Spatial and host-plant partitioning between coexistingBemisia ...

Popul Ecol (2012) 54:261–274 DOI 10.1007/s10144-012-0303-z

ORIGINAL ARTICLE

Spatial and host-plant partitioning between coexisting Bemisia tabaci cryptic species in Tunisia Dounia Saleh • Asma Laarif • Ce´cile Clouet Nathalie Gauthier

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Received: 9 July 2011 / Accepted: 5 January 2012 / Published online: 31 January 2012 Ó The Society of Population Ecology and Springer 2012

Abstract The whitefly Bemisia tabaci is a species complex including at least 24 morphologically indistinguishable species among which the Mediterranean (Med) and Middle East-Asia Minor I (MEAMI) species containing the biotypes commonly known as Q and B, respectively. These B and Q biotypes (hereafter referred to as MEAMI and Med species) are the most invasive agricultural pests of the B. tabaci complex worldwide. The spread of MEAMI and more recently of Med species into regions already invaded by other B. tabaci populations has been frequently seen to lead to their displacement by Med species. In Tunisia, in

Electronic supplementary material The online version of this article (doi:10.1007/s10144-012-0303-z) contains supplementary material, which is available to authorized users. D. Saleh INRA, UMR BGPI (CIRAD, INRA, SupAgro), TA A54-/K, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France A. Laarif Institution de la Recherche et de l’Enseignement Supe´rieur Agricole, Centre Re´gional de Recherche en Horticulture et Agriculture Biologique, BP57 Chott Meriem, 4042 Sousse, Tunisia C. Clouet INRA, UMR (INRA, IRD, CIRAD, SupAgro) Centre de Biologie pour la Gestion des Populations (CBGP), Campus international de Baillarguet, CS 30016, 34988 Montferrier-sur-Lez Cedex, France N. Gauthier (&) IRD, UMR (INRA, IRD, CIRAD, SupAgro) Centre de Biologie pour la Gestion des Populations (CBGP), Campus international de Baillarguet, CS 30016, 34988 Montferrier-sur-Lez Cedex, France e-mail: [email protected]

contrast to usual observations in the Mediterranean basin, Med and MEAMI species have been seen to co-occur in the main crop producing regions. Based on fine population genetics and field spatial distribution analyses, we found that the co-existence of these two interacting species was based on habitat partitioning including spatial and hostplant partitioning. Although they co-occurred at larger spatial scales, they excluded one another at sample scale. We observed neither spatial overlapping nor hybridization between MEAMI and Med B. tabaci. Vegetable crops were the main hosts for MEAMI specimens while 99.1% of the B. tabaci collected on the ornamental, Lantana camara, were Med specimens. Different patterns of genetic diversity were observed between the two species, as well as among Med specimens sampled on the ornamental versus vegetables, with the highest genetic diversity found in Med B. tabaci sampled on L. camara. These findings lead us to focus our discussion on the role played by lantana, human pressure, and competition, in the spatial and genetic patterns observed in the whitefly B. tabaci. Keywords Genetic diversity
Habitat partitioning
Microsatellite loci
Mitochondrial DNA
Population genetics
Whitefly

Introduction Knowledge of the distribution patterns of interacting species is a crucial starting point for understanding their biogeographical patterns, community biodiversity and stability. In the case of invasive agricultural pests, spatial and resource patterns could provide a framework for understanding the dynamics of biological invasions and help develop management programmes.

