ORIGINAL RESEARCH

Diffusion of the Nearctic leafhopper Scaphoideus titanus Ballin Europe: a consequence of human trading activity

Sabrina Bertin Æ Carmela R. Guglielmino ÆNisrine Karam Æ Ludvik M. Gomulski ÆAnna R. Malacrida Æ Giuliano Gasperi

Received: 21 July 2006 / Accepted: 22 December 2006 / Published online: 23 January 2007� Springer Science+Business Media B.V. 2007

Abstract Scaphoideus titanus Ball is a Nearctic leaf-

hopper that was introduced for the first time in Europe

probably at the beginning of the 20th century. In

Europe, this species is a specialist on cultivated

grapevines and is of great economic importance as the

vector of Flavescence doree (FD), a Grapevine Yel-

lows disease caused by Candidatus Phytoplasma vitis.

The Random Amplified Polymorphic DNA (RAPD)

technique was employed to obtain genetic information

about the diffusion and the structure of S. titanus

populations. Two American and 14 European popula-

tions were analysed. A total of 188 reproducible bands,

obtained from three arbitrary primers, were considered

to assess the amount and the pattern of genetic varia-

tion within and among leafhopper populations.

American populations showed high levels of intra-

population polymorphism and dissimilarity and

appeared to be the most isolated of all the tested

samples. The results confirm the historical role of

American samples as the sources for the more recently

founded European populations. RAPD analyses

revealed a weak genetic structure of European samples

that could probably be explained invoking the human

role in their diffusion. The non-natural spreading of

S. titanus across Europe is in fact attributable to the

exchange of grapevine canes and grafts carrying eggs

that the insect laid under the bark to overwinter.

Keywords Diffusion � Genetic relatedness �Grapevine � Polymorphism � RAPD-PCR �Scaphoideus titanus

Introduction

Scaphoideus titanus Ball (Schvester et al. 1962, 1969) is

a Nearctic leafhopper belonging to the family Cicad-

ellidae, subfamily Deltocephalinae. It is a hemime-

tabolous and phloem-feeding insect, which is of great

economic importance as vector of Flavescence doree

(FD), a persistent disease of cultivated grapevines. The

FD, considered the most threatening among Grapevine

Yellows (GY) diseases in Europe (Boudon-Padieu

2003), is caused by Candidatus Phytoplasma vitis

(16Sr-V group) (Firrao et al. 2005), which is a phloem-

restricted bacterium.

In Europe, S. titanus is a specialist for cultivated

grapes (Vitis vinifera) (Vidano 1964), while in the ori-

ginal area it is also widespread on other Vitis spp. and

highly abundant on wild Vitis riparia (Vidano 1966;

Maixner et al. 1993). This leafhopper is univoltine: the

eggs are laid under the bark of 2-year-old wood, where

they overwinter (Vidano 1964).

In the original area, the species was found in the

USA and South Canada (Barnett 1976) and it was

probably introduced into Europe as eggs, uninten-

tionally transported on imported grapevine canes from

North America (Boudon-Padieu 1999). Scaphoideus

titanus was reported for the first time in 1958 in vine-

S. Bertin � N. Karam � L. M. Gomulski �A. R. Malacrida � G. Gasperi (&)Dipartimento di Biologia Animale, Universita di Pavia,Piazza Botta 9, 27100 Pavia, Italye-mail: gasperi@unipv.it

C. R. GuglielminoDipartimento di Genetica e Microbiologia, Universita diPavia, Via Ferrata 1, 27100 Pavia, Italy

123

Genetica (2007) 131:275–285

DOI 10.1007/s10709-006-9137-y

yards in South France (Bonfils and Schvester 1960) and

in 1960s it diffused towards Italy (1963 in Liguria,

Vidano 1964) and Western and Southern Switzerland

(Baggiolini et al. 1968; Clerc et al. 1997). In Italy it has

spread to almost every northern region where grape-

vines are cultivated (Alma 2002). In 1987 S. titanus was

found in Croatia and Slovenia (Gabrijel 1987; Seljak

2002) and in 2003 in Serbia (Duduk et al. 2003). More

recently the species was detected in 1995 in Spain

(Lavina et al. 1995; Batlle et al. 1997) and in 1999 in

North Portugal (Quartau et al. 2001). In most of these

areas FD became an epidemic disease and spread as

the leafhopper dispersed.

As vector of FD, S. titanus was the target of studies

to analyse the modality of phytoplasma acquisition and

transmission (Schvester et al. 1969; Alma et al. 1997;

Bressan et al. 2005a, 2006). The unique sources for

vector acquisition in the field appear to be FD-infected

grapevines, since no other source plants have been

identified for the natural acquisition of FD phytoplas-

ma by S. titanus (Bressan et al. 2006). The leafhopper

vector is involved in vine-to-vine phytoplasma trans-

mission and the incidence of FD disease may increase

exponentially over successive years (Bressan et al.

2006). All developmental feeding stages of S. titanus

may acquire FD phytoplasma, although a long latent

period associated with the multiplication and persis-

tence of the phytoplasma in the vector’s body, and

flight ability, make adults the most important life stage

involved in the spread of FD (Bressan et al. 2006).

