Flora 208 (2013) 538548

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Flora
journal homepage: www.elsevier.com/locate/flora

Habitat features and genetic integrity of wild grapevine Vitis vinifera L.

subsp. sylvestris (C.C. Gmel.) Hegi populations: A case study from Sicily
Giuseppe Garf a, , Francesco Mercati a , Ignazio Fontana a , Giuseppe Collesano b ,
Salvatore Pasta a , Giovanni G. Vendramin c , Roberto De Michele a , Francesco Carimi a
a

CNR Istituto di Genetica Vegetale, UOS Palermo, Corso Calatami 414, I-90129 Palermo, Italy
Regione Siciliana Dipartimento Interventi Infrastrutturali per lAgricoltura, Viale Regione Siciliana 4600, I-90145 Palermo, Italy
c
CNR Istituto di Genetica Vegetale, UOS Firenze, via Madonna del Piano 10, I-50019 Sesto Fiorentino - Firenze, Italy
b

a r t i c l e

i n f o

Article history:
Received 24 February 2013
Accepted 10 August 2013
Available online 12 September 2013
Keywords:
Biodiversity
Microsatellites-Simple Sequence Repeat
(SSR)
Plant dispersal
Population dynamics
Habitat-level patterns
Screes

a b s t r a c t
Wild grapevine represents a valuable genetic resource for both future breeding programmes of cultivated
grape and conservation of biological diversity in natural environments. In Sicily, the knowledge of this
species is quite scarce and fragmentary. Therefore, in order to assess the presence and the genetic quality
of wild grapevine in the island, eight populations from different locations were investigated. Their habitats
were characterized and the genetic diversity was measured by microsatellite markers in order to evaluate
possible 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 in
ooded areas. Grapevine populations growing in riparian habitats were characterized by low inter- and
intra-population variability. Conversely, the scree-type population proved to be the most compact and
distinctive, as well as the most genetically isolated. Interestingly, together with other two populations
from the northern mountain range of the island, this scree-type grapevine population was genetically
rather distant from local domestic accessions, suggesting a weak gene exchange with the cultivated
grapevines in Sicily. On the contrary, the other populations showed evidences of probable introgression events, as a result of either gene ow between domestic and wild plants, or of possible secondary
domestication/genetic improving processes, based on the use of native wild material.
2013 Elsevier GmbH. All rights reserved.

Introduction
Grapevine (Vitis vinifera L.) is one of the earliest domesticated
fruits, and economically one of the most important, especially for
its derived products wine and spirits (This et al., 2006). Despite several varieties have been selected during its millennial cultivation,
in modern times extensive diffusion of only a few clones has drastically reduced genetic diversity, thus increasing the risk of epidemic
diseases. With regard to that, the wild relative V. vinifera subsp.
sylvestris (C.C. Gmel.) Hegi could represent a quite valuable genetic
resource for future breeding programmes of cultivated grape, as
well as for conservation of biological diversity in natural environments.
At present time wild grapevine has become rather rare due
to several forms of human disturbance, such as habitat destruction and fragmentation, silvicultural practices, diffusion of exotic
pathogens (e.g. odium, phylloxera, mildew), improper management of natural environment, etc. (Arnold et al., 1998, 2005, 2010;

Corresponding author.
E-mail address: giuseppe.gar@igv.cnr.it (G. Garf).
0367-2530/$ see front matter 2013 Elsevier GmbH. All rights reserved.
http://dx.doi.org/10.1016/j.ora.2013.08.005

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 (usually including less than 10 individuals) still persist (Lacombe et al.,
2003) and it is even reported that in the whole Upper Rhine Valley
only 20 scattered plants nowadays survive (Arnold et al., 2005).
Owing to such critical status of conservation, in the 1980s wild
grapevine was included in the IUCN (International Union for Conservation of Nature) list of endangered European species (Arnold
et al., 2010), and several programmes for its legal protection and
reintroduction have been promoted thereafter in some European
countries (Arnold et al., 2005; 2010). Accordingly, Maul (2004)
emphasized that preservation of wild Vitis germplasm is a priority in the frame of the Council Regulation (EC) 870/2004, and
indicated some main items for its conservation and valorization,
such as assessment of residual sites in the wild, preparation of
geo-referenced plants inventory, propagation of residual populations and ex situ conservation. However, as observed by Zecca
et al. (2010), problems of genetic identity may arise when wild
and domestic plants have grown sympatrically for long periods
of time. Gene ow through natural hybridization events may
result in transfer of new genes into wild populations, with related

