Habitat features and genetic integrity of wild grapevine Vitis vinifera L. subsp. sylvestris (C.C. Gmel.) Hegi populations: A case study from Sicily

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abitat features and genetic integrity of wild grapevine Vitis vinifera L.ubsp. sylvestris (C.C. Gmel.) Hegi populations: A case study from Sicily

iuseppe Garfì a,∗, Francesco Mercati a, Ignazio Fontanaa, Giuseppe Collesanob,alvatore Pastaa, Giovanni G. Vendraminc, Roberto De Michelea, Francesco Carimia

CNR – Istituto di Genetica Vegetale, UOS Palermo, Corso Calatafimi 414, I-90129 Palermo, ItalyRegione Siciliana – Dipartimento Interventi Infrastrutturali per l’Agricoltura, Viale Regione Siciliana 4600, I-90145 Palermo, ItalyCNR – Istituto di Genetica Vegetale, UOS Firenze, via Madonna del Piano 10, I-50019 Sesto Fiorentino - Firenze, Italy

r t i c l e i n f o

rticle history:eceived 24 February 2013ccepted 10 August 2013vailable online xxx

eywords:iodiversityicrosatellites-Simple Sequence Repeat

SSR)lant dispersal

a b s t r a c t

Wild grapevine represents a valuable genetic resource for both future breeding programmes of cultivatedgrape and conservation of biological diversity in natural environments. In Sicily, the knowledge of thisspecies is quite scarce and fragmentary. Therefore, in order to assess the presence and the genetic qualityof wild grapevine in the island, eight populations from different locations were investigated. Their habitatswere characterized and the genetic diversity was measured by microsatellite markers in order to evaluatepossible relationships between genetic features and the ecological behaviour of populations.

With the exception of one population found in a scree-type habitat, all the others were present inflooded areas. Grapevine populations growing in riparian habitats were characterized by low inter- andintra-population variability. Conversely, the scree-type population proved to be the most compact and

opulation dynamicsabitat-level patternscrees

distinctive, as well as the most genetically isolated. Interestingly, together with other two populationsfrom the northern mountain range of the island, this scree-type grapevine population was geneticallyrather distant from local domestic accessions, suggesting a weak gene exchange with the cultivatedgrapevines in Sicily. On the contrary, the other populations showed evidences of probable introgres-sion events, as a result of either gene flow between domestic and wild plants, or of possible secondarydomestication/genetic improving processes, based on the use of native wild material.

ntroduction

Grapevine (Vitis vinifera L.) is one of the earliest domesticatedruits, and economically one of the most important, especially forts derived products wine and spirits (This et al., 2006). Despite sev-ral varieties have been selected during its millennial cultivation,n modern times extensive diffusion of only a few clones has drasti-ally reduced genetic diversity, thus increasing the risk of epidemiciseases. With regard to that, the wild relative V. vinifera subsp.ylvestris (C.C. Gmel.) Hegi could represent a quite valuable geneticesource for future breeding programmes of cultivated grape, asell as for conservation of biological diversity in natural environ-ents.At present time wild grapevine has become rather rare due

o several forms of human disturbance, such as habitat destruc-

Please cite this article in press as: Garfì, G., et al., Habitat features and g(C.C. Gmel.) Hegi populations: A case study from Sicily. Flora (2013), h

ion and fragmentation, silvicultural practices, diffusion of exoticathogens (e.g. oïdium, phylloxera, mildew), improper manage-ent of natural environment, etc. (Arnold et al., 1998, 2005, 2010;

∗ Corresponding author.E-mail address: [email protected] (G. Garfì).

367-2530/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.flora.2013.08.005

© 2013 Elsevier GmbH. All rights reserved.

Lacombe et al., 2003). Its decline, especially in central Europe,has further increased since the beginning of the 20th century.Throughout the entire Europe only few small populations (usu-ally including less than 10 individuals) still persist (Lacombe et al.,2003) and it is even reported that in the whole Upper Rhine Valleyonly 20 scattered plants nowadays survive (Arnold et al., 2005).Owing to such critical status of conservation, in the 1980s wildgrapevine was included in the IUCN (International Union for Con-servation of Nature) list of endangered European species (Arnoldet al., 2010), and several programmes for its legal protection andreintroduction have been promoted thereafter in some Europeancountries (Arnold et al., 2005; 2010). Accordingly, Maul (2004)emphasized that preservation of wild Vitis germplasm is a pri-ority in the frame of the Council Regulation (EC) 870/2004, andindicated some main items for its conservation and valorization,such as assessment of residual sites in the wild, preparation ofgeo-referenced plants’ inventory, propagation of residual popu-lations and ex situ conservation. However, as observed by Zecca

enetic integrity of wild grapevine Vitis vinifera L. subsp. sylvestristtp://dx.doi.org/10.1016/j.flora.2013.08.005

et al. (2010), problems of genetic identity may arise when wildand domestic plants have grown sympatrically for long periodsof time. Gene flow through natural hybridization events mayresult in transfer of new genes into wild populations, with related

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http://www.sciencedirect.com/science/journal/03672530

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mailto:[email protected]

dx.doi.org/10.1016/j.flora.2013.08.005

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ifficulties in taxonomic distinctiveness and the implementation offficient conservation strategies.

