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Phylogeographical structure and conservation genetics of wild grapevine. F. Grassi1,*, M. Labra2, S. Imazio3, R. Ocete Rubio4, O. Failla3, A. Scienza3 & F.
Conservation Genetics (2006) 7:837–845 DOI 10.1007/s10592-006-9118-9

 Springer 2006

Phylogeographical structure and conservation genetics of wild grapevine F. Grassi1,*, M. Labra2, S. Imazio3, R. Ocete Rubio4, O. Failla3, A. Scienza3 & F. Sala1,5 1

Botanical Garden, Department of Biology, University of Milan, via Celoria 26, 20133, Milan, Italy; Department of Environmental Science, University of Milano-Bicocca, P.zza della Scienza 1, 20126, Milan, Italy; 3Department of Crop Sciences, University of Milan, via Celoria, 2-201233, Milano, Italy; 4Laboratory of Applied Zoology, University of Sevilla, Avda Reina Mercedes, 641012, Sevilla, Spain; 5IBF, CNR via Celoria 26, 20133, Milan, Italy (*Corresponding author: Phone: +39-02-5031-4818; Fax: +39-02-50314764; E-mail: [email protected]) 2

Received 20 May 2005; accepted 3 January 2006

Key words: biodiversity, chloroplast microsatellite, conservation, phylogeography, populations, Vitis vinifera subsp. silvestris

Abstract The distribution of Vitis vinifera subsp. silvestris, the wild grapevine subspecies of Vitis vinifera L., has been dramatically reduced in its major sites of diffusion, at first by the spread, over the last 150 years, of pathogens from North America and, more recently, with fragmentation of habitat and disbranching by humans. In this work, 418 wild grapevine samples, belonging to 78 populations, were collected in their main Mediterranean distribution areas, including the Caucasus area, and the extent of their genetic variability evaluated by analysing plastid microsatellite DNA polymorphism. Results show low haplotype diversity value, with five haplotypes detected within the analysed populations. The highest within-population haplotypic diversity, with the presence of all five detected haplotypes, was found in the Caucasus regions and in the central regions of Italy. The distribution of all detected haplotypes suggests the Caucasian region as the possible center of origin of Vitis vinifera subsp. silvestris. A principal plastid lineage was found to be fixed in several locations, in the Northernmost European countries and in the Southern island of Sardinia. These results draw attention to two different refugium sites in the Mediterranean basin and suggest that conservation priority should be given to grapevine populations still preserved in hotspots of these regions.

Introduction Vitis vinifera subsp. silvestris is the wild subspecies of V. vinifera L., which includes the largely cultivated Vitis vinifera subsp. vinifera subspecies. The species is anemophilous, disperses seeds through birds and has a current distribution range from the South Atlantic coast of Europe to the Western Himalaya, where it persists at altitudes from sea level up to 900–1000 m of elevation (Hegi 1925). In former centuries, wild grapevine colonised a broad range of habitats and soils in the Mediterranean area and in a few sites in Central Europe (Figure 1). However, now-a-days it is only found

in permanently flooded areas (Anzani et al. 1990). In fact, the distribution of the wild grapevine has been dramatically reduced over the last 150 years, with the spread of pathogens from North America (phylloxera, oidium, mildew). Currently, other events impose further threats to its biodiversity; these include fragmentation of habitats, essentially due to intensive river management, and forest cutting and removal of other trees, on which wild grapevine frequently climb. Wild grapevines represent a unique, invaluable genetic resource for cultivated grapevines (Negrul 1938). Their disappearance from their natural habitat would be an irreversible loss for breeding

838

Figure 1. Map showing the distribution ranges of wild grapevine in the Mediterranean basin.

programs and for the environment. Recent studies of the residual wild grapevine sites in Europe warn that wild grapevine may be on the brink of extinction (Arnold 1998). In the 1980s this subspecies was added to the IUCN List of endangered European plants. Since 2000, it has been declared a critically endangered subspecies and is strictly protected in France (Lacombe et al. 2002), in the Czech Republic (Holub and Procha´zka 2000), in Spain (Ocete et al. 1999) and in Italy (Grassi et al. 2003a). Wild and cultivated grapevine plants have already been analysed at the nuclear microsatellite DNA level to map genes, as well as to catalogue biodiversity for evolutionary and conservation studies (Labra et al. 2002a; Rossetto et al. 2002; Kozjak et al. 2003; Grassi et al. 2003b; This et al. 2004). Molecular markers based on plastid DNA (cpDNA) analysis have been shown to be a superior tool to reconstruct species history (King and Ferris 1998), since cpDNA is characterised by a low mutation rate and no recombination. Furthermore, cpDNA analysis is more revealing in studies on genotype diffusion through seeds since organelle genomes, both plastid and mitochondria, are maternally inherited in most Angiosperms (Dumolin-Lape`gue et al. 1995). In particular, the discovery of polymorphic microsatellites in cpDNA, which feature variable numbers of mononucleotide repeats, provides novel opportunities to analyse the genetic structure of the population and address phylogeographical issues

