates (giving a starting list of 521 taxa).

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Despite pteridophytes may show a significant number of cases of PIPPs many of them are also ancient relicts and generally have long-distance dispersal ability; furthermore, because they are excluded from the literature on peripheral populations (Abeli et al., 2014), this group of plants was discarded.

Relict plant species such as paleotemperate and tropical plants were also excluded because they are likely to represent old biogeographical events and especially because they show a fragmented distribution across their whole distribution area.

Rarity is not a direct property to define a PIPP because anthropogenic extinction forces may render historical density patterns irrelevant (Channell & Lomolino, 2000); thus many taxa of conservation relevance were not eligible.

Finally, narrow endemic species were also excluded while Italian endemic species whose range extends across Apennines and/or Alps were retained.

In a second step, this list was further reduced, by considering only those species significantly isolated from the core populations (and from other populations of the same species that are furthermore peripheral, e.g. some species possess isolated populations along the Italian peninsula). Afterwards, a threshold based on the number of populations (max 5 populations) was applied with flexibility: because detailed knowledge is not equal across the species, and authors used different concepts to identify populations, in the following analysis, population was used as synonym of locality.

Highlighted species

From the list of PIPPs obtained, I restricted the inventory to those most representative species for each Operational Geographic Unit (see below) and by introducing an additional restriction: represented in Tuscany and surrounding areas by no more than 3 populations.

Data analysis

With the distribution data, a binary matrix was built (116 species × 10 geographic units), whose OGU (Operational Geographic Units) coincide with those proposed by Peruzzi et al. (2012). The matrix was subjected to cluster analysis (WPGMA), using the index of association of Baroni Urbani & Buser, particularly suitable for the purpose when the knowledge distribution taxa investigated are good, because it considers also co-absences but only if at least one positive attribute is shared (Biondi, 2006). The graphical

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representation of the composition based on the distribution of the taxa in each OGU is represented by a cartogram.

An attempt to draw indications on main characteristics of PIPPs and on the ecological determinants can be made with the help of life forms (Raunkiaer, 1937), chorotypes (Meusel et al., 1978) and Ellenberg’s indices (Ellenberg, 1974). Coding of life forms and chorotypes were obtained from Pignatti (1982), values of Ellenberg indices from Pignatti et al. (2005) and subsequent update (Guarino et al., 2012). Ellenberg’s indices (Eivs) can be divided in two subgroups of three and four indices respectively. The first three indices are linked to climatic variables: light regime (L), temperatures (T) and climate continentality (C); the remaining four deal with edaphic conditions: moisture of soils (U), pH (R), nutrients availability (N) and salt concentration (S). For the subsequent analysis S was excluded because of many 0 values (Pignatti et al., 2005). Furthermore, all records (species) with indeterminate values were discarded.

I calculated averages of the Eivsdespite not being appropriate in strict mathematical terms, but as shown by many authors (Diekmann, 2003). The echogram was thus derived from the averages of the relative Eivs.

To reveal the species pattern, their relation to the trait values and their relative position within the whole Italian flora, I chose a factorial analysis for mixed data (FAMD; Husson et al., 2010; Wildi, 2013). FAMD is a principal component method dedicated to explore data with both numerical (Eivs) and categorical variables (Life forms, Chorotypes). It can be seen roughly as a mix between PCA (Principal Components Analysis) and MCA (Multiple Correspondence Anaysis). This ensures a balance of both numerical and categorical variables in the analysis. Thus, a matrix of 4468 species × 8 factors (2 categorical, 6 numeric) was built.

RESULTS AND DISCUSSION

Regional overview: composition, distribution and chorotypes

The considered species are 116 (Annex 1) that represent about 3% of the whole Tuscan flora. Among them, 12 are Italian endemics, 30 Mediterranean, 7 Mediterranean-Montane, 18 Eurasiatic, 10 Atlantic, 20 Orophytic and 17 Boreal species. Among the Italian endemics, 3 show a Sardo-Corsican-Balearic distribution, 3 occur southwards up to southern Apennines, 1 is south Mediterranean and 4 Alpine.