In mainland Italy, knowledge of this particular taxon is overallather conspicuous and extensive data describing its distribution,onservation, phylogeographic structure and genetic diversity ineveral regions are available (Anzani et al., 1990; Arnold et al.,998; Grassi et al., 2003a,b, 2006, 2008; Arroyo-García et al.,006; De Mattia et al., 2008; Zecca et al., 2010). Conversely, infor-ation on Sicily is limited to few preliminary data (Collesano,

998; Mazzola et al., 2011; Bigini et al., 2012) and some floris-ic and phyto-sociological investigations (e.g. Gussone, 1842–1845;ojacono-Pojero, 1888–1889; Barbagallo et al., 1979; Marcenò et al.,985; Brullo and Spampinato, 1991; Tomaselli, 2003; Bazan et al.,006; Giardina et al., 2007; Minissale et al., 2008; Pasta et al., 2008).his is a major gap, especially when we consider the complex andtill debated domestication history of grape (Arroyo-García et al.,006; Grassi et al., 2003a,b, 2008; De Mattia et al., 2008; Mylest al., 2011), coupled to the position of Sicily in the centre of theediterranean area, at the crossway of migration routes of peoples

nd plants. Furthermore, this region boasts a very ancient andich tradition of viticulture practices (Carimi et al., 2010, 2011).rcheobotanical findings have shown that grape has been used byative populations since the Protohistoric Age onward (Collesano,998), as documented by macro-remains of wild-type fruits datingack to the 8th millennium BC from Grotta dell’Uzzo (NW Sicily:ostantini, 1989), and by pips ascribed to the 8th century BC fromorgantina (inner Sicily: Leighton, 1993, 1999), Selinunte and Mt.

olizzo near Salemi (W Sicily: Stika et al., 2008).Research on wild grapevine in Sicily could be of valuable con-

ern at both the regional and the international scale, as it couldrovide useful information for an optimal conservation strategyf this rather rare species, and in case contribute also to clar-fy its domestication history. Moreover, it should allow to set uphe basis for exploiting this germplasm in future breeding pro-rammes for cultivated grapes. In the present paper we analyzedight Sicilian populations of V. vinifera subsp. sylvestris sampled inifferent locations and habitats. The aim was to assess the pres-nce and distribution patterns of native wild grapevine in thesland, as well as its genetic integrity. Genetic variability was inves-igated by microsatellite analysis and the possible relationshipsetween genetic features and habitat diversification and/or geo-raphic provenance are discussed.

aterials and methods

he wild grapevine in Sicily: state of knowledge and relevance

Wild grapevine is considered a characteristic species of Saliciurpureae-Populetea nigrae, the phyto-sociological class whichroups all the riparian wet deciduous woodland and willowommunities of Euro-Siberian and Mediterranean regions (Rivas-artínez et al., 2002). This is also true for the Italian territory

cf. Biondi and Blasi, 2009, report cards concerning the EU habi-ats 91F0, 92A0 and 92C0, and references therein), where it is

ostly common in riverine forest communities characterized byure stands or mixed assemblages of poplars, willows, elms andshes thriving on wet fluvisols with high water-table (order Popule-alia albae) or in the usually flooded willow-wooded and shrubbyioneer communities of the lowest zone of rivers and streambedsorder Salicetalia purpureae). In addition, it takes part in theygrophilous mantle communities growing along the borders of


ater courses (order Prunetalia spinosae, class Rhamno-Prunetea:lasi et al., 2002).

Unfortunately there are still gaps and incongruence in thenowledge of putative wild grapevine distribution in the Italian

PRESS (2013) xxx–xxx

territory. For example, according to Conti et al. (2005, 2007) V.vinifera subsp. sylvestris is doubtfully present in Abruzzo and absentin Sicily as well as in the Italian peninsula from Latium southwards,with the only exception of Campania; conversely, Bigini et al. (2012)collected abundant material in Latium, Basilicata, Calabria and SESicily, but not in NE Italy, Umbria, Campania and Sardinia.

In Sicily, wild grapevine is mentioned among the locally rareand threatened species, although it does not figure within themost recent regional red lists (Conti et al., 1997; Raimondo et al.,2011). In agreement to its usual habitat, Sicilian populations of wildgrapevine seem to be strictly linked to riparian woodlands domi-nated by Populus spp., Salix spp. and Platanus orientalis L. Despitethe huge amount of papers concerning the forest and hygrophilouscommunities of the island, wild grapevine has been recorded onlyin few valleys of rivers, streams and creeks, most of the latter flow-ing within the canyons of the Hyblaean Plateau (Barbagallo et al.,1979; Brullo and Spampinato, 1991; Tomaselli, 2003; Minissaleet al., 2008; Garfì et al., 2011), where already in the first half of XIXcentury Gussone (1842–1845) mentioned its presence in the vicin-ity of the village of Cassaro (Anapo River Valley). Additional singlelocalities are known in the Peloritani Mts. (Brullo and Spampinato,1991), the Sicani Mts. (Bazan et al., 2006) and the Belice Riverwatershed (Pasta et al., 2008). In these areas, at least until the endof XIX century, wild grapevines were also present in mantle andforest communities, as testified by Lojacono-Pojero (1888–1889),who reported it in the hedges of northern Sicily and in the wood-lands of Nebrodi Mts. and Mt. Etna. However, the presence of thesepopulations has not been confirmed in recent times, and duringthe last decades wild grapevine seems to have become quite rarewithin most of these regional forest communities. In fact, in thepast decades its presence has been reported only for one site inthe Sicani Mts. by Marcenò et al. (1985) and for two localities onthe Madonie Mts. (Giardina et al., 2007), while no recent phytoso-ciological record is available neither for the woods dominated bymesic deciduous broadleaved trees (Brullo et al., 1996, 2000) norfor the thermophilous maquis-forests communities (Brullo et al.,2009; Gianguzzi et al., 2011) in the whole island.

Sampling sites and plant materials

Several surveys were carried out since 2007 in the main moun-tainous ranges and nature protected areas of Sicily, where naturalenvironment is still relatively well preserved. The exploration crite-ria were primarily based on the habitat features, focusing theinterest on damp areas and river/brook banks and taking intoaccount records from the literature and oral sources.

Considering the strong morphologic resemblance of wildgrapevine with the cultivated one, we tried to reduce as much aspossible the risk that plants deriving from naturalized grapevinecultivars or rootstocks were mistaken for wild grapevines, althoughit is believed that cultivated varieties have a low chance to survivein the wild (Grassi et al., 2003a). To this aim, the plant samplingstrategy was based on the main differentiating traits used to dis-tinguish wild from domesticated grapevines, as summarized inTable 1. Moreover, as a general rule we avoided sampling closeto either current vineyards or sites with evidence of past viticul-ture practices. Exceptions from the former issues were promptedby unequivocal citations from the literature reporting the occur-rence of wild grapevine or, sometimes, by oral information fromnative people. In those cases, we opted to evaluate a posteriori theactual genotype through molecular analysis.