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Based on the competitive exclusion principle, the traditional view of interacting species within a community is that species need sufficient genetic, phenotypic and ecological disparity to co-exist stably (Hardin 1960). If this were not the case, co-existence would not exist, implying that all but one species would become extinct or displaced during the course of time (Tilman 1982; Reitz and Trumble 2002; Neill et al. 2009; Crowder et al. 2010a). Species exclusion or displacement has often been shown to be mediated by a combination of factors (i.e., between-species variation in life history traits, reproductive interference, behavioural interactions, and the ability of the species to rapidly adapt to selection pressure). In agricultural systems, human activities including cultivation practices, the use of cultivated plants and pesticide treatments, create intense selection pressure on populations and can have a major influence on population demographics and on patterns of species distribution (Reitz and Trumble 2002; Crowder et al. 2010a). However, exclusion between interacting species can be avoided through a number of mechanisms. Habitat partitioning including spatial, temporal and resource partitioning allows species to escape or minimize competition, and consequently fosters co-existence. Moreover, co-occurrence/exclusion between interacting species cannot be defined without reference to spatial and/ or temporal scales: organisms that coexist at larger spatial scales may be able to do so because they are segregated at smaller scales and/or do not overlap at a temporal scale (Tilman 1982; Chesson 2000; Wang et al. 2002; Amarasekare 2003; Amarasekare et al. 2004; Leibold and McPeek 2006; Gro¨ning et al. 2007; Gilbert et al. 2008; Leisnham and Juliano 2009). Today, increasing attention is being paid to the co-existence of closely related species and their distribution in spatially structured environments because it is not clear whether they actually do display sufficient genotypic, phenotypic and ecological disparity to co-exist. Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is an ideal system to explore whether and/or how two or more closely related species could co-exist and/or exclude one another in agricultural systems. The whitefly B. tabaci is a complex containing at least 24 morphocryptic species (i.e., genetically distinct but morphologically indistinguishable), most of which are reproductively incompatible (Dinsdale et al. 2010; Elbaz et al. 2010; Xu et al. 2010; Wang et al. 2010; De Barro et al. 2011; Sun et al. 2011). Among the 24 B. tabaci putative species delineated by Dinsdale et al. (2010), the Mediterranean (Med) and the Middle East-Asia Minor I (MEAMI) species, have attracted particular attention in the past 30 years. The Med species, which includes what has been commonly known as the Q ‘biotype’ (hereafter Med) in addition to the J, L, Sub-Saharan silverleaf ‘biotypes’, and the MEAMI

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species, which contains what has been commonly known as the B ‘biotype’ (hereafter MEAMI) in addition to the B2 ‘biotype’ are of major economic importance worldwide. They are highly invasive, insecticide resistant, polyphagous and cause considerable damage to a wide range of vegetable and ornamental crops by feeding on phloem and thus transmitting harmful plant viruses (Brown 2007). The pest status of B. tabaci has reached international prominence since the 1980s due to spread of MEAMI species throughout the world. In many regions, its introduction— mostly due to human activities—has resulted in the displacement of some relatively innocuous, indigenous morphocryptic B. tabaci belonging to different putative species of the complex. More recently, the widespread invasion of MEAMI was followed by the invasion of Med (De Barro et al. 2011). The rapid spread of Med from its presumed area of origin in the Mediterranean basin to other parts of the world (Chu et al. 2006; Ueda and Brown 2006; De Barro et al. 2011) altered the composition of B. tabaci species in the regions that were invaded. In many locations worldwide, Med has become the predominant—or even the only—B. tabaci present. However, putative MEAMI and Med species have sometimes been reported to co-occur with other species of the B. tabaci complex in the field, and particularly in the Mediterranean basin. But after a period of co-existence, Med has often been reported to rapidly displace or eliminate the other B. tabaci species, including MEAMI (Tahiri et al. 2006; Simo´n et al. 2007; Tsagkarakou et al. 2007; Dalmon et al. 2008). Only a few Mediterranean countries including Tunisia have been reported to still ‘‘host’’ the highly competing Med and MEAMI B. tabaci, at least in some localities (Chermiti et al. 1997; Bel-Kadhi et al. 2008). Such a rare in natura situation provided a unique opportunity to increase our understanding of the factors and mechanisms of co-existence between B. tabaci species in the field. To this end, we analysed a wide field sample at successive spatial scales (i.e., country vs. region vs. site) of B. tabaci specimens collected on three major vegetable crops and on one ornamental species in the main crop producing regions of Tunisia. Since the B. tabaci complex consists of morphocryptic species, we chose a population genetic approach based on diagnostic and highly discriminatory molecular markers, cytochrome oxidase I (COI) gene and microsatellite markers (Fro¨hlich et al. 1999; Gauthier et al. 2008) to (1) clearly establish the status of the B. tabaci complex in Tunisia, i.e., determine whether several morphocryptic B. tabaci species really co-occur, (2) investigate whether significant genetic and distribution patterns exist between them, (3) identify the role of spatial scales and host-plant species in such patterns, and (4) identify the possible factors and mechanism which may have influenced this outcome in Tunisia.