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From the taxonomic point of view, 43 families (Asteraceae 13%, Cyperaceae 7%, Brassicaceae, Caryophyllaceae and Fabaceae 5%, etc.) and 93 genera (Carex 4%, Hieracium and Sedum 3%, Agrostis, Biscutella, Carduus, Eriophorum, Gentiana, Juncus, Lysimachia, Rhynchospora, Taraxacum, and Verbascum 2%) are represented (species/genus ratio of 1.2), indicating that the species are approximately equally represented in each genus. On the contrary, for the Tuscan endemic flora (Peruzzi et al., 2012), the species are over-represented in some genus (e.g. Centaurea 13% and Limonium 11%).

The majority of PIPPs (Fig. 1) are reported for the Tuscan Archipelago (31), the siliceous Apennines (23), the Apuan Alps (23), and the Tuscan plains (19). This pattern was already reported for the endemics by Peruzzi et al. (2012) and confirms the high biodiversity value of these areas (Blasi et al., 2011). The WPGMA cluster analysis (Fig. 2) shows 2 main groups: Tuscan Archipelago + southern Tuscany (A-C), in the second group central and northern (mountains) Tuscany (G-J); ophiolithic (E-F) and coastal (D) OGUs are characterized by few species and are shown in an isolated cluster.

Figure 1. Map of Tuscany with cartograms representing the percentage of chorotypes in each OGU. The diameter of circles is proportional to the number of PIPPs taxa in the OGU.

Central Tuscany and southern Tuscany show a balanced proportion of the different chorotypes. In contrast, the chorotypes are not equally distributed in the other areas. Indeed, the Apennines are characterized by boreal species, indicating that these mountains

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are clearly linked with northern regions and especially with the Alps (Foggi, 1990). The Apuan Alps are dominated by orophytic species, suggesting a linkage with the Mediterranean basin (Ferrarini, 1970), while the Tuscan Archipelago, given its palaeogeographical history, is characterized by west-Mediterranean species (Arrigoni et al., 2003; Carta et al., 2011a), but interestingly also an eurasiatic-orophytic species reached and survived in this area with a peripheral isolated population (Gagea bohemica, see Peruzzi et al., 2008).

Figure 2. WPGMA Cluster Analysis of the Operational Geographic Units.

Regional overview: biological forms

Whilst the endemics in Tuscany are mainly represented by hemicryptophytes (75%, Peruzzi et al., 2012), PIPPs show a good representation of the other life forms as well (Fig. 3). These data are similar to those of the whole Italian flora; thus, from this pattern it may be concluded that life forms do not explain the tendency to generate a PIPP. On the contrary, it has been argued that other life history attributes, like mating system and seed dispersal may cause a liability to form peripheral populations (Lesica & Allendorf, 1995). However, if analyzed within each single OGU, life form percentages clearly indicates an environmental selection: for example in the Tuscan Archipelago the therophytes dominate (29%), while on the Apennines hemicryptophytes are the most abundant (52%).

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T I G H Ch P

Figure 3. Life forms percentage of PIPPs in Tuscany.

Regional overview: Ellenberg indices and final assessment

Comparing ecograms for the Tuscan PIPPs and the Italian flora, the only relevant difference refers to continentality (C), indicating a significant presence of mediterranean and atlantic species in the PIPPs flora. From the multivariate analysis it is possible to gain slightly more information (Fig. 4). Each OGU clearly occupies a defined region of the graph: the Tuscan Archipelago species are characterized by thermophilous plants (with a significant contribution of annuals) and are reported in the left part of the graph; on the contrary the siliceous Apennines species are reported in the bottom right. The other areas show a degree of overlap, however a pattern is recognizable: for example, central Tuscany plants are mainly reported for the right part of the graph following the moisture gradient.

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Figure 4. Mixed data Factorial analysis for the Italian flora (gray points) and the Tuscan PIPPs: yellow circles (Tuscan Archipelago), red circles (southern Tuscany), green diamonds (central Tuscany), light blue squares (calcareous Apennines), blue squares (Apuan Alps), brown triangles (siliceous Appennines). Only numeric variables which contribute > 10% are reported.

Highlighted species

The most representative PIPPs in Tuscany are reported in Table 1. Among them, many are known since long time (e.g. Hypericum elodes, Drosera intermedia; see Caruel, 1860; Corti, 1955); while others have been found in recent years (e.g. Symphytum tanaicense,

Anthemis cretica subsp. columnae; see Peruzzi et al., 2001; Selvi, 2009).