At each investigated site the plants were more or less dispersed:


in certain locations the farthest were at some ten metres apart,whereas elsewhere the maximum distance reached several kilo-metres. Therefore, all the collected plants were arbitrarily groupedinto separate populations depending on the river watershed where

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Table 1Comparative morphology of wild and domesticated grapevine based on Olmo (1976).

Character Wild grapevine Domesticated grapevine

Mating system Dioecious HermaphroditeHabitat Humid soils Dry habitatsBerry shape Small, round or oblated Large and elongatedTrunk Often branches, slender, bark separated in very long thin strips Thick bark separates in wider and more-coherent stripsSeeds Small, rounded body, high width/length ratio (>0.70) Large, pyriform body, lower width/length ratio (<0.60)Fruit clusters Small, globular to conical, irregular set, berry maturity variable in cluster Large, elongated, compact to well-fitted, berry, uniform in maturityLeaves Small, usually deeply three-lobed. Petioles short and slender, dull aspects Large, many entire or with shallow sinuses, petiole thick, glabrous to

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ach group was found. Accordingly, we distinguished 8 popula-ions, overall including a total of 81 individuals (Table 2).

For each population we recorded the general vegetation pat-erns and the dominant species of the forest habitat, in addition tohe main topographical and hydraulic features of the stream flows.vidences of present or past land use and anthropic disturbancesere also described.

For all sampled plants, we annotated the geographical coor-inates and altitude through a sub-metric precision GPS devicemodel Trimble GeoXT; Trimble, Sunnyvale, CA, USA); the geodesicystem we used was WGS84. Moreover, we identified somettributes such as: plant sex (if assignable in the field), habit (climb-ng or creeping), position of the crown (canopy, overshadowed),earing tree species (if any), micro-habitat (topography, canopyover), trunk class size (less than 2 m long, from 2 to 10 m, morehan 10 m), and health state.

In order to evaluate more correctly the true genotypic identity ofhe wild plants and to highlight any possible relationships with cul-ivated grapevines, five most common Sicilian traditional cultivarsnamely Catarratto, Nero d’Avola, Inzolia, Perricone and Zibibbo)ere used for comparison. Plant material was obtained from the

ermplasm collection fields of the Institute of Plant Genetics – CNR,alermo (Italy).

NA extraction and microsatellite analysis

Total genomic DNA was extracted from young, fresh leaves col-ected in the field, ground into fine powder with liquid nitrogen andtored at −80 ◦C until use. The extraction was carried out followinghe CTAB method (Doyle and Doyle, 1990) and DNA was quantifiedn 1% agarose gels.

Samples were analyzed at six microsatellite loci (SSR – Sim-le Sequence Repeat), as often performed in similar works on wildrapevine (e.g., Grassi et al., 2003b; Arrigo and Arnold, 2007; Bartht al., 2009; Cunha et al., 2010). Therefore, the set of microsatellitearkers, i.e. VVS2 (Thomas and Scott, 1993), VVMD5, VVMD7 andVMD27 (Bowers et al., 1996), VrZAG62, and VrZAG79 (Sefc et al.,999), was selected as proposed by the GENRES 081 Project (Euro-ean Vitis Database: www.genres.de/vitis/vitis.htm) to allow theomparison of the resulting profiles with the available database.

PCR amplification was carried out using the Qiagen multiplexCR kit (Quiagen, Hilden, Germany) with the following conditions:5 min at 95 ◦C (HotStar Taq activation step) followed by 35 cyclesonsisting of 30 s at 94 ◦C (denaturation), 90 s at 50–56 ◦C (anneal-ng), 60 s at 72 ◦C (extension) and a final step for 30 min at 60 ◦C. Onef the primers of each pair was fluorescently labelled with FAM,OE or TAMRA. Each sample was amplified at least twice to cor-


ect possible mistyping or amplification errors. PCR products wereize-separated by capillary electrophoresis performed on a geneticutomatic sequencer (ABI Prism 3130; Applied Biosystems, Fosterity, CA, USA).

Data analysis

Allele size was estimated by comparing the fragment peaks withthe internal size standard, using the default method for band callingwith microsatellite and the expected repeat size. Electrophero-grams were verified visually using Gene Mapper v. 4.1 software.Several diversity parameters were estimated using GenAlEx 6.5(Peakall and Smouse, 2012): the number of alleles per locus (Na),the allele size range and the allele frequency, the observed (Ho) andexpected (He) heterozygosity (Nei, 1987); microsatellite screeningability was also based on the probability of identity (PI; Paetkauet al., 1995) and the polymorphic information content (Weber,1990), with the most effective microsatellite having high valuesof Ho and polymorphic information content, and low PI.

The genetic diversity within each group was estimated bycomparing number of alleles per locus (Na), effective number ofalleles (Ne), number of private alleles (Npa), allelic richness (AR),observed (Ho) and expected (He) heterozygosity (Nei, 1987) usingGeneALex 6.5 and FSTAT version 2.9.3.2 (Goudet, 2002) software.The intra-populational genetic variability was also analyzed bycalculating the inbreeding coefficient (FIS; Weir and Cockerham,1984) using the FSTAT software. Each locus and population wastested for deviations from Hardy-Weinberg equilibrium expec-tations with exact tests using Genepop v. 3.4 (Raymond andRousset, 1995). Genepop was used to examine linkage disequi-librium among all pairs of loci within each wild populations. AllGenepop analyses were performed using the following parameters:dememorization = 10000, number of batches = 100, and numberof iterations/batch = 1000. Significant positive FIS values indicateinbreeding (excess of homozygotes) or undetected null alleles,whereas significant negative values indicate excess of heterozygos-ity and then low inbreeding. Near zero values are expected underrandom mating.

ARLEQUIN v. 3.5.1.3 (Excoffier and Lischer, 2010) software wasemployed to estimate the fixation index FST (Weir and Cockerham,1984). FST was used to measure the divergence among the investi-gated grapevine populations. The significance of the differentiationbetween pairs of populations was tested using permutation proce-dures (10000 replicates).