G. Garf et al. / Flora 208 (2013) 538548

difculties in taxonomic distinctiveness and the implementation of

efcient conservation strategies.
In mainland Italy, knowledge of this particular taxon is overall
rather conspicuous and extensive data describing its distribution,
conservation, phylogeographic structure and genetic diversity in
several regions are available (Anzani et al., 1990; Arnold et al.,
1998; Grassi et al., 2003a,b, 2006, 2008; Arroyo-Garca et al.,
2006; De Mattia et al., 2008; Zecca et al., 2010). Conversely, information on Sicily is limited to few preliminary data (Collesano,
1998; Mazzola et al., 2011; Bigini et al., 2012) and some oristic and phyto-sociological investigations (e.g. Gussone, 18421845;
Lojacono-Pojero, 18881889; Barbagallo et al., 1979; Marcen et al.,
1985; Brullo and Spampinato, 1991; Tomaselli, 2003; Bazan et al.,
2006; Giardina et al., 2007; Minissale et al., 2008; Pasta et al., 2008).
This is a major gap, especially when we consider the complex and
still debated domestication history of grape (Arroyo-Garca et al.,
2006; Grassi et al., 2003a,b, 2008; De Mattia et al., 2008; Myles
et al., 2011), coupled to the position of Sicily in the centre of the
Mediterranean area, at the crossway of migration routes of peoples
and plants. Furthermore, this region boasts a very ancient and
rich tradition of viticulture practices (Carimi et al., 2010, 2011).
Archeobotanical ndings have shown that grape has been used by
native populations since the Protohistoric Age onward (Collesano,
1998), as documented by macro-remains of wild-type fruits dating
back to the 8th millennium BC from Grotta dellUzzo (NW Sicily:
Costantini, 1989), and by pips ascribed to the 8th century BC from
Morgantina (inner Sicily: Leighton, 1993, 1999), Selinunte and Mt.
Polizzo near Salemi (W Sicily: Stika et al., 2008).
Research on wild grapevine in Sicily could be of valuable concern at both the regional and the international scale, as it could
provide useful information for an optimal conservation strategy
of this rather rare species, and in case contribute also to clarify its domestication history. Moreover, it should allow to set up
the basis for exploiting this germplasm in future breeding programmes for cultivated grapes. In the present paper we analyzed
eight Sicilian populations of V. vinifera subsp. sylvestris sampled in
different locations and habitats. The aim was to assess the presence and distribution patterns of native wild grapevine in the
island, as well as its genetic integrity. Genetic variability was investigated by microsatellite analysis and the possible relationships
between genetic features and habitat diversication and/or geographic provenance are discussed.

Materials and methods

The wild grapevine in Sicily: state of knowledge and relevance
Wild grapevine is considered a characteristic species of Salici
purpureae-Populetea nigrae, the phyto-sociological class which
groups all the riparian wet deciduous woodland and willow
communities of Euro-Siberian and Mediterranean regions (RivasMartnez et al., 2002). This is also true for the Italian territory
(cf. Biondi and Blasi, 2009, report cards concerning the EU habitats 91F0, 92A0 and 92C0, and references therein), where it is
mostly common in riverine forest communities characterized by
pure stands or mixed assemblages of poplars, willows, elms and
ashes thriving on wet uvisols with high water-table (order Populetalia albae) or in the usually ooded willow-wooded and shrubby
pioneer communities of the lowest zone of rivers and streambeds
(order Salicetalia purpureae). In addition, it takes part in the
hygrophilous mantle communities growing along the borders of
water courses (order Prunetalia spinosae, class Rhamno-Prunetea:
Blasi et al., 2002).
Unfortunately there are still gaps and incongruence in the
knowledge of putative wild grapevine distribution in the Italian

539

territory. For example, according to Conti et al. (2005, 2007) V.

vinifera subsp. sylvestris is doubtfully present in Abruzzo and absent
in 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 SE
Sicily, but not in NE Italy, Umbria, Campania and Sardinia.
In Sicily, wild grapevine is mentioned among the locally rare
and threatened species, although it does not gure within the
most recent regional red lists (Conti et al., 1997; Raimondo et al.,
2011). In agreement to its usual habitat, Sicilian populations of wild
grapevine seem to be strictly linked to riparian woodlands dominated by Populus spp., Salix spp. and Platanus orientalis L. Despite
the huge amount of papers concerning the forest and hygrophilous
communities of the island, wild grapevine has been recorded only
in few valleys of rivers, streams and creeks, most of the latter owing within the canyons of the Hyblaean Plateau (Barbagallo et al.,
1979; Brullo and Spampinato, 1991; Tomaselli, 2003; Minissale
et al., 2008; Garf et al., 2011), where already in the rst half of XIX
century Gussone (18421845) mentioned its presence in the vicinity of the village of Cassaro (Anapo River Valley). Additional single
localities are known in the Peloritani Mts. (Brullo and Spampinato,
1991), the Sicani Mts. (Bazan et al., 2006) and the Belice River
watershed (Pasta et al., 2008). In these areas, at least until the end
of XIX century, wild grapevines were also present in mantle and
forest communities, as testied by Lojacono-Pojero (18881889),
who reported it in the hedges of northern Sicily and in the woodlands of Nebrodi Mts. and Mt. Etna. However, the presence of these
populations has not been conrmed in recent times, and during
the last decades wild grapevine seems to have become quite rare
within most of these regional forest communities. In fact, in the
past decades its presence has been reported only for one site in
the Sicani Mts. by Marcen et al. (1985) and for two localities on
the Madonie Mts. (Giardina et al., 2007), while no recent phytosociological record is available neither for the woods dominated by
mesic deciduous broadleaved trees (Brullo et al., 1996, 2000) nor
for 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 mountainous ranges and nature protected areas of Sicily, where natural
environment is still relatively well preserved. The exploration criteria were primarily based on the habitat features, focusing the
interest on damp areas and river/brook banks and taking into
account records from the literature and oral sources.
Considering the strong morphologic resemblance of wild
grapevine with the cultivated one, we tried to reduce as much as
possible the risk that plants deriving from naturalized grapevine
cultivars or rootstocks were mistaken for wild grapevines, although
it is believed that cultivated varieties have a low chance to survive
in the wild (Grassi et al., 2003a). To this aim, the plant sampling
strategy was based on the main differentiating traits used to distinguish wild from domesticated grapevines, as summarized in
Table 1. Moreover, as a general rule we avoided sampling close
to either current vineyards or sites with evidence of past viticulture practices. Exceptions from the former issues were prompted
by unequivocal citations from the literature reporting the occurrence of wild grapevine or, sometimes, by oral information from
native people. In those cases, we opted to evaluate a posteriori the
actual 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 kilometres. Therefore, all the collected plants were arbitrarily grouped
into separate populations depending on the river watershed where