Despite their disjunction, some species consist of large populations and show less ecological marginality. For example, Cneorum tricoccon show a distinct structural position within the ecosystem (Foggi et al., 2011). It is important to mention the possibility of important functional linkage of this species with other biological components of the ecosystem (e.g. lizards or mammals, see Traveset, 1995).

Other taxa show single, small populations (< 100 individuals or area of occupancy < 500 m2). For example Drosera intermedia is reported for a disjunct population at the Southern periphery of the species range, located near San Lorenzo a Vaccoli (Monte Pisano, Lucca). The population lives in mosaic patches under a forest canopy of maritime pine (Pinus

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pinaster Mill.). Despite the pauci-specific communities and degraded habitat, the presence of another PIPP in the same area, namely Rhyncospora alba, was confirmed (Carta & Vannucchi, 2011). This rare minute sedge allows to refer the pioneer community with D. intermedia to the association Drosero intermediae - Rhynchosporetum albae (Allorge & Denis 1923) Allorge 1926 (see Carta et al., 2011c). This association is typical for depressions on peat substrates attributable to the alliance Rhynchosporion (Biondi et al., 2012). According to De Ridder & Dhont (1987), plants of D. intermedia are reported to be cleistogamic in northern-Europe. Notably, alpine populations are not cleistogamic (M. Beretta in verbis); therefore, I speculate that this behavior is not a genetically fixed character and it could be an advantage in shady habitats.

Table 1. Highlighted species which show peripheral isolated populations in Tuscany. Habitat Directive column refers to the plant communities in which the plants are found.

Scientific name L if e fo rm C ho ro ty pe O G U N um be r of lo ca lit ie s 2n in T us ca ny re po rt ed in R ed L is ts or L oc al L aw s H ab ita t D ir ec tiv e

Anthemis cretica L. subsp. columnae (Ten.) Franzén H NW-MEDIT.-MONT. E 2 36 Yes

Astragalus muelleri Steud. & Hochst. H NE-MEDIT.-MONT. A-G 2 16 Yes

Brassica procumbens (Poir.) O.E.Schulz T SW-STENOMEDIT. C 1 18 Yes Cneorum tricoccon L. N NW-STENOMEDIT. C 2 ? Yes Drosera intermedia Hayne H SUBATLANT. G 1 20 Yes Yes Gentiana pneumonanthe L. subsp. pneumonanthe H EUROSIB. G 1 26 Yes Yes Horminum pyrenaicum L. H OROF. SW-EUROP. H 3 ? Yes

Hypericum elodes L. H W-EUROP. (ATL.) G 1 16 Yes Yes

Jacobaea incana (L.) Veldk. subsp. incana H ENDEM. ALP. J 1 40 Yes

Sedum brevifolium DC. C W-MEDIT.-MONT. C 1 ?

Silene catholica (L.) W.T. Aiton. H NE-MEDIT.-MONT. A-B 3 ? Silene suecica (Lodd.) Greuter & Burdet H (CIRCUM.)ART.ALP. J 3 ? Yes Symphytum tanaicense Steven H EUROP.-CAUCAS. G 2 40 Yes

Sedum brevifolium is reported for a single Tuscan disjunct population at the North-eastern periphery of the species range, on Elba Island (Monte Capanne). The plants grows in pioneer communities attributable to the Sedo-Scleranthion alliance, colonising superficial soils of siliceous rock surfaces. As a consequence of drought, this open vegetation is characterised by lichens, Crassulaceae and therophytes (A. Carta, unpublished data). Hypericum elodes L. is reported for a single disjunct population at the South-eastern periphery of the species range. Plants grow clustered forming 4 main compacted swards and few sub-nuclei patchily distributed over an area of 4000 m2_{in a shallow pools system}

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surrounded by deciduous woods. The community is referable to the association Ranunculo flammulae-Juncetum bulbosi Oberd. 1957, typical of amphibious short-lived perennial vegetation on the pool banks (Savio, 2010; Carta et al., 2011b; Biondi et al., 2012).