A cluster analysis of wild and domesticated populations was car-ried out using the DA genetic distance (Nei et al., 1983) accordingto the UPGMA (Unweighted Pair-Group Method with Arithmeticmean) algorithm. A dendrogram was generated using the softwarePopulations 1.2.31 (Langella, 2002) and visualized with Treeview1.6.6 (Page, 1996).

To identify the number of genetic groups in the wild populationsand to investigate their relationships with domesticated Siciliancultivars, the software Structure version 2.3.4 (Pritchard et al.,


2000) that employs a model-based Bayesian clustering method,was used. The estimate of the most likely number of genetic groups(Ks) was performed following Pritchard and Wen (2003) and thesimulation study by Evanno et al. (2005), which proposed an ad hoc

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xxx–xxxTable 2Population sizes and habitat conditions of the investigated wild grapevine occurrences in Sicily.

Geographical districts(Province)

No. ofpopulations

Population name(code)

Total no. of trees and (inbrackets) no. of treeswith length >10 m

Habitat Vegetation patterns anddominant trees/shrubs

Anthropic evidences Available literaturecontaining informationon the presence of wildgrapevines

Madonie Mts. (Palermo) 1 Castelbuono (CSB) 9 (2) Temporary river banks Riparian forest, canopy cover100%, h = 5–8 m. Salix pedicellata,Populus nigra, Laurus nobilis,Quercus ilex, Clematis cirrhosa,Hedera helix, Smilax aspera

Sporadic drafted, agedolive trees along thetrack to the samplingarea

Giardina et al. (2007)and Mazzola et al.(2011)

Nebrodi Mts. (Messina) 1 Stretta di Longi (LON) 7 (3) Permanent river banks Riparian forest, canopy cover100%, h = 8–10 m. Salixpedicellata, Ficus carica, Fraxinusangustifolia, Celtis australis,Clematis cirrhosa, Hedera helix,Rubus sp.

Grazing disturbanceevidences

Lojacono-Pojero(1888–1889)

Sicani Mts. (Agrigento) 1 Fiume Sosio (SOS) 13 (3) Permanent river banks Riparian forest, canopy cover100%, h = 8–15 m.Salix pedicellata, Quercus ilex, Q.virgiliana, Crataegus monogyna,Prunus spinosa, Pistaciaterebinthus, Viburnum tinus,Rhamnus alaternus, Hedera helix

Sporadic small patchesof abandoned fieldsalong the track to thesampling area

Marcenò et al. (1985)and Bazan et al. (2006)

Belice Valley (Trapani) 1 Riserva Zangara (ZAN) 8 (3) Permanent river banks Riparian forest, canopy cover70%, h = 8–12 m. Ulmus canescens,Salix pedicellata, Populus nigra,Ficus carica, Tamarix sp., Neriumoleander, Myrtus communis,Hedera helix

Large vineyard in thesurroundings;Small abandoned olivepatches inside thesampling area

Pasta et al. (2008)

Torto Valley (Palermo) 1 Riserva Boschi Favara eGranza (FAV)

17 (2) Scree-type deposits Pioneer communities, canopycover 40%, h = 2–4 m.Ficus carica, Rubus sp., Clematiscirrhosa, Quercus virgiliana (alongthe external border)

Sporadic small patchesof abandoned fieldsalong the track to thesampling area

Collesano (1998)

Iblei Mts.(Siracusa)

3 Riserva Pantalica eValle Anapo (PAN)

18 (0) Permanent river banks Riparian forest, canopy cover80%, h = 10–15 m. Salixpedicellata, Populus nigra, Ficuscarica, Platanus orientalis, Quercusilex, Fraxinus ornus, Rhamnusalaternus, Nerium oleander,Clematis cirrhosa

Small cultivatedpatches inside thesampling area;Large abandoned andactive cultivations inthe surroundings

Gussone (1842–1845);Barbagallo et al.(1979); Brullo &Spampinato (1991);Minissale et al. (2008);Tomaselli (2003)

Fiume Manghisi(MAN)

5 (0) Permanent river banks Riparian forest, canopy cover80%, h = 8–12 m. Salix pedicellata,Ficus carica, Platanus orientalis,Quercus ilex, Clematis cirrhosa,Hedera helix

Abandoned and activecultivations in thesurroundings

Cava Sturia(CST)

4 (4) Temporary river banks Maquis, canopy cover 100%,h = 6–8 m. Quercus ilex, Q.virgiliana, Ficus carica, Oleaeuropaea var. sylvestris, Fraxinusornus, Ailanthus altissima, Pistacialentiscus, P. terebinthus, Rhamnusalaternus, Phillyrea latifolia,Smilax aspera, Hedera helix

Abandoned and activecultivations in thesurroundings

Total 8 81

h = height.

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tatistic, �K. For each K (ranging from 1 to 10), 20 independent runs100000 burn-in, 1000000 Marchov Chain Monte Carlo) were car-ied out. All runs were performed with the admixture model addinghe sampling information to the input data (LocData = 1) accord-ng to Hubisz et al. (2009). The 20 runs were averaged using theoftware CLUMPP (CLUster Matching and Permutation Programme;akobsson and Rosenberg, 2007), and shown in histograms usinghe programme Distruct (Rosenberg, 2004).

esults

abitat-level patterns and individual features: somelements of peculiarity

The eight sampled populations were scattered throughout SicilyFig. 1 and Table 2): three populations were from the northern

ountain ranges, two from the southwestern district and threerom the southeastern hills. No population was found in the crop-ominated landscapes of the inner provinces of the island. Allopulations are located within protected areas or Natura 2000 sites,xcept populations CST and ZAN (for name coding, see Table 2). CSTnd ZAN populations were both within small isolated patches ofatural vegetation surrounded by cultivated areas, especially cerealrops (CST) and olive groves and vineyards (ZAN). Minor evidencesf ancient cultivations were also noticed in the surveyed areas foropulations SOS, CSB and PAN, but not in the close proximity of theampled plants.