Acquisition efficiency of FD phytoplasma by the leaf-

hopper vector is influenced by factors such as the sus-

ceptibility of grapevine cultivars, the developmental

nymph instar and probably the period of growing

season (Bressan et al. 2005b).

Vector movement and dispersal represent important

components of incidence and spread of phytoplasma

disease (Hoy et al. 1992; McClure 1980). The inability

of the leafhopper to disperse over long distances has

been documented (Lessio and Alma 2004, 2006): the

adults do not spread significantly outside the vineyard

and their movements within the vineyard appear to be

strongly influenced by the planting layout.

There is no question that human activities have

played the major role in the long-distance dispersal and

in introducing this vector into previously unoccupied

areas (Weintraub and Beanland 2006). However the

frequency and the patterns of long-range movements is

largely unknown. DNA markers may provide effective

tools to infer movements within and between leaf-

hopper populations and to identify possible sources of

introduction.

Given the dearth of molecular data for S. titanus, we

have used the random amplified polymorphic DNA

technique (RAPD, Williams et al. 1990; Welsh and

McClelland 1990), which amplifies DNA fragments

from any genome, to derive suitable genetic markers

for S. titanus. The obvious advantage of the RAPD

approach is the relatively easy assay of a large number

of marker loci across the entire genome (Lynch and

Milligan 1994). The limitations associated with RAPD

markers include variable reproducibility unless reac-

tion conditions are stringently controlled (Baruffi et al.

1995; Sebastiani et al. 2001), and a dominant mode of

inheritance (Black 1993). The dominant nature of

RAPDs precludes the direct estimation of allele fre-

quencies and can bias calculations of genetic diversity

and population differentiation (Lynch and Millingan

1994). However, different estimates for measuring

genetic similarity and/or dissimilarity have been

developed (Wang 2004; Ritland 2005; Kosman and

Leonard 2005). These estimates provide a rough eval-

uation of the genetic similarity at the different levels of

population hierarchy and are relevant as descriptive

tools.

This study regards 16 populations of S. titanus from

North America and from Europe and its principal aims

were to (i) assess the level of genetic variation of the

species; (ii) evaluate the level of population differen-

tiation and structuring; (iii) determine if there is a

relationship between the level of genetic variation and

the spread of the species during the last 50 years.

Materials and methods

Sample collection

Fifteen out of the 16 samples of S. titanus were field-

caught as adults during the summer of 2001 from the

following locations: 2 in USA (Valois and Geneva

Ontario, NY State), 1 in France (Ponteves), 6 in Italy

(Chatillon, Chambave, San Colombano, Corniglia,

Vignale, Treiso), 2 in Switzerland (Piano Magadino

and Castelrotto), 3 in Slovenia (Goriska Brda, Izola,

Nova Gorica), and 1 in Spain (Penedes). An additional

sample from France was obtained from eggs collected

in Moroges during the winter of 2005. The adults hat-

ched from these eggs were obtained as ethanol pre-

served specimens (Table 1; Fig. 1).

Also all the other samples were obtained as ethanol

preserved specimens and DNA was extracted from

each single individual of both sexes, according to the

method of Baruffi et al. (1995).

276 Genetica (2007) 131:275–285

123

RAPD-PCR amplification

DNA from 15–18 individuals per population was

amplified using three oligonucleotide random primers

of 24–26 bp in length (Baruffi et al. 1995; Sebastiani

et al. 2001)

494N (5¢-AGCGCTGTGAGAAAGATGAAA-

GAT-3¢);

D17 (5¢-CGAAACAAGCGTGCATGAGCCCG-

AA-3¢);

NP4 (5¢-CTAATGCAGGAGTCGCATAAGG-

GAGA-3¢).

Primer selection was based on the clarity and con-

sistency of the resulting banding patterns.

Amplification reactions were performed in a final

volume of 10 ll, containing 10 mM Tris–HCl (pH 8.3),

50 mM KCl, 2 mM MgCl2, 200 lM dNTPs, 30 lM

primer, 0.1% gelatin, 3–7 ng of template DNA and 0.2

units of Taq DNA polymerase (Roche).

Amplifications were performed using a program-

mable thermocycler (PTC-100 MJ-Research Inc.) using

the following protocol: an initial denaturing step at

94�C for 90 s and 45 cycles of denaturation at 94�C for

30 s, annealing at 45�C for 1 min, extension at 75�C

for 2 min. The samples were finally incubated at 75�C

for 10 min and 60�C for 10 min and stored at 4�C.

Aliquots of 5 ll of the PCR products were loaded

together with a DNA size marker (50-bp ladder,

Amersham) onto a 2.5% (w/v) agarose TBE gel (1%

SeaKem GTG Agarose plus 1.5% NuSieve GTG

Agarose). Electrophoresis was carried out at 5.5 V/cm

for 4 h; the gels were stained using 0.5 lg/ml (w/v) of

ethidium bromide and the bands were visualised using

UV light.

Duplicate PCR reactions on each individual tem-

plate DNA were performed to ensure the reproduc-

ibility of the individual DNA bands. The

reproducibility of this method has already been tested

in other organisms (Baruffi et al. 1995; Sebastiani et al.

2001). The faintest bands and bands that differed in

replicates were not considered; several bands were not

taken into account because they could not be distin-

guished clearly from neighbouring bands. A negative

control without genomic DNA was included in each

series of PCR amplifications to ensure that there was

no contamination.