540

G. Garf et al. / Flora 208 (2013) 538548

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

Wild grapevine

Domesticated grapevine

Mating system
Habitat
Berry shape
Trunk
Seeds
Fruit clusters
Leaves

Dioecious
Humid soils
Small, round or oblated
Often branches, slender, bark separated in very long thin strips
Small, rounded body, high width/length ratio (>0.70)
Small, globular to conical, irregular set, berry maturity variable in cluster
Small, usually deeply three-lobed. Petioles short and slender, dull aspects

Hermaphrodite
Dry habitats
Large and elongated
Thick bark separates in wider and more-coherent strips
Large, pyriform body, lower width/length ratio (<0.60)
Large, elongated, compact to well-tted, berry, uniform in maturity
Large, many entire or with shallow sinuses, petiole thick, glabrous to
drowny

each group was found. Accordingly, we distinguished 8 populations, overall including a total of 81 individuals (Table 2).
For each population we recorded the general vegetation patterns and the dominant species of the forest habitat, in addition to
the main topographical and hydraulic features of the stream ows.
Evidences of present or past land use and anthropic disturbances
were also described.
For all sampled plants, we annotated the geographical coordinates and altitude through a sub-metric precision GPS device
(model Trimble GeoXT; Trimble, Sunnyvale, CA, USA); the geodesic
system we used was WGS84. Moreover, we identied some
attributes such as: plant sex (if assignable in the eld), habit (climbing or creeping), position of the crown (canopy, overshadowed),
bearing tree species (if any), micro-habitat (topography, canopy
cover), trunk class size (less than 2 m long, from 2 to 10 m, more
than 10 m), and health state.
In order to evaluate more correctly the true genotypic identity of
the wild plants and to highlight any possible relationships with cultivated grapevines, ve most common Sicilian traditional cultivars
(namely Catarratto, Nero dAvola, Inzolia, Perricone and Zibibbo)
were used for comparison. Plant material was obtained from the
germplasm collection elds of the Institute of Plant Genetics CNR,
Palermo (Italy).

DNA extraction and microsatellite analysis

Total genomic DNA was extracted from young, fresh leaves collected in the eld, ground into ne powder with liquid nitrogen and
stored at 80 C until use. The extraction was carried out following
the CTAB method (Doyle and Doyle, 1990) and DNA was quantied
on 1% agarose gels.
Samples were analyzed at six microsatellite loci (SSR Simple Sequence Repeat), as often performed in similar works on wild
grapevine (e.g., Grassi et al., 2003b; Arrigo and Arnold, 2007; Barth
et al., 2009; Cunha et al., 2010). Therefore, the set of microsatellite
markers, i.e. VVS2 (Thomas and Scott, 1993), VVMD5, VVMD7 and
VVMD27 (Bowers et al., 1996), VrZAG62, and VrZAG79 (Sefc et al.,
1999), was selected as proposed by the GENRES 081 Project (European Vitis Database: www.genres.de/vitis/vitis.htm) to allow the
comparison of the resulting proles with the available database.
PCR amplication was carried out using the Qiagen multiplex
PCR kit (Quiagen, Hilden, Germany) with the following conditions:
15 min at 95 C (HotStar Taq activation step) followed by 35 cycles
consisting of 30 s at 94 C (denaturation), 90 s at 5056 C (annealing), 60 s at 72 C (extension) and a nal step for 30 min at 60 C. One
of the primers of each pair was uorescently labelled with FAM,
JOE or TAMRA. Each sample was amplied at least twice to correct possible mistyping or amplication errors. PCR products were
size-separated by capillary electrophoresis performed on a genetic
automatic sequencer (ABI Prism 3130; Applied Biosystems, Foster
City, CA, USA).

Data analysis
Allele size was estimated by comparing the fragment peaks with
the internal size standard, using the default method for band calling
with microsatellite and the expected repeat size. Electropherograms were veried 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 ) and
expected (He ) heterozygosity (Nei, 1987); microsatellite screening
ability was also based on the probability of identity (PI; Paetkau
et al., 1995) and the polymorphic information content (Weber,
1990), with the most effective microsatellite having high values
of Ho and polymorphic information content, and low PI.
The genetic diversity within each group was estimated by
comparing number of alleles per locus (Na ), effective number of
alleles (Ne ), number of private alleles (Npa ), allelic richness (AR),
observed (Ho ) and expected (He ) heterozygosity (Nei, 1987) using
GeneALex 6.5 and FSTAT version 2.9.3.2 (Goudet, 2002) software.
The intra-populational genetic variability was also analyzed by
calculating the inbreeding coefcient (FIS ; Weir and Cockerham,
1984) using the FSTAT software. Each locus and population was
tested for deviations from Hardy-Weinberg equilibrium expectations with exact tests using Genepop v. 3.4 (Raymond and
Rousset, 1995). Genepop was used to examine linkage disequilibrium among all pairs of loci within each wild populations. All
Genepop analyses were performed using the following parameters:
dememorization = 10000, number of batches = 100, and number
of iterations/batch = 1000. Signicant positive FIS values indicate
inbreeding (excess of homozygotes) or undetected null alleles,
whereas signicant negative values indicate excess of heterozygosity and then low inbreeding. Near zero values are expected under
random mating.
ARLEQUIN v. 3.5.1.3 (Excofer and Lischer, 2010) software was
employed to estimate the xation index FST (Weir and Cockerham,
1984). FST was used to measure the divergence among the investigated grapevine populations. The signicance of the differentiation
between pairs of populations was tested using permutation procedures (10000 replicates).
A cluster analysis of wild and domesticated populations was carried out using the DA genetic distance (Nei et al., 1983) according
to the UPGMA (Unweighted Pair-Group Method with Arithmetic
mean) algorithm. A dendrogram was generated using the software
Populations 1.2.31 (Langella, 2002) and visualized with Treeview
1.6.6 (Page, 1996).
To identify the number of genetic groups in the wild populations
and to investigate their relationships with domesticated Sicilian
cultivars, 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 the
simulation study by Evanno et al. (2005), which proposed an ad hoc