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Materials and methods Bemisia tabaci field sampling Bemisia tabaci adults were sampled from 2006 to 2008 on four host-plant species in two major vegetable producing regions in Tunisia; the eastern part of central and northern Tunisia including Cap Bon and the Sahel, the southern part of Tunisia including Kebili, Tozeur and their surrounding areas (Fig. 1). Twenty-eight samples were collected from three major vegetable crops (tomato, Solanum

lycopersicum; aubergine, S. melongena; courgette, Cucurbita pepo) and one ornamental (lantana, Lantana camara), which has become a dominant component of natural and agricultural ecosystems. One sample consisted of individuals of B. tabaci collected from several individual plants of the same host-plant species growing in a restricted site (i.e., a field or a vegetable production system, a home-plusgarden unit or in the case of L. camara, from nearby vegetable crops) (Table 1). Different sites (i.e., 2–5 per host plant) were sampled in each region (Table 1). Adult specimens in the same sample (i.e., sampled at the same

Tak1-T Gro1-L

2L 1T ChM1-L Sou1-L Mon1-L Teb2-L, 1-T, 2-E, 3-E, 4-Z Nak1-T, 2-T Bek1-T, 2-Z

Toz1-L, 2-T

Deb1-Z Keb1-L, 2-T

2E

3E

4Z

EHa1-E Lim1-T, 2-Z Baz1-T, 2-T, 3-E, 4-E

Dou1-L, 2-Z

TUNISIA

ALGERIA LIBYA

Fig. 1 Map of Tunisia with the sample collection sites (black triangles). The code for each sample is listed in Table 1. The percentages of Med (in grey) and MEAMI specimens (in black) at each collection site are shown in pie charts

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Table 1 Description of the samples and number of specimens analysed (N) by genotyping using the nine microsatellite loci or only the two diagnostic loci (BtIs1.2, BtIs1.9), and sequencing (676-bp COI fragment) of the 28 Bemisia tabaci samples collected in Tunisia Geographic origin Region

Location

Host-plant Acronym

Name

a

Site

Collection date

Genetic analysis

Month-year

Genotyping 9M

Sequencing (N = 43)