Detection of Flavescence doree phytoplasma

in vector populations

The DNAs from the same individuals used for RAPD-

PCR analysis were also checked for the possible pres-

ence of Flavescence doree phytoplasma DNA (Can-

didatus Phytoplasma Vitis 16Sr-V). Diagnostic PCRs

were performed using the following primers, that were

designed on the partial sequence of Flavescence doree

phytoplasma strain FD70 16S rRNA gene (Accession

Number AY197643; Lee et al. 2004):

FDf141 forward (5¢-GGAAACGGTTGCTAA-

GACTGGATA-3¢);

Table 1 Field collected samples of Scaphoideus titanus

Country Location Coordinates

USA (NY state) Valois 42�32¢N 76�52¢EGeneva Ontario 42�86¢N 76�98¢E

France Moroges 46�44¢N 04�40¢EPonteves 43�33¢N 06�01¢E

Italy Chatillon 45�44¢N 07�36¢EChambave 45�44¢N 07�32¢ESan Colombano 45�10¢N 09�29¢ECorniglia 44�07¢N 09�42¢EVignale 44�54¢N 08�24¢ETreiso 44�41¢N 08�05¢E

Switzerland Piano Magadino 46�08¢N 08�51¢ECastelrotto 45�59¢N 08�50¢E

Slovenia Goriska Brda 46�03¢N 13�30¢EIsola 45�32¢N 13�39¢ENova Gorica 45�56¢N 13�39¢E

Spain Penedes 41�20¢N 01�41¢E

12 - Castelrotto

U.S.A 1 - Valois

3 - Moroges4 - Ponteves

France

Italy

5 - Chatillon6 - Chambave

9 - Vignale8 - Corniglia7 - San Colombano

10 - Treiso

Switzerland

Slovenia

16 - PenedesSpain

(New York State)

14

11 - Piano Magadino

13 - Goriska Brda14 - Izola15 - Nova Gorica

810 95 6 7

1112

13153

4

New York State

1

2

16

2 - Geneva OntarioFig. 1 Geographic locationsof the sampling sites ofScaphoideus titanus analyzedin this study

Genetica (2007) 131:275–285 277

123

FDr1391 reverse (5¢-TCGGGTATTGCTAAC

TTTCGTGGT-3¢).

This primer pair, which yields a product of 1250 bp,

was used in direct PCR assays. Reaction products were

diluted 1:100 with sterile water and used as templates

in nested PCR, driven by R16(V)F1/R1 primer pair

that is specific for group V phytoplasmas (Lee et al.

1994; amplicon size 1100 bp). DNA amplifications

were performed in a total reaction volume of 25 ll; the

reaction mixture contained as template 1 ll of

extracted DNA or the diluted first amplification

product, 20 mM Tris–HCl (pH 8.0), 50 mM KCl,

1.5 mM MgCl2, 200 lM dNTPs, 1 lM of each primer

and 1 unit of Taq DNA polymerase (Invitrogen). The

conditions for amplification consisted of a pre-dena-

turation step at 94�C for 120 s, 35 cycles of denatur-

ation at 94�C for 60 s, annealing at 63�C in direct or

50�C in nested PCR for 90 s, extension at 72�C for

120 s and a final extension step at 72�C for 10 min.

A positive control (DNA extracted from an indi-

vidual of S. titanus known to be infected by FD) and a

negative control (without DNA) were included in each

PCR reaction.

Amplification products were analysed by electro-

phoresis on a 1% (w/v) agarose TAE gel stained using

0.5 lg/ml (w/v) of ethidium bromide and visualised

under UV light.

RAPD data analysis

For statistical analysis of RAPD data, the amount

and distribution of variability of the RAPD profiles

obtained from 283 individuals using the three primers

(NP4, D17, 494N) were considered. Under the

assumption that variation in banding patterns repre-

sents allelic segregation at homologous and inde-

pendent dominant loci, each locus was treated as a

two-allele system corresponding to the presence/ab-

sence of the amplified band. At each locus, the plus

allele (presence of a band) dominates over the null

allele (unamplified with PCR); therefore individuals

with a given amplified band are homozygous or het-

erozygous for a dominant allele whereas individuals

without that amplified band are homozygous reces-

sive. Each leafhopper was scored at each locus as 1

(present) or 0 (absent) across all polymorphic loci to

create a binary matrix. A locus was considered

polymorphic if the frequency of the most common

allele did not exceed 0.99. Bands with the same

molecular weight were assumed to represent homol-

ogous loci.

The presence/absence data were used to obtain

estimates of genetic relatedness between pairs of

individuals. Two estimates of pairwise relatedness have

been considered: ‘‘Dissimilarity index’’ (D), evaluated

according with Nei and Li (1985), and the ‘‘Similarity

estimator’’ (SM, Wang 2004). SM estimator has been

found to have consistently a small mean squared

deviation (MSD) over wide types of data (number of

loci, allele frequencies, sample size) (Wang 2004). The

two indices, calculated by the RAPDPLOT program

(Black 1995) and the MER (V3) program (Wang

2004), are respectively based on phenotypic data

(D) and on estimated allele frequencies (SM).