The altitudinal gradient was between 300 and 400 m a.s.l. forost populations. The highest population was FAV, ranging from

80 to 904 m a.s.l., whereas the lowest was ZAN, situated almostntirely at about 120 m a.s.l.

The habitat conditions of the investigated sites were rather dif-erent (Table 2). With the exception of FAV, all the other populations


rew along streams flowing inside gullies or open valleys with nar-ow or wide bottom. Plants were rooted in close proximity to thetream beds in ‘narrow-bottom’ valleys, and up to some ten metrespart in the other locations. The streams were usually permanent;

e investigated populations.

only CSB grew along a temporary rivulet, whereas almost all plantsfrom CST were found inside a nearly constantly dry river bed. FAVwas the only population not restricted to stream banks. It residedin a wide, humid niche few thousand-square metres large, lyingon a north-facing forested slope and surmounted by sub-verticalrocky cliffs, delimited at their base by scree-type stony depositswith coarse blocks of various size, among which vine plants werethriving.

In all sites, the forest cover was usually represented by thickriparian vegetation, 8–15 m high, dominated by Salix pedicellataDesf., Populus nigra L., Ficus carica L., Nerium oleander L. and someclimbing vines such as Clematis cirrhosa L. and Hedera helix L. inaddition to wild grapevine. Platanus orientalis L. was a typical ele-ment in two of the Iblei Mts. populations, whereas Ulmus canescensMelville was rather abundant only at ZAN; moreover, Quercus ilexL. and Fraxinus ornus L. were often observed, too. Together with Q.virgiliana (Ten.) Ten. and several Mediterranean evergreen sclero-phyllous shrubs, they characterized the maquis-type forest habitatof CST. Once again, FAV was quite peculiar, since the forest habitatwas represented by sparse pioneer communities with Ficus car-ica, Rubus ulmifolius and Clematis cirrhosa, whose canopy neverexceeded the height of 4 m.

The number of sampled individuals per population ranged from4 to 18. Their intra-population spatial distribution was quite diver-sified, varying from clustered (FAV and, at less extent, ZAN) tovery scattered (PAN). In the cluster pattern, the maximum inter-individual distance did not exceed 50 m, whereas in the scatteredpattern the farthest plants were spaced up to 4.6 km. The major-ity of the plants collected in the wild showed the most relevantdistinctive morphological traits of wild grapevine; among them:dioecy, black small fruits in loose clusters and small, rounded pips.However, we also observed some plants showing morphologicalcharacteristics of domesticated grapevine (i.e. hermaphroditism,and large leaves with shallow sinuses) mainly in the population


PAN. Although the presence of male and female plants was recordedin most populations, it was not possible to identify the gender of allthe sampled individuals due to difficulties in detecting flowers orto inaccessibility due to their high crown. Overall, a large amount

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ig. 2. Large creeping grapevine at FAV. The lush mosses indicate a notable amountf moisture in the habitat.

f vines measured between two and 10 m. Moreover, all popula-ions, except MAN and PAN, contained plants of notable size, with

trunk up to 20 m long. The biggest plants were found at LON (baseiameter 20 cm; length about 22 m) and FAV (base diameter 18 cm;

ength about 20 m). At CST all sampled plants were more than 10 mong. Seedlings were almost absent, except at LON and CSB whereust very few saplings were observed.

The habit was climbing for nearly all plants and the crown of theiggest plants usually reached the forest canopy; only at FAV creep-

ng plants were prevailing, including the most sizeable individuals,oo (Fig. 2). Support trees belonged to many species, without specialreferences between deciduous (e.g. willows, poplars, ashes, figs)r evergreen (e.g. holm oaks). Moreover, the shortest vines climbedven on Mediterranean sclerophyllous shrubs such as Phillyrea lat-folia L. and Rhamnus alaternus L. Almost all grape plants lookedealthy, without any evidence of typical grapevine pathogens suchs oïdium, phylloxera or mildew. At SOS and FAV, one large andhree small plants, respectively, looked rather stunted and exhib-ted a reduced crown, probably suffering for overshadowing orutrient competition.

uclear microsatellite diversity

The 81 samples analyzed at 6 microsatellite loci showed 72 dif-erent multi-locus profiles. The remaining 9 individuals withoutny private loci probably originated from clonal duplication. A totalf 59 alleles were detected with an average of 9.8 alleles for locusranging from 8 to 12).

The most variable loci were VVMD7 and VrZAG62, with 12 and


1 alleles, respectively. Twenty-five out of 59 alleles had a fre-uency lower than 5% (data not shown) while for VrZAG79 thereas a predominant allele (248 bp) with a frequency around 40%.

he Ho values were quite different among loci (from 0.444 to 0.931);


the He values ranged from 0.762 to 0.828 with an average of 0.806(Table 3). The ability of each microsatellite locus to distinguishamong genotypes (polymorphic information content) varied from0.723 to 0.781; the probability of identity (PI) between two geno-types harbouring different alleles at the analyzed loci ranged from0.053 to 0.076, while the calculated value for all loci was rela-tively low (4.98 × 10−8). The VVMD27 locus resulted the best fordiscriminating among our samples (polymorphic information con-tent = 0.781 and PI = 0.053).

Intra- and inter-population diversity

The genetic diversity estimates for each wild population arereported in Table 4. The total number of alleles (Na) ranged from 20(CST and FAV) to 43 (PAN), and the effective number of alleles (Ne)from 13.4 to 25.9. Observed (Ho) and expected (He) heterozygosityvaried between 0.500 (LON) and 0.778 (PAN) and from 0.543 (FAV)to 0.761 (PAN), respectively. In addition, the allelic richness (AR)ranged from 2.8 (FAV) to 4.3 (PAN), confirming a rather conspicuousrate of diversity in the whole germplasm collection.