2DM

Accession number

Cap Bon Grombalia

Gro1-L

Lantana

Plastic tunnel

Sep-2008

30

3

HM807538-39-40

Takelsa

Tak1-T

Tomato

Plastic tunnel

Nov-2008

24

3

HM807541-42-43

Monastir

Mon1-L

Lantana

Open yard

Apr-2006

30

2

HM807544-45

Teboulba

Teb2-L

Lantana

Open yard

Sep-2006

31

1

HM807546

Sousse

Sou1-L

Lantana

House-yard

Sep-2006

19

4

HM807547-48-49-50

Chott Meriem

ChM1-L

Lantana

Near plastic tunnel

Oct-2007

30

1

HM807551

Teboulba

Teb1-T

Tomato

Plastic tunnel

Jan-2007

33

1

HM807552

Nakharia

Nak1-T

Tomato

Open field

Jan-2007

30

1

HM807553

Nakharia

Nak2-T

Tomato

Plastic tunnel

Apr-2007

30

1

HM807554

Bekalta

Bek1-T

Tomato

Plastic tunnel

Apr-2007

30

1

HM807555

Teboulba

Teb2-E

Aubergine

Open field

Nov-2006

30

1

HM807556

Teboulba Bekalta

Teb3-E Bek2-Z

Aubergine Courgette

Open field Plastic tunnel

Nov-2006 Mar-2007

30 23

1 1

HM807557 HM807558

Teboulba

Teb4-Z

Courgette

Open field

Oct-2007

22

1

HM807559

Sahel

Kebili Kebili

Keb1-L

Lantana

Open field

Sep-2006

30

1

HM807560

Douz

Dou1-L

Lantana

House-yard

Oct-2007

30

3

HM807561-62-63

Bazma

Baz1-T

Tomato

Plastic tunnel

Apr-2007

30

1

HM807564

Bazma

Baz2-T

Tomato

Plastic tunnel

May-2007

30

1

HM807565

Kebili

Keb2-T

Tomato

Plastic tunnel

May-2007

30

3

HM807566-67-68

Limagues

Lim1-T

Tomato

Plastic tunnel

May-2007

28

1

HM807569

Bazma

Baz3-E

Aubergine

Plastic tunnel

May-2007

30

1

HM807570

Bazma

Baz4-E

Aubergine

Open field

Nov-2006

30

2

HM807571-72

Limagues

Lim2-Z

Courgette

Plastic tunnel

May-2007

27

1

HM807576

Douz

Dou2-Z

Courgette

Plastic tunnel

Apr-2007

24

3

HM807573-74-75

Debabcha

Deb1-Z

Courgette

Plastic tunnel

Apr-2007

27

1

HM807577

Tozeur

Toz1-L

Lantana

Field

Nov-2008

22

1

HM807578

Tozeur

Toz2-T

Tomato

Plastic tunnel

Nov-2008

25

1

HM807579

El Hamma

EHa1-E

Aubergine

Plastic tunnel

Sep-2008

28

1

HM807580

Tozeur

9M, 9 microsatellite loci; 2DM, 2 diagnostic microsatellite loci a

Lantana (Lantana camara), tomato (Solanum lycopersicum), aubergine (S. melongena), courgette (Cucurbita pepo)

site) were placed alive in a tube filled with 70% ethanol and stored at -20°C. As B. tabaci is a complex of haplodiploid species, only females were analysed. A total of 783 females were analysed to determine their species status, their distribution and prevalence at different spatial scales (i.e., throughout the country, in the two regions, at the 28 sites) and on different host plants in the field. Among the 783 females, 654 were genotyped using all microsatellite loci to infer B. tabaci population structure and their patterns of diversity as a possible function of geographical location and/or host-plant species.

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DNA protocols Total genomic DNA was extracted from each individual female using the Nonidet P-40-based protocol (Delatte et al. 2005). Diagnostic microsatellite loci A preliminary examination of diagnostic loci was performed by genotyping insects from rearings of well-characterised Med and MEAMI B. tabaci species and from field

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samples. The insects were assigned to Med or MEAMI according to the match between their COI sequences and GenBank COI sequences, and their COI–RFLP restriction patterns (Bosco et al. 2006; Tsagkarakou et al. 2007). For loci BtIs1.2 and BtIs1.9 (Gauthier et al. 2008), mean allele sizes were, respectively (312.7 ± 3.9, 271.6 ± 2.8 bp) for the Med specimens and (337.5 ± 5.1, 279.6 ± 5.3 bp) for the MEAMI specimens (N. Gauthier and D. Saleh, unpublished data). Microsatellite polymerase chain reaction (PCR) amplification and genotyping The 654 B. tabaci females collected in the Sahel and Kebili regions (the 23 samples collected in 2006–2007) were genotyped using nine microsatellite loci previously described in Gauthier et al. (2008). The other 129 females collected in the Cap Bon and Tozeur regions (five samples collected in 2008) were screened using only two microsatellite loci (BtIs1.2 and BtIs1.9) out of the nine that distinguished MEAMI and Med specimens (Table 1). PCR reactions of the three multiplex microsatellite sets were performed as described in Gauthier et al. (2008) with slight modifications [Table S1 in Electronic Supplementary Material (ESM)]. Diluted PCR products were run on a Megabace 1000 DNA sequencer using the ET400R size standard. Alleles were scored using the Megabace Genetic Profiler Software and manually confirmed.

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Data analysis Hardy–Weinberg and linkage testing, marker polymorphism Putative linkage disequilibrium (LD) and deviation from Hardy–Weinberg equilibrium (HWE) between each pair of microsatellite loci measured with the FIS estimator (Weir and Cockerham 1984) were estimated using the ‘‘HW exact test’’ and a Markov chain protocol (GENEPOP software: Raymond and Rousset 1995). To correct for multiple testing, the P value considered to be significant was adjusted from 0.05 by sequential Bonferroni correction for both LD and HWE tests. When deviation from HWE was detected, departure from panmixia due to the presence of null alleles or/and scoring errors was estimated (MICROCHECKER program: van Oosterhout et al. 2004; FreeNA package: Chapuis and Estoup 2007). Standard indices of genetic variability for each microsatellite locus [number of alleles, allele size range, mean observed (Ho) and unbiased expected heterozygosities (He) (Nei 1978)] (GENEPOP: Raymond and Rousset 1995) and genetic variations between the forty-three 676 bp COI fragments obtained [number of haplotypes, polymorphic sites, haplotype (h) and nucleotide (p) diversities and their standard deviations] were evaluated (DNAsp package: Rozas et al. 2003). Inferring population structure: Bayesian and phylogenetic approaches