The presence/absence data were directly subjected

to analysis of molecular variance (AMOVA; Excoffier

et al. 1992), in ARLEQUIN Ver. 2.000 software

(Schneider et al. 2000), in order to describe the distri-

bution of genetic variability in the populations hierar-

chy. The significance of each variance component

was tested using a non-parametric permutation test

(Excoffier et al. 1992), consisting of 1023 random

permutations.

Allele frequencies were estimated using the

RAPDBIOS 2.0 program (Black 1995), assuming that

genotypes are in Hardy–Weinberg equilibrium and

applying the Lynch and Milligan (1994) correction.

On the basis of the derived gene frequencies, the

following analyses were performed. The degree of

polymorphism detected by each primer for the total

sampled leafhoppers were calculated using the pro-

gram BIOSYS-2 (modified from Swofford and

Selander 1981). Cavalli-Sforza and Edwards’s (1967)

Chord measures (DCE) were calculated between pairs

of populations, using the RAPDDIST program

(Black 1995). As this distance measure is correlated

to the pairwise fixation index (FST) (Cavalli-Sforza

et al. 1994), the effect of geographical distance on

genetic differentiation of populations was tested: we

regressed DCE/(1 – DCE) between pairs of popula-

tions to the natural logarithm (ln) of the geographical

distance between the pairs (Rousset 1997). The sig-

nificance of this relationship was assessed using the

Mantel test (Mantel 1967) with 100,000 random per-

mutations, in the ISOLDE routine within GENEPOP

3.4 program (Raymond and Rousset 1995). The

RAPDDIST program was also used to estimate the

Nei’s (1978) unbiased genetic distances among pop-

ulations. A consensus tree was derived from 1000

bootstrap resamplings (PHYLIP 3.57C package; Fel-

senstein 1995). A principal coordinate analysis

(PCoA; Gower 1966) was performed on the allele

frequencies to visualize the geometric relationship

among leafhopper populations in a two-dimensional

graph. PCoA analysis was conducted using the

NTSYS-PC program (Rohlf 1993).

278 Genetica (2007) 131:275–285

123

Results

RAPD patterns

The amplification of 283 individual DNAs from 16

populations with the three arbitrary primers (494N,

D17, NP4) resulted in a total of 188 interpretable and

reproducible bands. The RAPD patterns obtained

from the amplification of male and female genomic

DNA did not show differences. As assessed in all the

leafhopper individuals assayed, the fragment sizes

varied from 120 to 1300 bp. An average of 62.67 ± 4.04

(SD) bands were amplified per primer. The percentage

of polymorphism detected by each primer ranged be-

tween 33.33 ± 11.35 for NP4 to 44.19 ± 5.13 for 494N.

Out of the 188 bands, only four (NP4 – 345 bp, NP4

– 610 bp, D17 – 660 bp, 494N – 430 bp) were mono-

morphic over all individuals. Moreover, the 494N

– 410 bp band appears to be specific for the two Amer-

ican populations (Geneva Ontario and Valois) (Fig. 2).

Detection of Flavescence doree phytoplasma

in vector populations

Three individuals, all Italian, of S. titanus out of the 283

tested (1.06%) for the 16 populations, resulted FD

phytoplasma infected (2 individuals from Vignale and

1 individual from San Colombano populations). Com-

paring RAPD patterns of these three individuals with

RAPD patterns of the remaining individuals, no bands

discriminating between FD positive and non-positive

leafhoppers was observed. Therefore the presence of

FD DNA did not affect the resulting RAPD patterns

and the subsequent population analysis of S. titanus.

RAPD variability within populations

As shown in Table 2, considering all three primers,

a similar mean number of fragments per individual was

scored within each population (xi), with an average

value of 22.03 ± 0.916.

High intra-population heterogeneity levels for

RAPD variation were observed for all the geographic

samples. An average of 93.00 ± 3.374% of fragments

scored for each population were polymorphic. The two

estimators of intra-population differentiation, i.e. the

dissimilarity index D and the similarity index

SM, which are negatively correlated (non-parametric

Wilcoxon’s Signed-Ranks Test: P < 0.001), testify a

high level of variability.

All the polymorphism and differentiation estimates

indicate that the American populations, Valois and

Geneva Ontario are the most polymorphic. The

European populations were rather heterogeneous

within each country, both in terms of polymorphism

and intra-population differentiation.

Population structure of S. titanus

Phenotypic AMOVA analysis was performed in order

to test the significance of the partitioning of genetic

variance between the group of American populations

and the group including the fourteen European popu-

lations (Table 3A). A significant 12.77% of total vari-

ance was attributable to differences between the two

groups. When only the European populations were

considered (Table 3B), the value of the variance

component among the groups, corresponding to the

five European countries, was very low (1.86%) and not

significant (P = 0.165). In both analyses most of the

variance was significantly attributable to within popu-

lation variability (71.99 and 81.30%, respectively).