in plant species (Provan et al. 1999). This has already been shown in the case of diverse plant species (Dumolin-Lape`gue et al. 1997; Marchelli et al. 1998; Fineschi et al. 2000). In a previous paper, we verified the genetic structure of 12 wild grapevine populations collected in Italy and Spain and gained knowledge of their genetic isolation and inbreeding (Grassi et al. 2003a). In the present work, the analysis was extended to 78 populations collected in their main Mediterranean distribution areas, including the Caucasus area, and DNA analysis was performed by using cpDNA microsatellite markers. This analytical approach was intended to shed light on (i), the phlylogeographic structure of wild grapevine in the Mediterranean area (including the Caucasus area), (ii) the rate of seed flow among populations and (iii) the geographic structure of present day wild grapevine genetic resources. Materials and methods A total of 418 individuals of Vitis vinifera subsp. silvestris, belonging to 78 wild grapevine populations (Table 1), was analysed. We used the mating system to discriminate wild grapevines from domesticated grapevines. Wild grapevine is dioecious, while cultivated grapevines are hermaphrodite and self-pollinating. Hermaphroditism was a crucial trait selected by ancient farmers to assure fruit production. All the populations were collected in areas that are distinctive for wild grapevine habitats:

839 Table 1. The 78 populations of wild grapevine collected in different geographical locations Popolation France Hendave Hendave Hendave Corsica - Sartene, Loreto Corsiaca - Sartene, Avene Corsica - Ajaccio Valence Spain Rio Esca - Huesca Rio Ver - Huesca Navarra Cadiz Ciudad Real Menorca Guipuzcoa La Mirilla Rivera del Hueznar Siviglia Malaga P N. Don˜ana P N. Don˜ana a Rocinas P N. Don˜ana Acebro`n Rio Murtiga Arrovo Peguera Arrovo Nateruela El Chorreadero Pantano de los Hurones Jaen Italy Liguria - Val Grande Liguria - Castiglione Toscana - Grosseto Toscana - Parco Uccellina Toscana - Sticciano Toscana - Ringhiere Toscana - Muro Toscana - La Sterza, Era river Lombardia - Po river Lombardia - Bosco S. Negri Emilia Romagna - Mesola Sardegna - M. Arcosu Sardegna - Querceto Sardegna - Rio Antas Sardegna - Urruos Sardegna - Orroa de Saide Sardegna - Ristalu Sardegna - Sabarva Roma - Tolfa Frosinone Campania

Code

N

F1 F2 F3 F4 F5 F6 F7

2 7 2 7 6 2 3

E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13 E14 E15 E16 E17 E18 E19 E20