MtDNA PCR amplification and sequencing Confirming B. tabaci species status also involved COI fragment gene sequencing of at least one individual per sample. PCR amplifications were conducted in a 25-ll volume containing 2.5 ll of one DNA template, 19 Qiagen amplification buffer, 2 mM MgCl2, 0.16 lM of dNTPs, 0.5 lM of each primer (Fro¨hlich et al. 1999), one unit of Taq polymerase (Qiagen), and ultrapure water. PCR reactions were performed as follows: an initial denaturing step of 5 min at 94°C, 34 cycles comprising a 1-min denaturing step at 94°C, a 1-min annealing step at 55°C, a 1-min elongation step at 72°C, and a final 10-min elongation step at 72°C. Both strands were directly sequenced with amplification primers using the Big-Dye Terminator Sequencing Kit (Applied Biosystems Inc.) and an ABI 3730XLÒ DNA analyser (Beckman Coulter Genomics). The resulting consensus sequences were aligned before being deposited in GenBank (Accession no. HM807538– HM807580) and analysed. A total of 43 B. tabaci out of the 783 analysed with at least the two diagnostic microsatellite loci (Table 1) were partially sequenced revealing a shared 676-bp COI amplified fragment.

STRUCTURE software v2.3 (Pritchard et al. 2000) delineates clusters on the basis of individual genotypes at multiple loci using a Bayesian approach implemented in a Markov chain Monte Carlo (MCMC) algorithm. The most likely value of K (number of genetic populations) and quantification Q (individual assignment) of how likely each individual is to belong to each genetic population was estimated using the admixture model with correlated allele frequencies, and using (or not) prior location information (Hubisz et al. 2009). For each run, a burn-in period of 8 9 105 iterations followed by 8 9 105 MCMC iterations was applied. Log-likelihood estimates [lnP(D)] were calculated for K = 1–20. Five replicates were performed to test the consistency of the results. The lowest value of K that best expressed the population structure was taken as the best estimate (Pritchard et al. 2000, Fig. S1 in ESM). The data set was then sub-divided into the defined K populations and reanalysed by STRUCTURE with K = 1 to K = 6, using prior sample information (LocPrior = 1) and five runs each to delineate possible sub-populations. We also examined the value of r, which parameterizes the amount of information carried by the locations. Values of

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r [ 1 indicate that either there is no population structure or that the structure is independent of the location. As a complementary way of distinguishing population structure, phylogenetic COI-based trees were built using neighbor-joining (NJ) genetic distance and character-based maximum likelihood (ML) methods (PAUP*: Swofford 2002). For ML, the jMODELTEST (Posada 2008) was used to determine the most suitable model of DNA substitution. Based on the Akaike information criterion (AIC), the TIM3 ? I ? G model was determined as the statistically appropriate model for the 81-COI sequence set (c = 0.603) (see below). For NJ analysis, GTR distances, i.e., the closest distance to the TIM model, were used. Phylogenetic analyses were performed using a heuristic search with a tree-bisection-reconnection (TBR) random branch-swapping algorithm. Branch supports were assessed by bootstrap resamplings (1000 for NJ, 500 for ML). Phylogenetic trees were constructed with 43 COI sequences from the present study, 36 homologous sequences from different B. tabaci species retrieved from GenBank, and rooted with two homologous sequences from B. afer (AJ784260, AF418673). Levels and factors of genetic differentiation To assess the level of genetic differentiation among the resulting genetic populations (K), and between the 23 samples analysed with all nine loci, corrected pairwise estimators of FST values were calculated from each microsatellite dataset potentially harbouring null alleles following 1000 bootstrap replications across loci (FreeNA: Chapuis and Estoup 2007). The significance of genotypic differentiation between pairs of samples was assessed using Fisher’s exact test (GENEPOP: Raymond and Rousset 1995). In addition, hierarchical analyses of molecular variance (AMOVA) were performed (ARLEQUIN: Excoffier et al. 2005) to separately test for the respective significance of B. tabaci status, sampling site and host plant components in the genetic structure observed. These tests were performed on the individuals from the previous 23 samples with all nine microsatellite loci and also without the two diagnostic loci to estimate their possible impact when the significance of the other factors was assessed. Associated P values were calculated with 1000 permutations. The test for geographical structure was supplemented by an isolation-bydistance analysis using the previous 23 samples. The relationship between genetic and geographic distances was assessed using a regression of FST/(1 - FST) values against the log(geographical distance between each sampling site). The significance of the correlation between the two data matrices was assessed using a Mantel test with 1000 permutations (GENEPOP: Raymond and Rousset 1995).