Table 2
Population sizes and habitat conditions of the investigated wild grapevine occurrences in Sicily.
No. of
populations

Population name
(code)

Total no. of trees and (in

brackets) no. of trees
with length >10 m

Habitat

Vegetation patterns and

dominant trees/shrubs

Madonie Mts. (Palermo)

Castelbuono (CSB)

9 (2)

Temporary river banks

Nebrodi Mts. (Messina)

Stretta di Longi (LON)

7 (3)

Permanent river banks

Sicani Mts. (Agrigento)

Fiume Sosio (SOS)

13 (3)

Permanent river banks

Belice Valley (Trapani)

Riserva Zangara (ZAN)

8 (3)

Permanent river banks

Torto Valley (Palermo)

Riserva Boschi Favara e

Granza (FAV)

17 (2)

Scree-type deposits

Riserva Pantalica e
Valle Anapo (PAN)

18 (0)

Permanent river banks

Fiume Manghisi
(MAN)

5 (0)

Permanent river banks

Cava Sturia
(CST)

4 (4)

Temporary river banks

Riparian forest, canopy cover

100%, h = 58 m. Salix pedicellata,
Populus nigra, Laurus nobilis,
Quercus ilex, Clematis cirrhosa,
Hedera helix, Smilax aspera
Riparian forest, canopy cover
100%, h = 810 m. Salix
pedicellata, Ficus carica, Fraxinus
angustifolia, Celtis australis,
Clematis cirrhosa, Hedera helix,
Rubus sp.
Riparian forest, canopy cover
100%, h = 815 m.
Salix pedicellata, Quercus ilex, Q.
virgiliana, Crataegus monogyna,
Prunus spinosa, Pistacia
terebinthus, Viburnum tinus,
Rhamnus alaternus, Hedera helix
Riparian forest, canopy cover
70%, h = 812 m. Ulmus canescens,
Salix pedicellata, Populus nigra,
Ficus carica, Tamarix sp., Nerium
oleander, Myrtus communis,
Hedera helix
Pioneer communities, canopy
cover 40%, h = 24 m.
Ficus carica, Rubus sp., Clematis
cirrhosa, Quercus virgiliana (along
the external border)
Riparian forest, canopy cover
80%, h = 1015 m. Salix
pedicellata, Populus nigra, Ficus
carica, Platanus orientalis, Quercus
ilex, Fraxinus ornus, Rhamnus
alaternus, Nerium oleander,
Clematis cirrhosa
Riparian forest, canopy cover
80%, h = 812 m. Salix pedicellata,
Ficus carica, Platanus orientalis,
Quercus ilex, Clematis cirrhosa,
Hedera helix
Maquis, canopy cover 100%,
h = 68 m. Quercus ilex, Q.
virgiliana, Ficus carica, Olea
europaea var. sylvestris, Fraxinus
ornus, Ailanthus altissima, Pistacia
lentiscus, P. terebinthus, Rhamnus
alaternus, Phillyrea latifolia,
Smilax aspera, Hedera helix

Iblei Mts.
(Siracusa)

Total

Anthropic evidences

Available literature
containing information
on the presence of wild
grapevines

Sporadic drafted, aged

olive trees along the
track to the sampling
area

Giardina et al. (2007)

and Mazzola et al.
(2011)

Grazing disturbance
evidences

Lojacono-Pojero
(18881889)

Sporadic small patches

of abandoned elds
along the track to the
sampling area

Marcen et al. (1985)

and Bazan et al. (2006)

Large vineyard in the

surroundings;
Small abandoned olive
patches inside the
sampling area

Pasta et al. (2008)

Sporadic small patches

of abandoned elds
along the track to the
sampling area

Collesano (1998)

Small cultivated
patches inside the
sampling area;
Large abandoned and
active cultivations in
the surroundings

Gussone (18421845);
Barbagallo et al.
(1979); Brullo &
Spampinato (1991);
Minissale et al. (2008);
Tomaselli (2003)

G. Garf et al. / Flora 208 (2013) 538548

Geographical districts
(Province)

Abandoned and active

cultivations in the
surroundings

Abandoned and active

cultivations in the
surroundings

81

h = height.
541

542

G. Garf et al. / Flora 208 (2013) 538548

Fig. 1. Geographical location of the investigated populations.

statistic, K. For each K (ranging from 1 to 10), 20 independent runs

(100000 burn-in, 1000000 Marchov Chain Monte Carlo) were carried out. All runs were performed with the admixture model adding
the sampling information to the input data (LocData = 1) according to Hubisz et al. (2009). The 20 runs were averaged using the
software CLUMPP (CLUster Matching and Permutation Programme;
Jakobsson and Rosenberg, 2007), and shown in histograms using
the programme Distruct (Rosenberg, 2004).