We also tested for Hardy–Weinberg predictions in order to eval-uate the amount of inbreeding in each population. The FIS valuesdiffered widely between loci in each population, ranging from 0.271(CST) to −0.243 (FAV). FAV was also the only population with asignificant value at P < 0.05. The pairwise genetic differentiation(FST) (Table 5) was highest between FAV and the other popula-tions, with maximums in respect of CSB (0.360) and LON (0.365).On the contrary, PAN showed the lowest mean value, with min-imums in respect of CST (0.045) and MAN (0.006), the other twopopulations belonging to the same geographic district. When wildgrapevine germplasm is compared to the cultivated group (DOM),LON and FAV showed the highest FST values (0.185 and 0.184,respectively) and MAN the minimum (0.000). All pairwise FST val-ues where significantly greater than 0 (P < 0.05) except for the threepairs PAN-MAN, ZAN-SOS and MAN-DOM (Table 5).

The cluster analysis of the populations did not match with theirgeographic origin (Fig. 3). According to the genetic distance, 3 clus-ters (named A, B and C) can be clearly distinguished. Cluster Aincluded CSB and LON, two populations in the northernmost andbest preserved forest areas; cluster B comprised FAV, the only scree-type population; finally, cluster C included the remaining wildpopulations as well as the group of cultivated varieties. A similarpattern was also obtained in cluster analysis using all individualgenotypes instead of populations (data not shown), although FAVdisclosed a singular structure, as it was the only population con-taining 3 sub-groups (ranging from 2 to 6 plants) with identicalgenotype, most probably of clonal origin.

The most likely number of genetic groups (K) of wild grapevinesand their relationships with cultivated germplasm was identifiedby Structure software. A preliminary analysis was carried out withno prior locus information assumption. Results showed a peak atK = 3, but no clear distinction emerged neither among wild groupsnor between wild and cultivated accessions. Only the FAV popu-lation showed a homogeneous profile that differed from all theother accessions. In order to clarify the distribution of all geno-types across different groups, an additional analysis was performedproviding geographic information to the input data in Structureanalysis according to Pritchard (2010) (cf. Material and methods).This approach maintained the K value to 3, confirming the partitionof the investigated germplasm into three most probable geneticpools (Fig. 4). The FAV population was the most distinguished


group, whilst individuals from ZAN, CST, MAN and PAN clusteredwith the domestic accessions. CSB, LON and, partially, SOS, mostlyshowed a genetic profile different from both FAV and the cultivatedpool.

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Table 3Diversity estimates at the 6 microsatellite loci analyzed for the 72 wild genotypes.

Locus Na Allele size range (bp) He Ho PI PIC

VVS2 9 127–151 0.805 0.931 0.0639 0.7533VVMD5 9 216–240 0.810 0.722 0.0611 0.7601VVMD7 12 231–265 0.809 0.467 0.0600 0.7619VVMD27 8 176–190 0.828 0.875 0.0528 0.7815VrZAG62 11 171–199 0.822 0.583 0.0530 0.7795VrZAG79 10 228–256 0.762 0.444 0.0759 0.7235Mean 9.8 – 0.806 0.670 – 0.7599All Loci 59 – – – 4.98E-08 –

Na: number of alleles per locus; He: expected heterozygosity; Ho: observed heterozygosity; PI: probability of identity; PIC: polymorphic information content.

Table 4Genetic diversity estimates in wild grapevine populations (in brackets the average value per locus).

Population Na Ne Npa Ho He AR FIS

CSB 27 (4.5) 17.2 (2.9) 1 (0.17) 0.556 0.606 3.4 0.141LON 21 (3.5) 13.4 (2.2) 1 (0.17) 0.500 0.517 3.0 0.110PAN 43 (7.2) 25.9 (4.3) 4 (0.67) 0.778 0.761 4.3 0.007MAN 27 (4.5) 20.0 (3.4) 2 (0.33) 0.700 0.607 4.0 −0.043CST 20 (3.3) 17.0 (2.8) 1 (0.17) 0.542 0.625 3.3 0.271SOS 36 (6.0) 20.1 (3.4) 1 (0.17) 0.667 0.691 3.8 0.076ZAN 28 (4.7) 21.3 (3.5) 1 (0.17) 0.708 0.699 3.9 0.054FAV 20 (3.3) 16.1 (2.7) 1 (0.17) 0.708 0.543 2.8 −0.243*

Na: total number of alleles; Ne: effective number of alleles; Npa: number of private alleles; Ho: observed heterozygosity; He expected heterozygosity; FIS: inbreeding coefficient;AR: average allelic richness.*Significant at the P < 0.05 level.

Table 5Pairwise Fst values obtained from the nuclear microsatellite markers of the investigated accessions.

CSB LON PAN MAN SOS CST ZAN FAV DOMCSB –LON 0.205* –PAN 0.123* 0.152* –MAN 0.150* 0.186* 0.006 –SOS 0.205* 0.223* 0.115* 0.106* –CST 0.070* 0.121* 0.045* 0.050* 0.127* –ZAN 0.232* 0.247* 0.088* 0.117* 0.073 0.113* –FAV 0.360* 0.365* 0.187* 0.181* 0.287* 0.249* 0.253* –

*

D

Hw

sn

DOM 0.145* 0.185* 0.037* 0.000

Significant at the P < 0.05 level.

iscussion

abitat patterns and populations dynamics of the Sicilianild grapevine


Despite millennia of human occupation and land use, Sicilytill maintains a number of natural areas hosting valuable rem-ants of plant biodiversity such as wild grapevine, and the present

Fig. 3. UPGMA dendrogram constructed using

0.103* 0.092* 0.108* 0.184* –

investigation unexpectedly revealed a rather conspicuous abun-dance of putative wild grape in many and diversified parts of theisland.

Consistently with most citations from the literature (e.g., Oceteet al., 1999; Arrigo and Arnold, 2007), all the investigated Sicilian


populations appeared to be related to damp conditions. On theother hand, it is known from the literature (Laguna Lumbreras,2003; Arnold et al., 2005) that differences in moisture availability

the DA genetic distance (Nei et al., 1983).