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Patterns of genetic diversity and distribution Standard indices of genetic variability across all nine loci [mean number of alleles, FIS, FST, Ho, He] were estimated for each sample and the genetic populations identified for each region and host-plant species whatever the collection date (GENEPOP: Raymond and Rousset 1995). The prevalence of each genetic population identified at each sampling site, region and host-plant species was illustrated by the mean allele size (±SE) observed in the BtIs1.2 and BtIs1.9 loci across all individuals, and percentages. G tests of independence (a = 5%) were performed to test whether the observed proportions of each genetic population were independent of the host-plant species and regions.

Results Hardy–Weinberg and linkage testing, marker polymorphism After applying the Bonferroni correction, no evidence of significant LD remained (5/828 tests with P \ 6.10-5). As a result, the nine microsatellite loci segregated independently. Significant deviations from HWE were expressed at five loci out of the nine used (Table S1 in ESM) and all samples (Table 2) through moderate to extremely high FIS values (0.19 \ FIS/LOCUS \ 0.77; data not shown; 0.19 \ FIS/SAMPLE \ 0.50, Table 2) and significant heterozygote deficiencies (Tables S1–S2 in ESM). The heterozygote deficiencies did not result from significant frequencies of scoring errors (i.e., few stuttering errors for four loci and some samples) and null alleles (10.5% with f \ 0.20). All microsatellite loci were polymorphic. The number of alleles per locus ranged from five to 33 with the two diagnostic loci (BtIs1.2 and BtIs1.9) being the most polymorphic. A total of 117 alleles were identified across the 654 unrelated specimens we genotyped. Apart from species identification, no diagnostic allele associated with the species of host plant or with the region was identified. The 43 COI fragments sequenced revealed six haplotypes with overall high genetic diversity (h = 0.726 ± 0.047, p = 0.027 ± 0.001) and polymorphism (5.8% with 95% of parsimony informative sites). The six haplotypes consisted of two distinct and very well supported groups with 4.9–5.4% of nucleotide differences between the two groups and \1% within each group (Fig. 2b). Identification of genetically distinct populations Based on Bayesian clustering analyses using (mean r = 1.72) prior information on location (or not), two genetic populations (K1 and K2) were identified: for K = 1

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Table 2 Genetic characteristics and species prevalence (%) of the Tunisian samples analysed using the nine microsatellite loci or only the two diagnostic loci for which the reference mean allele sizes for the Med and MEAMI species were added Sample

9 loci  A

The Northern region

The Southern region

FIS

He (SE)

Ho (SE)

BtIs1.2 (bp) (m ± SE)

BtIs1.9 (bp) (m ± SE)

Percentage of Med/MEAMI

Gro1-L

317.0 ± 9.6

270.3 ± 1.9

96.7/3.3

Tak1-T

334.4 ± 13.2

279.8 ± 4.3

16.6/83.4

Mon1-L

5.33

0.41

0.498 (0.202)

0.297 (0.213)*

319.8 ± 10.9

270.0 ± 3.2

100/0

Teb2-L

5.44

0.24

0.440 (0.240)

0.337 (0.289)*

316.4 ± 9.1

270.2 ± 1.8

100/0

Sou1-L

4.00

0.32

0.450 (0.255)

0.307 (0.262)*

315.9 ± 9.0

271.2 ± 2.9

94.7/5.3

ChM1-L Teb1-T

4.89 3.56

0.22 0.48

0.419 (0.199) 0.186 (0.213)

0.326 (0.243)* 0.098 (0.151)*

314.0 ± 4.1 339.7 ± 10.5

270.2 ± 1.6 281.6 ± 0.8

100/0 0/100

Nak1-T

2.89

0.50

0.192 (0.238)

0.097 (0.174)*

341.5 ± 13.3

281.3 ± 1.2

0/100

Bek1-T

3.22

0.30

0.173 (0.190)

0.122 (0.115)*

336.8 ± 7.7

281.8 ± 1.3

0/100

Nak2-T

2.89

0.33

0.144 (0.243)

0.096 (0.161)*

339.0 ± 10.3

281.7 ± 1.2

0/100

Teb2-E

2.67

0.38

0.153 (0.241)

0.097 (0.147)*

337.7 ± 5.8

281.0 ± 2.7

0/100

Teb3-E

2.89

0.31

0.120 (0.185)

0.083(0.130)*

338.2 ± 7.8

281.3 ± 2.8

0/100

Bek2-Z

3.22

0.26

0.208 (0.264)