Results
Habitat-level patterns and individual features: some
elements of peculiarity
The eight sampled populations were scattered throughout Sicily
(Fig. 1 and Table 2): three populations were from the northern
mountain ranges, two from the southwestern district and three
from the southeastern hills. No population was found in the cropdominated landscapes of the inner provinces of the island. All
populations are located within protected areas or Natura 2000 sites,
except populations CST and ZAN (for name coding, see Table 2). CST
and ZAN populations were both within small isolated patches of
natural vegetation surrounded by cultivated areas, especially cereal
crops (CST) and olive groves and vineyards (ZAN). Minor evidences
of ancient cultivations were also noticed in the surveyed areas for
populations SOS, CSB and PAN, but not in the close proximity of the
sampled plants.
The altitudinal gradient was between 300 and 400 m a.s.l. for
most populations. The highest population was FAV, ranging from
880 to 904 m a.s.l., whereas the lowest was ZAN, situated almost
entirely at about 120 m a.s.l.
The habitat conditions of the investigated sites were rather different (Table 2). With the exception of FAV, all the other populations
grew along streams owing inside gullies or open valleys with narrow or wide bottom. Plants were rooted in close proximity to the
stream beds in narrow-bottom valleys, and up to some ten metres
apart in the other locations. The streams were usually permanent;

only CSB grew along a temporary rivulet, whereas almost all plants
from CST were found inside a nearly constantly dry river bed. FAV
was the only population not restricted to stream banks. It resided
in a wide, humid niche few thousand-square metres large, lying
on a north-facing forested slope and surmounted by sub-vertical
rocky cliffs, delimited at their base by scree-type stony deposits
with coarse blocks of various size, among which vine plants were
thriving.
In all sites, the forest cover was usually represented by thick
riparian vegetation, 815 m high, dominated by Salix pedicellata
Desf., Populus nigra L., Ficus carica L., Nerium oleander L. and some
climbing vines such as Clematis cirrhosa L. and Hedera helix L. in
addition to wild grapevine. Platanus orientalis L. was a typical element in two of the Iblei Mts. populations, whereas Ulmus canescens
Melville was rather abundant only at ZAN; moreover, Quercus ilex
L. and Fraxinus ornus L. were often observed, too. Together with Q.
virgiliana (Ten.) Ten. and several Mediterranean evergreen sclerophyllous shrubs, they characterized the maquis-type forest habitat
of CST. Once again, FAV was quite peculiar, since the forest habitat
was represented by sparse pioneer communities with Ficus carica, Rubus ulmifolius and Clematis cirrhosa, whose canopy never
exceeded the height of 4 m.
The number of sampled individuals per population ranged from
4 to 18. Their intra-population spatial distribution was quite diversied, varying from clustered (FAV and, at less extent, ZAN) to
very scattered (PAN). In the cluster pattern, the maximum interindividual distance did not exceed 50 m, whereas in the scattered
pattern the farthest plants were spaced up to 4.6 km. The majority of the plants collected in the wild showed the most relevant
distinctive 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 morphological
characteristics 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 recorded
in most populations, it was not possible to identify the gender of all
the sampled individuals due to difculties in detecting owers or
to inaccessibility due to their high crown. Overall, a large amount

G. Garf et al. / Flora 208 (2013) 538548

543

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 distinguish
among genotypes (polymorphic information content) varied from
0.723 to 0.781; the probability of identity (PI) between two genotypes harbouring different alleles at the analyzed loci ranged from
0.053 to 0.076, while the calculated value for all loci was relatively low (4.98 108 ). The VVMD27 locus resulted the best for
discriminating among our samples (polymorphic information content = 0.781 and PI = 0.053).

Intra- and inter-population diversity

Fig. 2. Large creeping grapevine at FAV. The lush mosses indicate a notable amount
of moisture in the habitat.

of vines measured between two and 10 m. Moreover, all populations, except MAN and PAN, contained plants of notable size, with
a trunk up to 20 m long. The biggest plants were found at LON (base
diameter 20 cm; length about 22 m) and FAV (base diameter 18 cm;
length about 20 m). At CST all sampled plants were more than 10 m
long. Seedlings were almost absent, except at LON and CSB where
just very few saplings were observed.
The habit was climbing for nearly all plants and the crown of the
biggest plants usually reached the forest canopy; only at FAV creeping plants were prevailing, including the most sizeable individuals,
too (Fig. 2). Support trees belonged to many species, without special
preferences between deciduous (e.g. willows, poplars, ashes, gs)
or evergreen (e.g. holm oaks). Moreover, the shortest vines climbed
even on Mediterranean sclerophyllous shrubs such as Phillyrea latifolia L. and Rhamnus alaternus L. Almost all grape plants looked
healthy, without any evidence of typical grapevine pathogens such
as odium, phylloxera or mildew. At SOS and FAV, one large and
three small plants, respectively, looked rather stunted and exhibited a reduced crown, probably suffering for overshadowing or
nutrient competition.
Nuclear microsatellite diversity
The 81 samples analyzed at 6 microsatellite loci showed 72 different multi-locus proles. The remaining 9 individuals without
any private loci probably originated from clonal duplication. A total
of 59 alleles were detected with an average of 9.8 alleles for locus
(ranging from 8 to 12).
The most variable loci were VVMD7 and VrZAG62, with 12 and
11 alleles, respectively. Twenty-ve out of 59 alleles had a frequency lower than 5% (data not shown) while for VrZAG79 there
was a predominant allele (248 bp) with a frequency around 40%.
The Ho values were quite different among loci (from 0.444 to 0.931);