Notably, Anthemis cretica subsp. columnae is reported for two isolated populations at the northwest distribution limit of the taxon. At these sites A. cretica is found at only ca. 300 and 500 m a.s.l., well below its usual altitudinal range above 1200 m. Such findings reveal the capacity of this species to penetrate into mediterranean habitats and to tolerate the anomalies of ultramaphic substrates; further they underscore the important role as refugia that the ophiolitic “islands” of NW Italy have for taxa usually growing at higher altitudes in areas with a dry, continental climate (Selvi, 2009).

Challenges for the future and conclusions

In the last two decades, much attention was given to the endemic flora, particularly from the conservation point of view (Sposimo & Castelli, 2005). However, although many endemic taxa are under threat (Peruzzi et al., 2012; Foggi et al., 2014), stenochoria itself not necessarily prefigures extinction, and other criteria are required to set conservation priorities at the regional level (Bacchetta et al., 2012).

Despite detailed palaeogeographic studies integrated with molecular dating are absent, the biogeographical linkages of these taxa are evident. Two types of PIPPs were recognized. 1) climatic relicts which have persisted despite episodes of great stress, irregular recruitment, and a low carrying capacity imposed by their habitat. This is the case of many PIPPs in the Apennines and in Central Tuscany, because they experience a different climate than the core populations. They could survive under particular micro-environmental conditions. However, they may face the impact of climate change and/or competitive species which may reduce seedling recruitment. 2) Other PIPPs in Tuscany seem to show less ecological marginality, as they could be found under similar climatic conditions and similar landscape structures compared to the whole species’ range conditions. This is the case of many West-Mediterranean species that reach the eastern border of their distribution in the Tuscan Archipelago.

The presence of many disjunct populations of different geographical origins, highlights the importance of Tuscany for the survival of PIPPs, thanks to its environmental complexity (e.g. orography, lithology and bioclimate). As a consequence of the different biogeographical influences, the proportion of the different chorotypes is rather balanced.

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Interestingly, the areas where PIPPs could survive correlates with the level of endemism, where possibly relict-like taxa differentiated.

About 50% of the PIPPs in Tuscany are assessed using IUCN criteria, however, a deep knowledge is far to be reached. More precisely, further attempts are absolutely required to fill the lack of a) detailed distribution and demography data, b) functional traits (e.g. breeding system, seed biology) to assess conservation priorities of peripheral populations that are likely to generate future evolutionary diversity (Lesica & Allendorf, 1995).

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C

HAPTER

3

3.1 S

EED DORMANCY VARIATION AMONG

H

YPERICUM ELODES POPULATIONS

3.2. L

ABORATORY GERMINATION AND SEED

STORAGE OF

R

ANUNCULUS BAUDOTII

3.3. S

EED ECOLOGY IN THREE

C

ROCUS SPECIES

3.1. Originally submitted as: Carta A., Probert R., Puglia G., Peruzzi L., Bedini G. Seed dormancy variation in Hypericum elodes L. (Hypericaceae) is related to local climate: under review.

3.2 Originally published as: Carta A., Bedini G., Foggi B., Probert R. 2012. Laboratory germination and seed bank storage of Ranunculus baudotii seeds from the Tuscan Archipelago. Seed Science

and Technology 40: 11–20.

3.3 Originally published as: Carta A., Probert R., Moretti M., Peruzzi L., Bedini G. 2014. Seed dormancy and germination in three Crocus ser. Verni species (Iridaceae): implications for evolution of dormancy within the genus. Plant Biology. DOI:10.1111/plb.12168

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SEED DORMANCY VARIATION IN HYPERICUM ELODESL.(HYPERICACEAE)

IS RELATED TO LOCAL CLIMATE

ABSTRACT

Seed dormancy and germination characteristics may vary within species in response to various factors. Knowledge of such variation is crucial to understand plant evolution and adaptation to environmental changes. In this study the correlation of climate and population genetic distance with seed dormancy patterns and germination behaviour were investigated in an Atlantic-European soft-water pools specialist, Hypericum elodes, across its distribution range.

Laboratory germination experiments were performed (1) to investigate the effect of temperature and light on germination; and (2) to measure dormancy in seeds collected from different populations. The effect of local climate on dormancy was modelled. The genetic distances of the investigated populations were estimated using Inter Simple Sequence Repeats (ISSR) PCR, and their correlation with the dormancy patterns was analysed.