Pairwise Cavalli-Sforza and Edwards’s genetic dis-

tances (DCE, 1967) were derived in order to estimate

the genetic relationships between the American and

European populations and within and among Euro-

pean countries. In general (Table 4) no very high DCE

estimates were observed in all the comparisons (from

0.055 to 0.143). As expected, the largest DCE values

occurred with all the comparisons involving the two

American populations, especially with French (DCE

range: 0.125–0.143) and Spanish samples (DCE range:

0.137–0.142). In Europe, low values of differentiation

may be found in the comparisons between countries,

whilst higher or comparable values may be observed

within each country. For example, between Italian and

Slovenian samples DCE values ranged from 0.055 to

0.106, while DCE values vary from 0.068 to 0.109 within

Fig. 2 494N RAPD patterns in individuals from two populationsof S. titanus: San Colombano (Italy) and Valois (USA). A specificfragment (494N – 410 bp) is present only in American speci-mens; a monomorphic fragment (494N – 430 bp) is present inboth populations. M = 50 bp ladder

Genetica (2007) 131:275–285 279

123

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ep

op

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lym

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mv

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er

the

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8fr

ag

me

nts

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po

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280 Genetica (2007) 131:275–285

123

Italian group and from 0.066 to 0.092 within Slovenian

group. Between Slovenian and Swiss samples DCE

values ranged from 0.059 to 0.109, while the value

between the two Swiss populations is 0.096.

It is clear from these data that there is absence of

isolation by distance in Europe. In fact, the correlation

between the matrix of DCE/(1 – DCE) and the natural

logarithm of the pairwise geographical distances is

significant only if American populations are included

(Spearman Rank correlation coefficient r = 0.011;

Mantel P = 0.005).

A good description of the complex pattern of pop-

ulation relationships is shown in the Neighbour Joining

(NJ) consensus tree derived from Nei’s genetic dis-

tances (Fig. 3) and in the Principal Component Anal-

ysis (PCoA) (Fig. 4), both performed on RAPD-

derived allelic frequency data. In the NJ consensus

tree, obtained from 1000 bootstrap resamplings of the

data set, after the first statistically significant separation

of the two American populations, the only one division

supported by more than 50% of the resamplings sep-

arates the Italian sample of Vignale from the Spanish

sample of Penedes. The other branches separate a

heterogeneous mix of European samples.

The PCoA plot (Fig. 4) confirms the major separation

of American from European samples along the first PCo

axis (27% of variation). The European populations are

scattered along the second PCo axis (14% of variation)

without any apparent connection with the country of

origin and the historical chronology of S. titanus invasion.

Along this axis the two French populations from Moroges

and Ponteves have the extreme values.

Discussion

We have used RAPD markers as a descriptive tool for

inferences about DNA variability and population

structure of the leafhopper S. titanus, a species for

which little genetic knowledge is available.

The degree of polymorphism revealed by these

markers is extensive: more than 85% of fragments

scored per population are polymorphic, demonstrating

that there is a vast reservoir of hidden nuclear DNA

polymorphism in this species. On the basis of this

variability, the RAPD technique appears to be ade-

quate to reveal the level of intra- and inter-population

divergence in order to provide insights into the pattern

of leafhopper colonization.

Genetic structure and inferences on dispersal

of S. titanus

Among the North-American genus Scaphoideus, S. tit-

anus is so far the only species which has become

established in Europe, probably through the introduc-

tion of planting material initially in South-Western

France (Boudon-Padieu 2002). In agreement with the

Nearctic origin of the species, the two American pop-

ulations analysed in this study result clearly differen-

tiated from the European populations and were shown

to possess local private alleles and the highest levels of

intra-population genetic variability. In its home range

S. titanus has been found to be associated not only with

V. vinifera but also with different native Vitis species

(Vidano 1966; Maixner et al. 1993). This capacity to

adapt to different host plants may increase the dis-

persion of the species, favouring the maintenance of

high population size and high genetic variability (Nei

et al. 1975). The wide reservoir of genetic variation in

the leafhopper native populations may constitute the

background of adaptive processes during colonization.

Upon its arrival in Europe, this species settled pro-

gressively since the 1960’s in several countries from

West to East, into a climatic area with cold winter and

relatively long summer. In these areas, S. titanus

Table 3 Analysis of molecular variance (AMOVA) among Scaphoideus titanus samples

Source of variation d.f. SSD Variance components % Variation P-value

(A) between European and American samplesEuropean versus American 1 153.03 1.70 12.77 < 0.008Among populations within the two groups 14 636.52 2.03 15.24 < 0.001Within populations 267 2562.00 9.59 71.99 < 0.001Total 282 3351.55 13.33

(B) among five groups of European populations: France, Italy, Switzerland, Slovenia and SpainAmong five European countries 4 210.92 0.21 1.86 = 0.165Among populations within countries 9 388.68 1.92 16.84 < 0.001Within populations 233 2163.17 9.28 81.30 < 0.001Total 246 2762.77 11.42

d.f., degrees of freedom; SSD, sum of squared deviation; % of variation, percentage of total variance contributed by each component;P-value, probability value obtained by significant test (1023 permutations).

Genetica (2007) 131:275–285 281

123

appears to be well adapted to V. vinifera and it seems

that its biological cycle could adjust to all climates

compatible with vineyard cultivation (Boudon-Padieu

2002).