The genetic diversity estimates for each wild population are

reported 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 ) heterozygosity
varied 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), conrming a rather conspicuous
rate of diversity in the whole germplasm collection.
We also tested for HardyWeinberg predictions in order to evaluate the amount of inbreeding in each population. The FIS values
differed widely between loci in each population, ranging from 0.271
(CST) to 0.243 (FAV). FAV was also the only population with a
signicant value at P < 0.05. The pairwise genetic differentiation
(FST ) (Table 5) was highest between FAV and the other populations, with maximums in respect of CSB (0.360) and LON (0.365).
On the contrary, PAN showed the lowest mean value, with minimums in respect of CST (0.045) and MAN (0.006), the other two
populations belonging to the same geographic district. When wild
grapevine 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 values where signicantly greater than 0 (P < 0.05) except for the three
pairs PAN-MAN, ZAN-SOS and MAN-DOM (Table 5).
The cluster analysis of the populations did not match with their
geographic origin (Fig. 3). According to the genetic distance, 3 clusters (named A, B and C) can be clearly distinguished. Cluster A
included CSB and LON, two populations in the northernmost and
best preserved forest areas; cluster B comprised FAV, the only screetype population; nally, cluster C included the remaining wild
populations as well as the group of cultivated varieties. A similar
pattern was also obtained in cluster analysis using all individual
genotypes instead of populations (data not shown), although FAV
disclosed a singular structure, as it was the only population containing 3 sub-groups (ranging from 2 to 6 plants) with identical
genotype, most probably of clonal origin.
The most likely number of genetic groups (K) of wild grapevines
and their relationships with cultivated germplasm was identied
by Structure software. A preliminary analysis was carried out with
no prior locus information assumption. Results showed a peak at
K = 3, but no clear distinction emerged neither among wild groups
nor between wild and cultivated accessions. Only the FAV population showed a homogeneous prole that differed from all the
other accessions. In order to clarify the distribution of all genotypes across different groups, an additional analysis was performed
providing geographic information to the input data in Structure
analysis according to Pritchard (2010) (cf. Material and methods).
This approach maintained the K value to 3, conrming the partition
of the investigated germplasm into three most probable genetic
pools (Fig. 4). The FAV population was the most distinguished
group, whilst individuals from ZAN, CST, MAN and PAN clustered
with the domestic accessions. CSB, LON and, partially, SOS, mostly
showed a genetic prole different from both FAV and the cultivated
pool.

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G. Garf et al. / Flora 208 (2013) 538548

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

Na

Allele size range (bp)

He

Ho

PI

PIC

VVS2
VVMD5
VVMD7
VVMD27
VrZAG62
VrZAG79
Mean
All Loci

9
9
12
8
11
10
9.8
59

127151
216240
231265
176190
171199
228256

0.805
0.810
0.809
0.828
0.822
0.762
0.806

0.931
0.722
0.467
0.875
0.583
0.444
0.670

0.0639
0.0611
0.0600
0.0528
0.0530
0.0759

4.98E-08

0.7533
0.7601
0.7619
0.7815
0.7795
0.7235
0.7599

Na : number of alleles per locus; He : expected heterozygosity; Ho : observed heterozygosity; PI: probability of identity; PIC: polymorphic information content.
Table 4
Genetic diversity estimates in wild grapevine populations (in brackets the average value per locus).
Population

Na

Ne

Npa

Ho

He

AR

FIS

CSB
LON
PAN
MAN
CST
SOS
ZAN
FAV

27 (4.5)
21 (3.5)
43 (7.2)
27 (4.5)
20 (3.3)
36 (6.0)
28 (4.7)
20 (3.3)

17.2 (2.9)
13.4 (2.2)
25.9 (4.3)
20.0 (3.4)
17.0 (2.8)
20.1 (3.4)
21.3 (3.5)
16.1 (2.7)

1 (0.17)
1 (0.17)
4 (0.67)
2 (0.33)
1 (0.17)
1 (0.17)
1 (0.17)
1 (0.17)

0.556
0.500
0.778
0.700
0.542
0.667
0.708
0.708

0.606
0.517
0.761
0.607
0.625
0.691
0.699
0.543

3.4
3.0
4.3
4.0
3.3
3.8
3.9
2.8

0.141
0.110
0.007
0.043
0.271
0.076
0.054
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 coefcient;
AR: average allelic richness.
*Signicant at the P < 0.05 level.

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

CSB
LON
PAN
MAN
SOS
CST
ZAN
FAV
DOM

CSB

0.205*
0.123*
0.150*
0.205*
0.070*
0.232*
0.360*
0.145*

LON

PAN

MAN

SOS

CST

ZAN

FAV

DOM

0.152*
0.186*
0.223*
0.121*
0.247*
0.365*
0.185*

0.006
0.115*
0.045*
0.088*
0.187*
0.037*

0.106*
0.050*
0.117*
0.181*
0.000

0.127*
0.073
0.287*
0.103*

0.113*
0.249*
0.092*

0.253*
0.108*

0.184*

*Signicant at the P < 0.05 level.

Discussion
Habitat patterns and populations dynamics of the Sicilian
wild grapevine
Despite millennia of human occupation and land use, Sicily
still maintains a number of natural areas hosting valuable remnants of plant biodiversity such as wild grapevine, and the present

investigation unexpectedly revealed a rather conspicuous abundance of putative wild grape in many and diversied parts of the
island.
Consistently with most citations from the literature (e.g., Ocete
et al., 1999; Arrigo and Arnold, 2007), all the investigated Sicilian
populations appeared to be related to damp conditions. On the
other hand, it is known from the literature (Laguna Lumbreras,
2003; Arnold et al., 2005) that differences in moisture availability

Fig. 3. UPGMA dendrogram constructed using the DA genetic distance (Nei et al., 1983).