Whilst seed germination requirements were similar among populations, seed dormancy varied considerably and this variation was related to local climate but not to genetic differentiation. Lower winter temperature and summer precipitation at the population sites predicted higher dormancy in the seeds, whereas higher summer temperature and winter precipitation predicted for low dormancy. Our results suggest that seed maturation environment plays a substantial role to explain the dormancy variation, thus highlighting the great adaptive potential of physiological dormancy.

INTRODUCTION

Seed traits are recognized as decisive adaptations of spermatophytes that are subjected to natural selection and optimize plant fitness (Fenner & Thompson, 2005; Donohue et al., 2010). Among these traits, seed dormancy is considered one of the main factors determining the adaptive value of germination (Finch-Savage & Leubner-Metzger, 2006; Venable, 2007; Donohue et al., 2010). Thus, ascertaining how seed dormancy is determined by - and responds to - local environmental signals is crucial to understand how this trait affects other plant life history stages (Toorop et al., 2012; Eberhart & Tielbörger, 2012; de Casas et al., 2012), whether it may have contributed to micro-evolutionary patterns (Donohue et al., 2010; Volis & Bohrer, 2013) and if plant regeneration will be

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affected by environmental changes (Walck et al., 2011; Ooi et al., 2012; Engelbrecht et al., 2013; Sommerville et al., 2013).

Upon dispersal, seeds may show total (i.e. no germination under any conditions) or conditional dormancy (i.e. germination under a restricted set of conditions) (Baskin & Baskin, 2004). Apart from genetic origin, the level of primary dormancy in seeds is determined by several factors such as maternal environment during maturation, age of the mother plant during maturation and position of the seed on the plant (Andersson & Milberg, 1998; Fenner & Thompson, 2005). In response to certain environmental signals (the so-called dormancy breaking factors), seeds gradually lose dormancy and their range of germination conditions broadens until eventually they become non-dormant. This stimulus can be related to the the season of major risk for the seedling in many species (Vleeshouwers et al., 1995; Mattana et al., 2012; Wagmann et al., 2012). Seed dormancy is therefore a good candidate for variation among seed collections from different sites (Andersson & Milberg, 1998; Schütz & Rave, 2003; Lampei & Tielbörger, 2010), and across species’ distribution range (Skordilis & Thanos, 1995; Wagmann et al., 2012). Local climate may act on dormancy through selection pressures on germination timing and/or post-germination phases. Thus, heritable dormancy differences, through ecotypes, may result from long-term effects arising from sustained climatic differences among sites (Hufford & Mazer, 2003; Fernández-Pascual et al., 2013). Indeed, a great source of variation within a species may arise from peripheral populations: because they are likely to experience different physical factors compared to the populations in the core distribution area, they represent an important adaptive potential for a species (Holt & Keitt 2005; Abeli et al., 2014). Finally, the impact of the mother plant environment on seed maturation (known as short-term effect) is also thought to be responsible for seed dormancy/germination variation (Fenner & Thompson, 2005).

Investigations considering intraspecific variation across the distribution range are important to improve comparative analyses across taxa (Frederiksen et al., 2005) and biodiversity forecasts in future ecological scenarios (Lavergne et al., 2010). In this work, we studied variation in seed germination and dormancy in a shallow soft-water pools specialist: Hypericum elodes L. (Hypericaceae), an Atlantic-European perennial herb. Soft-waters are notable category of freshwater habitats because of their biodiversity values and ecosystem services function. They have become increasingly rare resulting in the disappearance of the species from many sites (Arts, 2002) and protected by the European Council Directive 92/43/EEC of 21 May 1992 (Habitats Directive). Hypericum elodes is a

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good representative of this vulnerable habitat (Bilz et al., 2011) and since it has a spread distribution range it is a good candidate for studying variation in seed dormancy. Improved understanding of how key species adapt to local climate will contribute to the European Strategy for Plant Conservation (ESPC). Here, we investigated the hypotheses that patterns of intra-specific variation in seed dormancy level at dispersal and receptiveness to dormancy-breaking factors (1) may be explained by the local climate at the population sites, and (2) are correlated with population genetic differentiation.

Figure 1. Distribution map of Hypericum elodes, redrawn from Meusel et al. (1978): continuous distribution range (grey region), isolated patches (grey circles), sampled populations (black circles).