0.154 (0.255)*

338.3 ± 9.4

282.7 ± 2.8

0/100

Teb4-Z

2.44

0.28

0.195 (0.251)

0.142 (0.198)*

339.0 ± 10.7

281.6 ± 1.1

0/100

Keb1-L

5.00

0.36

0.526 (0.242)

0.341 (0.249)*

314.6 ± 6.1

269.5 ± 3.4

100/0

Dou1-L

5.22

0.26

0.471 (0.238)

0.350 (0.252)*

317.0 ± 9.5

270.1 ± 1.3

100/0

Baz1-T

1.89

0.38

0.096 (0.202)

0.059 (0.111)*

340.1 ± 6.6

281.8 ± 0.9

0/100

Baz2-T

2.11

0.43

0.104 (0.242)

0.059 (0.144)*

340.8 ± 10.0

281.7 ± 1.0

0/100

Keb2-T

5.67

0.29

0.513 (0.228)

0.365 (0.230)*

315.2 ± 6.2

270.9 ± 2.8

96.7/3.3

Lim1-T

2.78

0.35

0.133 (0.220)

0.087 (0.157)*

340.7 ± 9.9

281.6 ± 1.2

0/100

Baz3-E Baz4-E

2.67 5.33

0.38 0.36

0.132 (0.224) 0.444 (0.199)

0.082 (0.132)* 0.288 (0.185)*

343.3 ± 11.1 315.2 ± 7.3

281.7 ± 1.23 270.4 ± 3.2

0/100 96.7/3.3

Lim2-Z

2.22

0.15

0.187 (0.215)

0.159 (0.205)*

337.1 ± 3.6

281.3 ± 2.0

0/100

Dou2-Z

4.11

0.19

0.428 (0.238)

0.345 (0.249)*

315.1 ± 7.3

270.5 ± 3.1

91.7/8.3

Deb1-Z

2.11

0.26

0.158 (0.253)

0.117 (0.179)*

Toz1-L

341.6 ± 8.3

281.8 ± 2.0

0/100

317.8 ± 10.1

270.1 ± 1.0

100/0

Toz2-T

316.3 ± 7.1

271.1 ± 2.2

100/0

EHa1-E

317.8 ± 8.9

270.3 ± 0.9

100/0

Ref. Med

312.7 ± 3.9

271.6 ± 2.8

100/0

Ref. MEAMI

337.5 ± 5.1

279.6 ± 5.3

0/100

Data of the prevalent species of B. tabaci are in bold  mean number of alleles per locus; He, unbiased expected heterozygosity; Ho, observed heterozygosity; m ± SE, mean allele size ± standard A, error (bp); SE, standard error * Significant heterozygote deficiency after Bonferroni correction (P \ 0.05)

to K = 3, the mean lnP(D) increased significantly (from 11269.5 to -7391.7) before reaching a plateau at a higher K (from -7186.5 to -6732.1) (Fig. S1a in ESM). When K [ 2, little information was gained by adding populations (Fig. S1b in ESM). All individuals were assigned to K1 or K2 with a high probability (Q C 0.80). K1 consisted of 249 individuals from L. camara samples, Keb2-T, Baz4-E and Dou2-Z, and K2 consisted of 405 individuals mainly from the other 14 samples (i.e., except one specimen each from Sou1-L, Keb2-T, Baz4-E and two specimens from Dou2Z). No potential hybrid specimens of K1 and K2 appeared

(Fig. 2a). No sub-populations were identified within K1 and K2. Regarding the phylogenetic trees, the existence of only two genetic populations of Tunisian B. tabaci (K1 and K2) was strongly supported (Fig. 2b). The GenBank sequences that showed the highest nucleotide identity with Tunisian B. tabaci individuals were from the two invasive MEAMI and Med species, which is consistent with results obtained with the mean allele size at loci BtIs1.2 and BtIs1.9 (Table 2). As a result, K1 represents Med individuals and K2 represents MEAMI individuals. The MEAMI Tunisian