G. Garf et al. / Flora 208 (2013) 538548

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

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

and/or watercourse regime may notably affect the distribution and

dynamics of grapevine populations, especially considering that
ooded environments prevent the diffusion of the root-hosted
homoptera Phylloxera (syn. Dactylosphaera vastatrix), therefore
favouring the persistence of wild grapevine populations along
stream banks.
In Sicily, the water ow of permanent large streams, as at PAN,
MAN and SOS, most probably facilitated the long-distance dispersion of propagation material, like seeds or broken branches,
promoting the establishment of new grape populations along the
alluvial network (Arrigo and Arnold, 2007; Arnold et al., 2010).
In these cases, plants were rooted on the stream banks even at
some ten metres afar from the river edge. On the contrary, the
quite ephemeral regime of the CST rivulet allowed the growth of
grapevines along the bottom of the gully, where underground water
resources were crucial for its survival. The population from FAV is
remarkable under many aspects. The habitat type here is devoid
of streams, and moisture is probably compensated by both elevation (about 900 m a.s.l.) and the northern aspect of the slope,
as suggested by the extreme abundance and luxuriance of moss
cover (Fig. 2). Moreover, as for the ooded areas, scree habitats are
also mentioned as unfavourable habitat to Phylloxera (Arrigo and
Arnold, 2007).
The scree-type habitat of FAV and the related ecological conditions likely also affect intra- and inter-population spread and
connectivity. Screes are isolated environments and, lacking the
propagation role of hydraulic dynamics, in this context plants dispersal more strictly depends on interactions between faunal and
plant communities. As described for various Mediterranean habitats (Herrera, 1989; Matas et al., 2010) and in the oodplain forest
of the Upper Seine Valley (Arnold et al., 2010), wild grape dispersal
could be favoured by mammals (e.g. carnivores like red fox, stone
marten, etc.) and several birds (mostly frugivorous passerines).
Bird communities in riparian woodlands are usually richer than
in non-riverine conifer and broadleaved forests (Dronneau, 2007),
probably due to the higher architectural complexity of the riparian
forests resulting from the specic dynamics of riverside habitats.
Diversity of woody plant species, high vertical stratication, horizontal heterogeneity, abundance of large vines and vigour of plant
growth are the most striking features of these habitats, offering to
avian communities a much wider spectrum of refuges and trophic
resources. Many birds use riparian corridors as migration pathways, thereby connecting different areas (Dcamps et al., 1987).
As a result, many plants are dispersed by birds (especially Turdidae) migrating along the riparian zones (van Dorp and Kalkhoven,
1988). Besides, plant movements or exchanges may increase at
nodes where riparian and other corridors, such as roads, railways
and hedgerows, intersect (Riffel and Gutzwiller, 1996). Overall,
in the present study-case it is suggested that the combination of
river dynamics and zoochory favours gene ow of wild grapevine
germplasm among all the riparian populations, whereas the screetype population FAV is maintained ecologically isolated. Likewise,

545

the small area size of population FAV might also have contributed
to its divergence.
As recorded in many central European countries (cf. Arnold et al.,
1998, 2005, 2010; Lacombe et al., 2003) also silvicultural practices might have had a detrimental role in conservation of wild
grapevine populations. But in Sicilian populations, forest management history, as well, can account for the relative abundance of
grape sizeable plants. As a rule, large-scale disturbances, especially
clear-cuttings, can notably inuence diffusion and development of
climbing vines (Schnitzer and Bongers, 2002). Silvicultural activities either create clearances which favour their establishment,
or conversely, especially in short rotation forest systems, can
indirectly control their sizeable growth, since vines are periodically removed with timber trees. In the investigated sites, even
the best preserved forest areas are actually aged coppices, no
longer exploited for fuel wood and charcoal production since at
least the 1960s. As observed for ivy vines at the Monte Carcaci
Nature Reserve (Garf and Ficarrotta, 2003), stops in clear-cuttings
involved the arising of vegetation dynamics that led to changes
in oristic and, above all, in structural features of forest stands.
Accordingly, this improved the chances that grapevine plants might
age undisturbed together with their trellis trees and grow remarkably up to the forest canopy. In addition, the scarcity of grapevine
seedlings can be similarly explained by the low-light availability in
the understory owed to the thick canopy covering the sites (Arnold
et al., 2010). A particular situation occurs only at FAV, where a
proper forest canopy is not present and the prevailing creeping
habit of grapevine there could notably enhance the probability of
clonal propagation. All these issues must be carefully evaluated in
the denition of a proper strategy of in situ conservation.
Genetic structure of populations: inferences to their integrity
The complexity deriving from the historical and extensive cultivation of its domesticated relative in Sicily can represent a potential
major threat to the genetic integrity of indigenous wild grapevine
populations. As a matter of fact, none of the sampled sites could
actually be considered completely exempted from evidences of
human activities, especially ZAN and all the populations from
the Iblei district. Accordingly, more or less intensive introgression
could be expected between domestic and wild germplasm.
Molecular analyses showed that the six microsatellites investigated were able to discriminate the sampled plants (Arrigo and
Arnold, 2007; Grassi et al., 2003b, 2008; Cunha et al., 2010). High
level of polymorphic information content along with lower PI
strengthened the effectiveness of microsatellites in distinguishing
among genotypes. However, the PI assessing was higher than values at which a microsatellite is considered hyper-polymorphic in
grapevine (Sefc et al., 2001). A rather valuable gene diversity was
detected in wild grapevine populations in spite of their small size.
This can be partly related to the mating system of this dioecious and
outbreeding plant (Grassi et al., 2003b). On the other hand, mean
FIS values indicated that, among the populations analyzed, only FAV
deviated from HardyWeinberg equilibrium, showing an excess of
heterozygosity. Single or reiterated events of selection, as well as
migration and/or mutation could be invoked to explain such a situation, but current data do not allow us to be biased towards one or
another factor. In general, the low allelic richness within wild populations provides evidence for a potential genetic bottleneck effect.
Allelic richness, being more heavily inuenced by rare alleles than
expected heterozygosity, is commonly regarded as a more relevant
criterion for measuring loss of diversity due to genetic bottlenecks
(Nei et al., 1975).
The observed pattern was also supported by the differentiation estimates. According to the FST values, FAV appears as the
most differentiated population among all the investigated ones,