MATERIAL AND METHODS

Seed material

Hypericum elodes is a perennial herb with an Atlantic-European distribution (Fig. 1). It is a characteristic species of acid pool fringe shallow-water swards, a habitat which is inundated during the larger part of the year and temporarily emerged in summer (Murphy, 2002).

Although little information is available regarding the reproductive biology of H. elodes, this species has a high vegetative reproduction capacity and shows a mixed mating system (A. Carta et al., in prep.). The flowers are borne in a lax panicle that is irregularly corymbose (composed by 5-7 flowers). Flowering starts in July, proceeds during summer, and ripe seeds are dispersed in September-October. We observed seedlings in the field in July-August, so we assume that emergence occurs in summer, after the cold season has

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ended. Fruits contain ca. 30 seeds each. The seeds are dark, small (c. 0.6-0.8 mm long), ovoid-cylindrical or ellipsoid, with a ribbed scalariform testa sculpturing (Meseguer & Sanmartín, 2012), which is a common trait in the clade comprising the Old Word monotypic section Tripentas (only including H. elodes; see Robson, 2012) and mainly New World sections (Nürk et al., 2012). Seed mass (c. 0.06 mg) is similar to that of other Hypericum species (Royal Botanic Gardens, Kew, 2008). The embryo belongs to Martin’s dwarf category (Martin, 1946).

Seeds (capsules) were collected from around 60 healthy plants for each population (Table 1; Fig. 1) at the time of natural dispersal (September-October 2012) when the capsules’ colour change from green to brownish. Ripe fruits were spread out in the laboratory to allow them to open and let the seeds fall outside.

We collected from random positions within the inflorescences to minimize the possible effect of position on seed maturation. After collection, the seeds spent a three week period at moderate humidity (approx. 20 °C, 50 % RH) to encourage maturation of immature seeds before being cleaned and used in the germination experiments.

During field visits we estimated population sizes by counting mature individuals (ramets) in 5 × 5 m plots and multiplying the density (number of individuals / m2) by the covered surface (Table 1).

Table 1. Description of the study sites. Tw, mean winter temperature (°C); Ts, mean summer temperature (°C); P, annual precipitation (mm); Pw%, Ps%, respectively the contribution of winter and summer precipitation to the total annual precipitation; Ns, population size (reproductive individuals); Ng, sample size for the genetic analysis.

Site Coordinates Tw Ts P Pw% Ps% Ns Ng

S.Rossore (ITA) 43°44'33"N, 10°20'32"E 7.5 22.0 894 27.6 13.4 375 11 Étang Blanc (FRA-S) 43°42'32"N, 01°21'22"W 7.7 19.3 1298 30.5 18.9 892 14 Petit Étang Neuf (FRA-N) 48°40'27"N, 01°43'24"E 3.5 16.4 639 25.3 24.6 2975 15 Crawley (UK) 51°05'35"N, 00°13'59"W 3.8 15.9 655 25.1 23.0 562 10 Bad Bentheim (GER) 52°15'40"N, 07°06'29"E 2.0 16.1 785 24.0 28.5 4375 12

Germination experiments

Each treatment consisted of four samples of 25 seeds for each population. Experiments were conducted in temperature (± 1 °C) and light (40 µmol m-2s-1) controlled conditions using a 12 h daily thermo- (for alternating temperatures) and photo-period (= light hereafter). Light was provided during the warm phase for the alternating temperature

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regimes by white fluorescent tubes. All experiments were carried out in 90 mm diameter Petri dishes containing 1% distilled water agar.

A complete 2 × 2 × 3 factorial design was used to test the interactive effects of temperature regime (constant vs. alternating), mean temperature and cold stratification on germination. Seeds were exposed to temperature conditions simulating seasonal temperatures in the collecting areas where diurnal alternating temperatures (20/10, 25/15 and 30/20 °C) simulated conditions at the margins of water bodies, and constant temperatures (15, 20 and 25 °C) simulated germination under flooded conditions. Seeds were cold-wet stratified for 0, 3, 6, 12, 18 weeks at 5 °C in the light.