A question arises about the modality of diffusion in

Europe of this species, which is characterized by a very

restricted range of mobility. It is known that S. titanus

does not disperse significantly beyond 24 m from the

vineyard, suggesting that the leafhopper can colonise

by its own means only nearby vineyards (Lessio and

Alma 2004, 2006). This characteristic, together with its

univoltine reproductive behaviour, would suggest

a population structure reflecting the geographical

distance and/or the chronology of the first records of

the species in Europe. On the contrary, our RAPD

data show an almost complete lack of isolation by

distance from France (which probably was the first

colonized country in Europe) to Slovenia and Spain

(which are the most recent colonized among our con-

sidered samples), reflecting a complex genetic frag-

mentation among populations, both within and

between countries. It seems likely that such scattered

pattern in the distribution of genetic diversity derives

from independent introduction events of S. titanus

within and between the different European areas. TheTa

ble

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Chambave

Castelrotto

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Ponteves

Chatillon

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Vignale

Penedes

Corniglia

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Treiso

Nova Gorica

Geneva Ontario

Valois

Moroges

Fig. 3 Neighbour Joining consensus tree based on Nei’s geneticdistances among 16 populations of S. titanus. Only bootstrapvalues greater than 50% (out of 1000 replications) are reported

282 Genetica (2007) 131:275–285

123

differentiation observed between the two French

samples may be related to the longer history of colo-

nization in France.

Thus a scenario involving a first ancient introduction

into Europe, followed by independent and mixed col-

onization events within and between European coun-

tries, may be the most parsimonious conclusion.

On this basis we may explain, for example, the differ-

entiations and similarities observed among and within

Italian and Slovenian samples and between Slovenian

and Swiss samples. Therefore, it is possible that this

scenario is associated with a concomitant effect of the

low mobility of the species and of the wide-range and

long-distance propagation by commercial exchange of

planting material (canes, grafts, scions and rootstocks)

carrying eggs that the leafhopper laided under the bark

and between the bud scales to overwinter (Boudon-

Padieu 2002; Weintraub and Beanland 2006).

Inferences on the diffusion of S. titanus as potential

vector of Flavescence doree

Scaphoideus titanus is not an serious pest insect by

itself, but it causes major concerns in Europe as vector

of Flavescence doree (FD), a GY disease caused by a

phytoplasma which has been decreed a quarantine

organism in the European Community (Boudon-Pa-

dieu 2002). Vitis vinifera, the host plant for S. titanus, is

also the reservoir of FD phytoplasma. The leafhopper

acquires the phytoplasma from infected host plants and

spreads the disease within the vineyard by feeding

activity, playing consequently an important role in the

tri-trophic relationship insect vector–phytoplasma–

plant. The exclusive vine-feeding activity of the insect

vector is sufficient for FD spread into vineyards, even if

the phytoplasma is initially present with a low inci-

dence (Boudon-Padieu 2002). Considering the large

quantity of plant material produced and exchanged

commercially, it is evident that rigorous quality con-

trols on this material are imperative, both for the

presence of phytoplasma infections and for the pres-

ence of leafhopper eggs.

Acknowledgements This study is part of the Project ‘‘Biodiv-ersita della Vite Coltivata in Italia e sua Protezione dai Parassiti’’funded by the ‘‘Fondazione Bussolera-Branca’’ in Mairano diCasteggio, Pavia, Italy. We are indebted to Elisabeth BoudonPadieu (Biologie et Ecologie des Phytoplasmes, UMR PlanteMicrobe Environnement, INRA of Dijon), to Alberto Alma,Simona Palermo and Domenico Bosco (Di.Va.P.R.A. Entomo-logia e Zoologia applicate all’Ambiente ‘‘Carlo Vidano’’, Uni-versita di Torino), and to Piero Attilio Bianco (Istituto diPatologia Vegetale, Universita di Milano), for supplying thewild-collected specimens of Scaphoideus titanus and for helpfuldiscussion about the leafhopper biology. We thank the severalunknown people from France, Spain, Slovenia, Swiss and USAwho provided invaluable assistance in the collection of wildmaterial. We also thank anonymous reviewers for helpful com-ments on the manuscript.

References

Alma A (2002) Diffusione di Scaphoideus titanus Ball in Italia. In:Canova A (ed) Atti Giornate Fitopatologiche, Baselga diPine, Italy, 7–11 April 2002. CLUEB, Bologna, Italy, pp 51–54

Alma A, Bosco D, Danielli A, Bertaccini A, Vibio M, Arzone A(1997) Identification of phytoplasmas in eggs, nymphs andadults of Scaphoideus titanus Ball reared on healthy plants.Insect Mol Biol 6(2):115–121

Baggiolini M, Canevascini V, Caccia R, Tencalla Y, Sobrio G(1968) Presence dans le vignoble du tessin d’une cicadellenearctique nouvelle pour la Suisse, Scaphoideus littoralisBall (Homoptera: Jassidae), vecteur possible de la Flaves-cence doree. Mitt Schwei Entomol Gesell 60:270–275

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2

Moroges

Vignale

Penedes

Castelrotto

Izola

CornigliaSan ColombanoValois

Geneva Ontario

Ponteves

Nova GoricaChantillon

ChambaveGoriska BrdaTreiso

Piano Magadino

PCoA

axi

s 2

(13.