546

G. Garf et al. / Flora 208 (2013) 538548

with the greatest distance with respect to CSB and LON. This result
is quite consistent with the assumptions about habitat patterns
and connectivity discussed above. Accordingly, the most effective
connecting role of both uvial dynamics and birds communities
support the idea of a most intense gene ow within the three populations from the Iblei district (PAN, MAN and CST), which showed
the lowest FST by far.
When compared to the domestic accessions, interestingly FAV
together with LON appear also the populations genetically most
distant from them, highlighting a most probable weak gene
exchange with the local cultivated grapevines considered in this
study. On the contrary, low FST values between domestic varieties and wild provenances from PAN and, at less extent, CST,
MAN and ZAN suggest that past introgression events could have
occurred. This hypothesis appears likely if we consider that the
PAN area, corresponding to the current Nature Reserve of Pantalica e Valle dellAnapo, represents the centre of development of
ancient civilizations dating back to the Bronze Age (ca. XIII century
BC: Leighton, 1999) that expanded to the whole Iblei Mts region.
The segregation of FAV, as well as the weak relationships of
LON and CSB with all the other populations, were conrmed by the
UPGMA cluster analysis and the results obtained from Structure at
K = 3. Overall, the comparative analysis of results from the different
approaches prompts some speculations about the genetic distinctiveness of the investigated germplasm. Populations in relatively
pristine forest areas, such as CSB and LON, most probably could be
retained as the closest to pure wild grape lineages. FAV, although
different from CSB and LON in the Structures data representation,
could also be referred to wild-like genotype, as supported by FST
values. Its uniqueness can be possibly related to its geographic and
ecological isolation that strongly limited gene ow with the other
populations. Consequently, it could represent one of the most interesting germplasm pools among all the investigated ones, although
such an assumption needs to be conrmed by comparison with a
higher number of Sicilian cultivated varieties.
The situation of the other populations is less straightforward.
Two opposite hypotheses can explain our results. ZAN, PAN, MAN
and CST may have largely resulted from introgression events
occurred during the last millennia. This is rather likely for the
Ibleis district populations, where the land has a long history of
anthropic use, as well as for ZAN, still lying within a territory
of renowned ancient viticulture. In these areas the current wild
populations could be descendants of several generations of interbreeding, due to pollen ow between domestic and wild plants,
with consequent partial loss of genetic attributes by the native wild
gene pool (Di Vecchi-Staraz et al., 2009). A different hypothesis
assumes that the most ancient Sicilian cultivated varieties derive
from one or more events of secondary domestication or genetic
improving, based on the use of indigenous wild germplasm. This
scenario seems supported by several evidences on the existence of
secondary domestication centres in the west Mediterranean (Grassi
et al., 2003a,b; Cunha et al., 2010; Zecca et al., 2012; De Andrs et al.,
2012), but additional investigation is needed to corroborate such
an assumption.

Conclusions
The present research represents the rst most comprehensive contribution to the knowledge of Sicilian wild grapevine
germplasm. At rst, results of the exploration campaign suggested
that the actual richness of this species in Sicily is much more conspicuous than expected, so the eight surveyed populations are
but a small sample of the total plant stock in the island. Moreover, data analyses provided some valuable indications about the
genetic quality of the plant material and the implications with the

habitat features, whose signicance could go beyond the narrow

local concern. Overall, most of the investigated populations showed
a fair genetic diversity that somehow seems to be depending on
the habitats functional processes and the level of their ecological connectivity. On the other side, the genetic afnities that some
populations shared with local domestic varieties triggered intriguing assumptions that could assign to Sicily a role of concern in the
history of grape domestication, whereas the most singular populations should be accounted as potential sources of precious gene
material for future programmes of genetic improving of current
cultivars.
Anyway, more accurate investigations must be still extended
to several aspects. For instance, a comparison with a larger panel
of Sicilian and international grapevine cultivars, could notably add
information on the actual nature of the local V. sylvestris gene pool
and the intensity of possible introgression events between wild and
cultivated grapevines. Besides, with regard to territory exploration,
more surveys must be carried out and extended to a variety of habitats other than riverbanks, especially including screes or marshes,
wherein the existence of additional distinctive genetic diversity can
be assumed.
In the conservation perspective several aspects must be taken
into account. Agricultural policy of recent years has involved the
increase of vineyard areas in many parts of the region. This could
enhance the risk for genetic integrity of wild grape, due to the
potential increase of gene ow from domestic to wild germplasm
(Zecca et al., 2010). Only the most isolated populations or those
growing in nature protected areas could possibly be retained as in
relatively safe conditions. But considering the species dynamics
(e.g. poor regeneration by sexual reproduction, dispersion mechanisms) coupled to the current situation of general lack/improper
forest management in Sicily, in situ conservation should not disregard a monitoring activity of the most prominent populations,
and the implementation of assisting measures in case of plants dieback or decline. In any case, ex situ conservation always retains a
major role in order to preserve the most valuable genotypes, so that
installation of germplasm collection elds in the region is highly
advisable.
Acknowledgements
We are very grateful to Paolo Uccello, Giuseppe Clementi,
Giuseppe Traina, Giovanni Giardina and Pietro Zangh for eld
assistance and for providing personal data on the exact location
of several investigated wild grapevine populations. The study was
supported by funding of PSR 20072013, Sottomisura 214/2
Preservazione della biodiversit: Centri pubblici di conservazione.
Progetto DDS n. 1264 Recupero, conservazione e valorizzazione
del germoplasma di vitigni minori (Vitis vinifera ssp. vinifera) e vite
selvatica (Vitis vinifera ssp. sylvestris) in Sicilia.
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