A second 2 × 4 factorial experiment was used to test the effects (and their interaction) of the following dormancy breaking treatments: cold stratification for 12 weeks (5 °C), warm stratification at 20 or 25 °C for 30 days, soaking in tap water for 24 h. These effects were tested as single treatments and after the following combinations: fresh (or cold-stratified) + warm soaked; fresh (or cold-stratified) + soaked. The incubation temperature in this experiment was 25/15 °C for all treatments. We chose this temperature condition, because preliminary tests showed that this may be regarded as optimal for germination in this species. We considered warm stratification because it simulated seasonal conditions experienced by seeds in the wild: early-autumn flooded conditions and, if previously cold-wet stratified, late spring flooded condition. Soaking in tap water was used because experimental studies on seed germination in other Hypericum species (Çırak et al., 2007) showed that this treatment may be effective as dormancy-breaking agent.

Seeds were also tested in continuous darkness at 25/15 °C with or without being cold stratified for 12 weeks in the dark. Darkness was achieved by wrapping Petri dishes in aluminium foil; Petri dishes were then kept in the dark for the duration of the experiment. Germination was defined as radicle emergence from the testa by at least 1 mm; germinated seeds were counted and removed every five days. At the end of the experiments (6 weeks), the number of ungerminated but viable seeds was determined by a cut test; defective seeds (i.e. empty, damaged and infected) were excluded from subsequent calculations.

Seed germination follows a binomial distribution and so lacks the properties of linearity and additivity. We analysed the effects of treatments on final germination (patterns of dormancy variation) in the five populations by fitting factorial generalized linear mixed models (GLMMs, logit link function, binomial distribution) to the germination data with incubation conditions (mean temperature and temperature regimes) and pre-treatments (cold-stratification, warm-stratification, water soaking) as fixed predictors, and population

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as a random factor (Bolker et al., 2008). The following parameters were contrasted: fresh vs. stratified, fresh vs. water soaked, alternating temperature vs. constant. Mean temperature was included as continuous variable. All calculations were analysed using the R environment for statistical computing (R Development Core Team, 2013).

Climatic models

To determine if dormancy variation could be predicted from the local environment of the populations, we fitted factorial generalized linear models (GLMs, logit link function, binomial distribution; separately per fresh/ 12-weeks-stratified seeds) to germination results obtained at 25/15 °C (found to be optimal), using the local values of temperature and precipitation as predictors (Table 1). Seasonal precipitation data were transformed as percentage on the total annual precipitation because plants do not only respond to rainfall quantity, but at a regional scale, where variation in mean annual precipitation among sites is low, plant species may be more sensitive to the seasonality of rainfall than to its total annual amount (Volis & Bohrer, 2013). Climatic data of the collecting sites were downloaded at a spatial resolution of 1 km2 from the WorldClim website (http://www.worldclim.org), as documented by Hijmans et al. (2005).

We also included population size in the models to control demographic effects in germinability. The explanatory variables were not significantly correlated according to the Pearson test (P > 0.05). In a first step, two full models, including all main factors and interactions, were computed. In a second step, the full models were fitted using a forward/backwards stepwise variable selection procedure with Akaike’s information criterion.

Genetic analysis

Genetic analyses were conducted using seedlings of each population randomly chosen from germinated seeds (Table 1). To obtain a suitable amount of plant tissue for DNA extraction, each seedling was grown until at least 0.8 mg of weight and then placed along with a stainless steel bead (Qiagen, GmbH) into a 2.0 ml propylene tube, chilled in liquid nitrogen and disrupted using Qiagen Tissue Lyser (Qiagen, GmbH). The obtained tissue powder was used for DNA extraction as reported by Cullings (1992) with some modifications (keeping sample during precipitation at - 80°C for 1 day and extending centrifuge time to 30 min. after DNA precipitation in order to increase yields). DNA quantity and quality were assessed by using a Nanodrop spectrophotometer (Nanodrop

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Technologies, Oxfordshire, UK). Population genotyping was performed using Internal Simple Sequence Repeat (ISSR) markers. In particular, eight different ISSRs 3’ or 5’ anchored primers (synthesized by Eurofins MWG Operon, Ebersberg, Germany) were used including two primers in the same reaction in order to increase the number of bands (Table 2). Each 25 µL reaction volume contained 25 ng DNA template, 2.5 µL of 10 × PCR buffer

(100 mM Tris–HCl, 15 mM MgCl2, 500 mM KCl), 5% of DMSO, 0.5 µL of dNTPs (10

mM each), 0.5 µL of each primer (10 mM), 1 unit of Solis HotStart Taq DNA polymerase (Solis BioDyne, Tartu, Estonia). The amplification reactions were carried out using a Quanta biotech SI-96 Thermal Cycler (Quanta Biotech Ltd, Surrey, UK) with the following PCR profile: 95°C for 15 min; 35 cycles at 94° for 30 sec, specific annealing temperature for each primer (Table 1) for 1 min, 72°C for 1 min; and final extension at 72°C for 10 min.