91%

)

PCoA axis 1 (26.92%)

U.S.AFrance (1958)Italy (1963)Switzerland (1968)Slovenia (1987)Spain (1995)

Fig. 4 Principal componentanalysis (PCoA) of 16populations of S. titanus basedon RAPD data. The first andsecond axes explained 26.92and 13.91% of the totalvariation, respectively. Theyear of leafhopper detectionin the different countries isreported in the legend, inparenthesis

Genetica (2007) 131:275–285 283

123

Barnett DE (1976) A revision of the Nearctic species of thegenus Scaphoideus (Homoptera: Cicadellidae). Trans AmEntomol Soc 102:485–593

Baruffi L, Damiani G, Guglielmino CR, Bandi C, Malacrida AR,Gasperi G (1995) Polymorphism within and between pop-ulations of Ceratitis capitata: comparison between RAPDand multilocus enzyme electrophoresis data. Heredity74:425–437

Batlle A, Lavina A, Clair D, Larrue J, Kuszala C, Boudon-Padieu E (1997) Detection of Flavescence doree in grape-vine in Northern Spain. Vitis 36(4):211–212

Black WC IV (1993) PCR with arbitrary primers: approach withcare. Insect Mol Biol 2:1–6

Black WC IV (1995) FORTRAN programs for the analysis ofRAPD-PCR markers in populations. Colorado State Uni-versity, Fort Collins, CO

Bonfils J, Schvester D (1960) The leafhoppers (Homoptera:Auchenorrhynchas) and their relationship with vineyards inSouth-Western France. Ann Epiphyt 11(3):325–336

Boudon-Padieu E (1999) Grapevine phytoplasmas. In: Firstinternet conference on phytopathogenic Mollicutes, 24–29May 1999. http://web.uniud.it/phytoplasma//conf.html

Boudon-Padieu E (2002) Flavescence doree of the grapevine:knowledge and new developments in epidemiology, etiologyand diagnosis. In: Canova A (ed) Atti Giornate Fitopato-logiche, Baselga di Pine, Italy, 7–11 April 2002. CLUEB,Bologna, Italy, pp 15–34

Boudon-Padieu E (2003) The situation of grapevine yellows andcurrent research directions: distribution, diversity, vectors,diffusion and control. In: Proceedings of the 14th ICVGConference, Locorotondo, Italy, 12–17 September 2003

Bressan A, Larrue J, Boudon-Padieu E (2006) Patterns ofphytoplasma-infected and infective Scaphoideus titanusleafhoppers in vineyards with high incidence of Flavescencedoree. Entomol Exp Appl 119:61–69

Bressan A, Girolami V, Boudon-Padieu E (2005a) Reducedfitness of Scaphoideus titanus exposed to Flavescence doreephytoplasma. Entomol Exp Appl 115:283–290

Bressan A, Spiazzi S, Girolami V, Boudon-Padieu E (2005b)Acquisition efficiency of Flavescence doree phytoplasma byScaphoideus titanus Ball from infected tolerant or suscep-tible grapevine cultivars or experimental host plants. Vitis44:143–146

Cavalli-Sforza LL, A.Edwards WF (1967) Phylogenetic analysis:models and estimation procedures. Evolution 21:550–570

Cavalli-Sforza LL, Menozzi P, Piazza A (1994) The history andgeography of human genes. Princeton University Press,Princeton, NJ

Clerc L, Linder C, Gunthart H (1997) Premiere observation enSuisse romande de la cicadelle Scaphoideus titanus Ball(Homoptera: Jassidae), vecteur de la Flavescence doree dela vigne. Rev Suisse Vitic Arboric Hortic 29(4):245–247

Duduk S, Botti S, Ivanovic M, Dukic N, Bertaccini A (2003)Molecular characterization of a Flavescence doree phytopl-asma infecting grapevine in Serbia. In: Proceedings of the14th ICVG Conference, Locorotondo, Italy, 12–17 Septem-ber 2003

Excoffier L, Smouse PE, Quattro JM (1992) Analysis ofmolecular variance inferred from metric distances amongDNA haplotypes: application to human mitochondrial DNArestriction data. Genetics 131:479–491

Felsenstein J (1995) PHYLIP: Phylogeny inference package,Version 3.57 C. University of Washington, Seattle, WA

Firrao G, Gibb K, Streten C (2005) Short taxonomic guide to thegenus ‘Candidatus Phytoplasma’. J Plant Pathol 87(4):249–263

Gabrijel S (1987) Scaphoideus titanus Ball (= S. littoralis Ball), novistetnik vinove loze u Jugoslaviji. Zastita Bija 38(4):349–357

Gower JC (1966) Some distance properties of latent root andvector methods used in multivariate analysis. Biometrika53:325–338

Hoy CW, Heady SE, Koch TA (1992) Species composition,phenology and possible origins of leafhoppers (Cicadelli-dae) in Ohio vegetable crops. J Econ Entomol 85:2336–2343

Kosman E, Leonard KJ (2005) Similarity coefficients formolecular markers in studies of genetic relationshipbetween individuals for haploid, diploid and polyploidspecies. Mol Ecol 14(2):415–424

Lavina A, Batlle A, Larrue J, Daire X, Clair D, Boudon-PadieuE (1995) First report of grapevine bois noir phytoplasma inSpain. Plant Dis 79(10):1075