12 12 4 4 9 5 2 5 6 3 3 6 4 2 6 6 6 4 3 2

I1 I2 I3 I4 I5 I6 I7 I8 I9 I10 I11 I12 I13 I14 I15 I16 I17 I18 I19 I20 I21

3 2 7 12 6 6 3 3 1 4 8 17 11 12 13 5 6 2 13 7 2

I

1

II

III

3 4 1

1

IV

V

HD

Latitude, longitude

2 7 2 4 1 1 3

– – – 0.286 0.348 0.5 –

43 43 43 – – 41 –

10, 01 22¢ 10¢, 01 31¢ 19¢, 01 22

12 12 4 1 8 5 2

– – – 0.251 0.111 – – – – – – – – – 0.167 – 0.167 – 0.333 –

42 42 – – – – 42 37 37 – – 36 36 37 38 36 36 36 36 –

31¢, 00 90¢ 30¢, 00 70¢

– – 0.524 0.553 0.266 0.266 – 0.333 – 0.251 – – – – – – – – 0.449 0.142 1

44 – 42 42 42 – – – – 45 – 39 40 39 40 39 39 40 42 41 41

21¢, 09 43¢

3

5 6 3 3 6 4 2 5

1 6 5 4 2

1

1

2

2 4

1 1 2 2 1 1

7 1

3

1 1

3 2 4 6 4 4 3 2 1 3 8 17 11 12 13 5 6 2 2 6 1

1

90¢, 8 95¢

75¢, )02 74¢ 39¢, )06 09¢ 55¢, )05 42¢

52¢, 07¢, 08¢, 01¢, 22¢, 20¢, 49¢, 46¢,

)06 )06 )06 )06 )05 )05 )05 )05

23¢ 30¢ 32¢ 47¢ 38¢ 37¢ 29¢ 33¢

81¢, 10 95¢ 55¢, 11 21¢ 91¢, 11 18¢

12¢, 09 03¢ 18¢, 19¢, 40¢, 11¢, 59¢, 55¢, 12¢, 09¢, 60¢, 41¢,

08 09 08 09 09 09 09 11 12 13

90¢ 37¢ 50¢ 31¢ 70¢ 15¢ 30¢ 90¢ 82¢ 92¢

840 Table 1. (Continued) Popolation

Code

N

Calabria - Cosenza Calabria - Rossano Calabria - San Anargia Calabria - San Apollinare Calabria - San Zaccaria Molise - Isernia Basilicata - Sinni Basilicata - Tempa Martina Others Tunisia - Tabarka Tunisia - Cap Serrat Turkey - Guemulder Turkey - Fethye Turkey - Dalaman river Armenia Armenia Armenia Armenia - Talin Deutschland - Ketsch Deutschland - Dirmstein Deutschland - Koller Deutschland - Hoerdit Austria E`eska´ Republika - Bøeclav Russia - Kabardino Russia - Dagestan Russia - Majkop Azerbaijan Georgia - Kisi Afghanistan Turkmenistan Total

I22 I23 I24 I25 I26 I27 I28 I29

2 7 3 8 9 6 7 10

TU1 TU2 TK1 TK2 TK3 AR1 AR2 AR3 AR4 D1 D2 D3 D4 A1 C1 R1 R2 R3 AZ1 G1 AF1 TR1

2 1 4 2 2 8 4 8 2 8 2 2 2 16 6 2 4 4 4 2 1 4 418

I

II

III

1

2 1 1

3 1

1

1 1

IV 2 6 3 8 9 1 4 7

V

HD

Latitude, longitude 39 39 – – – 41 40 40

21¢, 16 28¢ 58¢, 16 70¢

2

– 0.142 – – – 0.433 0.476 0.356 0.5 – – – – 0.215 0.5 – – – – – – 0.201 – 0.5 0.333 0.296 0.584 – – 0.251

36 36 38 – – 41 41 40 – – 49 – 49 48 – – – – – – – –

57¢, 08 43¢ 60¢, 08 46¢ 03¢, 26 95¢*

1 4 2 2

2 1

6 2

1 8 2

1

8 2 2 2 12 6 1

1 1

3 2

12

302

4

1

3

1

24

2 1 1 68

3 12

62¢, 14 20¢ 25¢, 16 52¢* 20¢, 15 96¢

03¢, 44 54¢ 05¢, 45 07¢ 55¢, 45 11¢

61¢, 08 23¢ 03¢, 08 21¢ 10¢, 16 30¢*

The table reports population name, code, number of samples analysed in the population (N), haplotypes (group I–V of Table 1), haplotype diversity (HD) and geographic coordinate (*=Approx ±3¢ ).

wetlands and forests with a high degree of humidity, due to rivers and brooks, and with a massive presence of tree species such as elms, poplars, hawthorns, hornbeams and oaks, on which wild grapevines grow as lianas. Discriminant traits for wild grapes are listed in Table 2. The plant sampling strategy was the same for all populations and designed to prevent: (i) collection of individuals from the cultivated subspecies (Vitis vinifera subsp. vinifera L.) or from rootstock, and, (ii) collection of clones. Unfortunately, no material could be obtained from some regions (e.g. Greece). DNA extraction was performed on 1–2 cm long leaves harvested from rooted cuttings. Tissue

was frozen in liquid nitrogen and ground into a fine powder. Total DNA was extracted and purified as described by Labra et al. (2001). DNA was analysed at the following 15 microsatellite loci: ccmp2, ccmp3, ccmp4, ccmp6, ccmp7, and ccmp10 (Weising and Gardner 1999); NTcp6, NTcp7, NTcp8, NTcp9, NTcp12 (Provan et al. 1999); Wct2, Wct10, Wct11, Wct15 (Ishii et al. 2001). The amplification was performed by using PCRbeads Ready-to-go KIT (Amersham- Bioscience, Italy) starting from 10 ng of total DNA. PCR amplification was performed with the following thermal cycles: 3 min at 94 C; 35 cycles of denaturation (45 s at 94 C), annealing (30 s at 50 C)

841 Table 2. Comparative morphology of wild and domesticated grapevine based on Grassi (2003b)

Mating system Habitat Berry shape Trunk Seeds Fruit clusters Leaves

Wild grapevine

Domesticated grapevine

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 coberent strips Large, pyriform body, lower width/length ratio (