Afterwards, 10µl of PCR product was electrophoresed on 2.0% agarose gel stained with SyBr Safe DNA Gel Stain (Life Technologies, New York, USA) during 2.0 h at 100 V in 1Å~ TAE buffer and a 100 base-pair ladder (1 Kb Plus DNA ladder, Life Technologies) was used as molecular size marker. Gel was then visualized and photographed using a gel documentation system (BioDoc-It® Imaging System, CA).

Band analysis was carried out using Total Lab 100 (TotalLab Ltd, UK) and only non-overlapping and highly reproducible bands were considered for fragments detection. Amplification products on the gels had a variable length between 200 and 1200 bp.

We manually constructed a binary 0/1 matrix based on the absence/presence of the DNA bands. The total number of loci, their mean number per population (A), the number of polymorphic loci per population (P), the frequency of polymorphic loci (%P), Nei’s gene diversity (h) and genetic distance (Nei, 1972) among populations, were computed and subjected to UPGMA clustering with POPGENE 3.2 (Yeh et al., 1997). We used a Mantel permutation test using GenAlEx (Peakall et al., 2012) to verify whether genetic distances between pairs of populations were significantly correlated to corresponding straight-line geographical distances and to seed dormancy. Seed dormancy euclidean distance was computed from the predicted germination values at 25/15 °C obtained from GLMs. Further, Mantel test was also used to verify whether seed dormancy matrix was correlated to a climatic euclidean distance matrix (5 populations x 4 climate factors).

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Table 2. ISSR primers used in this study.

Primer combination Primer ID Sequence (5’-3’) Publication Annealing temperature (°C) I-32 (AGC)4C (Barcaccia, 2006)

1 I-29 (GT)6CA (Barcaccia, 2006) 49.8 I-50a _CCA(GCT) 4 (Barcaccia, 2006) 2 P-02 CTG(GT)8 (Huangfu, 2009) 46.2 I-18 GTGC(TC)7 (Barcaccia, 2006) 3 I-39a _(AGC) 4AC (Barcaccia, 2006) 47.0 P-01 GAG (CAA)5 (Huangfu, 2009)

4

I-34 (AGC)4GG (Barcaccia, 2006)

53.8

RESULTS

Germination experiments

Whilst germination requirements of seeds from the five populations were similar, there was considerable variation in seed dormancy across populations. Fresh seeds of H. elodes collected in Italy (ITA) and South France (FRA-S) showed relatively high germination at 25/15 °C (>30 %) and particularly at 30/20 °C (~80 %). On the contrary, fresh seeds germination of the other populations were significantly lower (P < 0.05) in all germination treatments except for seeds collected in Germany (GER) and incubated at 30/20 °C (Fig. 2).

Temperature regime (alternating vs. constant temperatures) was a highly positive significant term (P < 0.0001) in the GLMM model. The main effect of mean temperature was slightly positive significant (P < 0.05), while its interactive effect with the temperature regime was highly significant (P < 0.0001) with a positive coefficient. Cold stratification itself was a slightly positive significant term in the GLMM model (P < 0.05). Indeed, stratification promoted germination only at alternating temperatures as shown by the highly positive significant (P < 0.0001) interaction between temperature regime and stratification in the model. At the end of each period of cold-stratification (3, 6, 12, 18 weeks), seeds of all population gradually germinated at higher percentages. However, a 12-weeks period was not enough to break dormancy completely in most populations (Fig. 2). Germination of northern populations at 25/15 °C was further increased by extending stratification up to 18 weeks (FRA-N = 82%, UK = 67%, GER = 75%; data not shown). In contrast, after only 6 weeks of cold stratification, the germination of southern populations (ITA and FRA-S) reached 70% (data not shown).