Lee IM, Gundersen DE, Hammond RW, Davis RE (1994) Useof Mycoplasma-like Organism (MLO) group-specific oligo-nucleotide primers for nested-PCR assays to detect mixed-MLO infections in a single host plant. Phytopathology84:559–566

Lee IM, Martini M, Marcone C, Zhu SF (2004) Classification ofphytoplasma strains in the elm yellows group (16SrV) andproposal of ‘Candidatus Phytoplasma ulmi’ for the phytopl-asma associated with elm yellows. Int J Syst Evol Microbiol54:337–347

Lessio F, Alma A (2004) Dispersal patterns and chromaticresponse of Scaphoideus titanus Ball (Homoptera: Cicadel-lidae), vector of the phytoplasma agent of grapevineFlavescence doree. Agric For Entomol 6:121–127

Lessio F, Alma A (2006) Spatial distribution of nymphs ofScaphoideus titanus (Homoptera: Cicadellidae) in grapesand evaluation of sequential sampling plans. J Econ Ento-mol 99:578–582

Lynch M, Milligan B (1994) Analysis of population geneticstructure with RAPD markers. Mol Ecol 3:91–99

Maixner M, Pearson RC, Boudon-Padieu E, Caudwell A (1993)Scaphoideus titanus, a possible vector of Grapevine Yellowsin New York. Plant Dis 77:408–413

Mantel N (1967) The detection of disease clustering anda generalised regression approach. Cancer Res 27:209–220

McClure MS (1980) Spatial and seasonal distribution of leaf-hopper vectors of peach X-disease in Connecticut. EnvironEntomol 9:668–672

Nei M, Li WH (1985) Mathematical model for studying geneticvariation in terms of restriction endonucleases. Proc NatlAcad Sci USA 76:5269–5273

Nei M (1975) Molecular population genetics and evolution.North Holland, Amsterdam

Nei M (1978) Estimation of average heterozygosity and geneticdistance from a small number of individuals. Genetics89:583–590

Quartau, JA, Guimaraes JM, Andre G (2001) On the occurrencein Portugal of the Nearctic Scaphoideus titanus Ball(Homoptera: Cicadellidae), the natural vector of the grape-vine ‘‘Flavescence doree’’ (FD). In: Carlo Lozzia (ed)IOBC/wprs Bulletin, 24(7):273–276. Proceedings of themeeting of the Working group ‘‘Integrated Control inViticulture’’, Ponte de Lima, Portugal, 3–7 March 2000

Raymond M, Rousset F (1995) GENEPOP: population geneticssoftware for exact tests and ecumenicism. J Hered 86:248–249

Ritland K (2005) Multilocus estimation of pairwise relatednesswith dominant markers. Mol Ecol 14(10):3157–3165

Rohlf FJ (1993) NTSYS-PC. Numerical taxonomy and multi-variate analysis system, Version 1.80. Applied BiostatisticsInc, New York, NY

284 Genetica (2007) 131:275–285

123

Rousset F (1997) Genetic differentiation and estimation of geneflow from F-statistics under isolation by distance. Genetics145:1219–1228

Schneider S, Roessli D, Excoffier L (2000) Arlequin ver. 2.000:A software for population genetics data analysis. Geneticsand Biometry Laboratory, University of Geneva, Switzer-land

Schvester D, Moutous G, Carle P (1962) Scaphoideus littoralisBall (Homoptera: Jassidae), cicadelle vectrice de la Flaves-cence doree de la vigne. Rev Zool Agric Appl 91:118–131

Schvester D, Carle P, Moutous G (1969) Nouvelles donnees surla transmission de la flavescence doree de la vigne parScaphoideus littoralis Ball. Ann Zool Ecol Anim 1969:445–465

Sebastiani F, Meiswinkel R, Gomulski LM, Guglielmino CR,Mellor PS, Malacrida AR, Gasperi G (2001) Moleculardifferentiation of the Old World Culicoides imicola speciescomplex (Diptera, Ceratopogonidae), inferred using ran-dom amplified polymorphic DNA markers. Mol Ecol10:1773–1786

Seljak G (2002) Non-European Auchenorrhyncha (Hemiptera)and their geographic distribution in Slovenia. Acta EntomolSlov 10:97–101

Swofford DL, Selander RB (1981) BIOSYS-1: a FORTRANprogram for the comprehensive analysis of electronicdata in population genetics and systematics. J Hered72:281–283

Vidano C (1964) Scoperta in Italia dello Scaphoideus littoralisBall cicalina Americana collegata alla Flavescence doreedella vite. L’Italia Agric 101:1031–1049

Vidano C (1966) Scoperta della ecologia ampelofila del Cica-dellide Scaphoideus littoralis Ball nella regione nearticaoriginaria. Ann Fac Sci Agrarie Stud Torino 3:297–302

Wang J (2004) Estimating pairwise relatedness from dominantgenetic markers. Mol Ecol 13:3169–3178

Weitraub PG, Beanland L (2006) Insect vectors of phytoplasmas.Ann Rev Entomol 51:91–111

Welsh J, McClelland M (1990) Fingerprinting genomes usingPCR with arbitrary primers. Nucleic Acids Res 18:7213–7218

Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV(1990) DNA polymorphisms amplified by arbitrary primersare useful as genetic markers. Nucleic Acids Res 18:6531–6535

Genetica (2007) 131:275–285 285

123