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8 G. Garfì et al. / Flora xxx

Fig. 4. Genetic clustering of the investigated grape populations, inferred usingSe

adflhfs

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TRUCTURE (Pritchard et al., 2000). Each vertical bar represents an individual, andach colour/grey shadow a distinct gene pool.

nd/or watercourse regime may notably affect the distribution andynamics of grapevine populations, especially considering thatooded environments prevent the diffusion of the root-hostedomoptera Phylloxera (syn. Dactylosphaera vastatrix), therefore

avouring the persistence of wild grapevine populations alongtream banks.

In Sicily, the water flow of permanent large streams, as at PAN,AN and SOS, most probably facilitated the long-distance dis-

ersion of propagation material, like seeds or broken branches,romoting the establishment of new grape populations along thelluvial network (Arrigo and Arnold, 2007; Arnold et al., 2010).n these cases, plants were rooted on the stream banks even atome ten metres afar from the river edge. On the contrary, theuite ephemeral regime of the CST rivulet allowed the growth ofrapevines along the bottom of the gully, where underground wateresources were crucial for its survival. The population from FAV isemarkable under many aspects. The habitat type here is devoidf streams, and moisture is probably compensated by both ele-ation (about 900 m a.s.l.) and the northern aspect of the slope,s suggested by the extreme abundance and luxuriance of mossover (Fig. 2). Moreover, as for the flooded areas, scree habitats arelso mentioned as unfavourable habitat to Phylloxera (Arrigo andrnold, 2007).

The scree-type habitat of FAV and the related ecological con-itions likely also affect intra- and inter-population spread andonnectivity. Screes are isolated environments and, lacking theropagation role of hydraulic dynamics, in this context plants’ dis-ersal more strictly depends on interactions between faunal andlant communities. As described for various Mediterranean habi-ats (Herrera, 1989; Matías et al., 2010) and in the floodplain forestf the Upper Seine Valley (Arnold et al., 2010), wild grape dispersalould be favoured by mammals (e.g. carnivores like red fox, stonearten, etc.) and several birds (mostly frugivorous passerines).

ird communities in riparian woodlands are usually richer thann non-riverine conifer and broadleaved forests (Dronneau, 2007),robably due to the higher architectural complexity of the riparianorests resulting from the specific dynamics of riverside habitats.iversity of woody plant species, high vertical stratification, hori-ontal heterogeneity, abundance of large vines and vigour of plantrowth are the most striking features of these habitats, offering tovian communities a much wider spectrum of refuges and trophicesources. Many birds use riparian corridors as migration path-ays, thereby connecting different areas (Décamps et al., 1987).s a result, many plants are dispersed by birds (especially Turdi-ae) migrating along the riparian zones (van Dorp and Kalkhoven,988). Besides, plant movements or exchanges may increase atodes where riparian and other corridors, such as roads, railwaysnd hedgerows, intersect (Riffel and Gutzwiller, 1996). Overall,


n the present study-case it is suggested that the combination ofiver dynamics and zoochory favours gene flow of wild grapevineermplasm among all the riparian populations, whereas the scree-ype population FAV is maintained ecologically isolated. Likewise,


the small area size of population FAV might also have contributedto its divergence.

As recorded in many central European countries (cf. Arnold et al.,1998, 2005, 2010; Lacombe et al., 2003) also silvicultural prac-tices might have had a detrimental role in conservation of wildgrapevine populations. But in Sicilian populations, forest manage-ment history, as well, can account for the relative abundance ofgrape sizeable plants. As a rule, large-scale disturbances, especiallyclear-cuttings, can notably influence diffusion and development ofclimbing vines (Schnitzer and Bongers, 2002). Silvicultural activ-ities either create clearances which favour their establishment,or conversely, especially in short rotation forest systems, canindirectly control their sizeable growth, since vines are periodi-cally removed with timber trees. In the investigated sites, eventhe best preserved forest areas are actually aged coppices, nolonger exploited for fuel wood and charcoal production since atleast the 1960s. As observed for ivy vines at the Monte CarcaciNature Reserve (Garfì and Ficarrotta, 2003), stops in clear-cuttingsinvolved the arising of vegetation dynamics that led to changesin floristic and, above all, in structural features of forest stands.Accordingly, this improved the chances that grapevine plants mightage undisturbed together with their trellis trees and grow remark-ably up to the forest canopy. In addition, the scarcity of grapevineseedlings can be similarly explained by the low-light availability inthe understory owed to the thick canopy covering the sites (Arnoldet al., 2010). A particular situation occurs only at FAV, where aproper forest canopy is not present and the prevailing creepinghabit of grapevine there could notably enhance the probability ofclonal propagation. All these issues must be carefully evaluated inthe definition of a proper strategy of in situ conservation.

Genetic structure of populations: inferences to their integrity

The complexity deriving from the historical and extensive culti-vation of its domesticated relative in Sicily can represent a potentialmajor threat to the genetic integrity of indigenous wild grapevinepopulations. As a matter of fact, none of the sampled sites couldactually be considered completely exempted from evidences ofhuman activities, especially ZAN and all the populations fromthe Iblei district. Accordingly, more or less intensive introgressioncould be expected between domestic and wild germplasm.

Molecular analyses showed that the six microsatellites inves-tigated were able to discriminate the sampled plants (Arrigo andArnold, 2007; Grassi et al., 2003b, 2008; Cunha et al., 2010). Highlevel of polymorphic information content along with lower PIstrengthened the effectiveness of microsatellites in distinguishingamong genotypes. However, the PI assessing was higher than val-ues at which a microsatellite is considered hyper-polymorphic ingrapevine (Sefc et al., 2001). A rather valuable gene diversity wasdetected in wild grapevine populations in spite of their small size.This can be partly related to the mating system of this dioecious andoutbreeding plant (Grassi et al., 2003b). On the other hand, meanFIS values indicated that, among the populations analyzed, only FAVdeviated from Hardy–Weinberg equilibrium, showing an excess ofheterozygosity. Single or reiterated events of selection, as well asmigration and/or mutation could be invoked to explain such a situ-ation, but current data do not allow us to be biased towards one oranother factor. In general, the low allelic richness within wild popu-lations provides evidence for a potential genetic bottleneck effect.Allelic richness, being more heavily influenced by rare alleles thanexpected heterozygosity, is commonly regarded as a more relevantcriterion for measuring loss of diversity due to genetic bottlenecks


(Nei et al., 1975).The observed pattern was also supported by the differentia-

tion estimates. According to the FST values, FAV appears as themost differentiated population among all the investigated ones,

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ith the greatest distance with respect to CSB and LON. This results quite consistent with the assumptions about habitat patternsnd connectivity discussed above. Accordingly, the most effectiveonnecting role of both fluvial dynamics and birds communitiesupport the idea of a most intense gene flow within the three popu-ations from the Iblei district (PAN, MAN and CST), which showedhe lowest FST by far.

When compared to the domestic accessions, interestingly FAVogether with LON appear also the populations genetically mostistant from them, highlighting a most probable weak genexchange with the local cultivated grapevines considered in thistudy. On the contrary, low FST values between domestic vari-ties and wild provenances from PAN and, at less extent, CST,AN and ZAN suggest that past introgression events could have

ccurred. This hypothesis appears likely if we consider that theAN area, corresponding to the current Nature Reserve of “Pan-alica e Valle dell’Anapo”, represents the centre of development ofncient civilizations dating back to the Bronze Age (ca. XIII centuryC: Leighton, 1999) that expanded to the whole Iblei Mts region.

The segregation of FAV, as well as the weak relationships ofON and CSB with all the other populations, were confirmed by thePGMA cluster analysis and the results obtained from Structure at

= 3. Overall, the comparative analysis of results from the differentpproaches prompts some speculations about the genetic distinc-iveness of the investigated germplasm. Populations in relativelyristine forest areas, such as CSB and LON, most probably could beetained as the closest to pure wild grape lineages. FAV, althoughifferent from CSB and LON in the Structure’s data representation,ould also be referred to “wild-like” genotype, as supported by FSTalues. Its uniqueness can be possibly related to its geographic andcological isolation that strongly limited gene flow with the otheropulations. Consequently, it could represent one of the most inter-sting germplasm pools among all the investigated ones, althoughuch an assumption needs to be confirmed by comparison with aigher number of Sicilian cultivated varieties.

The situation of the other populations is less straightforward.wo opposite hypotheses can explain our results. ZAN, PAN, MANnd CST may have largely resulted from introgression eventsccurred during the last millennia. This is rather likely for theblei’s district populations, where the land has a long history ofnthropic use, as well as for ZAN, still lying within a territoryf renowned ancient viticulture. In these areas the current wildopulations could be descendants of several generations of inter-reeding, due to pollen flow between domestic and wild plants,ith consequent partial loss of genetic attributes by the native wild

ene pool (Di Vecchi-Staraz et al., 2009). A different hypothesisssumes that the most ancient Sicilian cultivated varieties deriverom one or more events of secondary domestication or geneticmproving, based on the use of indigenous wild germplasm. Thiscenario seems supported by several evidences on the existence ofecondary domestication centres in the west Mediterranean (Grassit al., 2003a,b; Cunha et al., 2010; Zecca et al., 2012; De Andrés et al.,012), but additional investigation is needed to corroborate suchn assumption.

onclusions

The present research represents the first most comprehen-ive contribution to the knowledge of Sicilian wild grapevineermplasm. At first, results of the exploration campaign suggestedhat the actual richness of this species in Sicily is much more con-


picuous than expected, so the eight surveyed populations areut a small sample of the total plant stock in the island. More-ver, data analyses provided some valuable indications about theenetic quality of the plant material and the implications with the

PRESS (2013) xxx–xxx 9

habitat features, whose significance could go beyond the narrowlocal concern. Overall, most of the investigated populations showeda fair genetic diversity that somehow seems to be depending onthe habitats’ functional processes and the level of their ecologi-cal connectivity. On the other side, the genetic affinities that somepopulations shared with local domestic varieties triggered intrigu-ing assumptions that could assign to Sicily a role of concern in thehistory of grape domestication, whereas the most singular popu-lations should be accounted as potential sources of precious genematerial for future programmes of genetic improving of currentcultivars.

Anyway, more accurate investigations must be still extendedto several aspects. For instance, a comparison with a larger panelof Sicilian and international grapevine cultivars, could notably addinformation on the actual nature of the local V. sylvestris gene pooland the intensity of possible introgression events between wild andcultivated grapevines. Besides, with regard to territory exploration,more surveys must be carried out and extended to a variety of habi-tats other than riverbanks, especially including screes or marshes,wherein the existence of additional distinctive genetic diversity canbe assumed.

In the conservation perspective several aspects must be takeninto account. Agricultural policy of recent years has involved theincrease of vineyard areas in many parts of the region. This couldenhance the risk for genetic integrity of wild grape, due to thepotential increase of gene flow from domestic to wild germplasm(Zecca et al., 2010). Only the most isolated populations or thosegrowing in nature protected areas could possibly be retained as inrelatively safe conditions. But considering the species’ dynamics(e.g. poor regeneration by sexual reproduction, dispersion mech-anisms) coupled to the current situation of general lack/improperforest management in Sicily, in situ conservation should not dis-regard a monitoring activity of the most prominent populations,and the implementation of assisting measures in case of plants die-back or decline. In any case, ex situ conservation always retains amajor role in order to preserve the most valuable genotypes, so thatinstallation of germplasm collection fields in the region is highlyadvisable.

Acknowledgements

We are very grateful to Paolo Uccello, Giuseppe Clementi,Giuseppe Traina, Giovanni Giardina and Pietro Zanghì for fieldassistance and for providing personal data on the exact locationof several investigated wild grapevine populations. The study wassupported by funding of PSR 2007–2013, Sottomisura 214/2 –Preservazione della biodiversità: Centri pubblici di conservazione.Progetto DDS n. 1264 “Recupero, conservazione e valorizzazionedel germoplasma di vitigni minori (Vitis vinifera ssp. vinifera) e viteselvatica (Vitis vinifera ssp. sylvestris) in Sicilia”.

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Documents

Habitat features and genetic integrity of wild grapevine Vitis vinifera L. subsp. sylvestris (C.C. Gmel.) Hegi populations: A case study from Sicily