123

268

Popul Ecol (2012) 54:261–274

a 1.0 0.8 0.6 0.4 0.2 0.0

K1 b B. afer B. afer

123

K2

Popul Ecol (2012) 54:261–274

269

b Fig. 2 Inference of population structure. a Results of Bayesian clustering analysis for the 23 samples; b NJ consensus tree based on GTR genetic distance, i.e., the closest genetic distance to the TIM model estimated by ML with jModelTest (TIM3 ? I ? G model, c = 0.603), showing the phylogenetic clusters among the 43 Tunisian B. tabaci 676-bp COI sequences, the 38 homologous sequences retrieved from GenBank from 36 B. tabaci, and 2 B. afer as outgroups. Bootstrap values (NJ/ML) over 60 are shown at the corresponding nodes. Abbreviations (sample acronym followed by the individual number in brackets) are as defined in Table 1. Sequences generated in this study are typed in black. K genetic population, Q1 western Mediterranean populations of the commonly named Q ‘biotype’, Q2 Middle Eastern populations of the commonly named Q ‘biotype’ (in Gueguen et al. 2010), B the commonly named B ‘biotype’, Med Mediterranean putative species, MEAM1 Middle EastAsia Minor 1 putative species (in Dinsdale et al. 2010)

COI-haplotypes). Likewise, samples with a prevalence of Med individuals displayed a higher level of genetic diversity than those with a prevalence of MEAMI individuals (Table 2). Within the same species, no significant difference in genetic diversity was linked with a particular region or host-plant species except in the case of B. tabaci sampled on L. camara. The mean number of alleles  G) in the pool of Med individuals sampled on observed (A L. camara was significantly higher than that of the other host plant species but observed heterozygosities were not significantly affected (Table 3).

specimens were closely related to common MEAMI specimens found worldwide. All the Tunisian Med specimens belonged to the Q1 group (western Mediterranean populations) as named in Gueguen et al. (2010). Some specimens were closely related to the Q1 B. tabaci commonly found in Mediterranean countries and in China, but most of the specimens we analysed appeared to be more typical of Tunisia (Fig. 2b).

In our survey (28 samples collected throughout Tunisia), 45.5 versus 54.5% (1 df, P \ 0.05) of the B. tabaci collected belonged to the Med and the MEAMI species, respectively. The two species were found in both regions of Tunisia whatever the collection date (Fig. 1; Tables 2, 3) but MEAMI specimens were significantly more frequent in the north and Med specimens in the south (Table 3). Their distribution at the sample scale was characterised by a high percentage of individuals belonging to either the Med or the MEAMI species (Fig. 1; Table 2), indicating little or no mixing between Med and MEAMI specimens at all the sites sampled in our survey. Regarding the host plant species, vegetables were found to contain both MEAMI and Med B. tabaci but with a significantly higher percentage of MEAMI specimens (Table 3). Tomato and courgette contained significantly more MEAMI individuals than aubergine. In contrast, whatever the region, the type of site and the collection date, except for two MEAMI/220 Med specimens, only Med specimens were found on the ornamental L. camara. The difference in distribution of MEAMI and Med specimens on the four host plants was highly significant (G = 453.5, 3 df, P \ 0.001) and did not follow a random distribution (Table 3).

Patterns and factors of genetic differentiation and diversity Regarding the 23 samples analysed with the nine microsatellite loci, a very high level of differentiation was revealed between the 249 Med individuals and the 405 MEAMI individuals (FST = 0.51) with FST values ranging from 0.31 to 0.58 depending on the samples (Table S2 in ESM). Low (FST-Q B 0.14) and low to moderate (0 B FSTB B 0.23) levels of genetic differentiation were revealed within the Med and MEAMI samples (Table S2 in ESM), even when they came from different regions and host plants (Table 3). AMOVA analyses of MEAMI and Med B. tabaci taken together and separately indicated that genetic variation was poorly structured as a function of the host plant (2.1% for MEAMI vs. 0.8% for Med) and geographical location (3.9% for MEAMI vs. 0.9% for Med) with most genetic variation explained by the species status (&28%) and the individuals themselves (&69%). This result is consistent with the results obtained using the Bayesian approach in which the value of r showed that no information was provided by the sampling location. Isolation of genetic populations and samples as a function of distance revealed no evidence of geographic structuring. Regarding the global genetic diversity of Med and MEAMI B. tabaci, all estimators of the Med species were significantly higher than those of the MEAMI species  Q = 9.8 vs. A  B = 7.9, Ho–Q (SE) = 0.332 ± 0.232 (A vs. Ho–B (SE) = 0.103 ± 0.140, P \ 0.05, number of

Patterns of spatial and host plant species distribution

Discussion Co-occurrence of two B. tabaci species in Tunisia Several species of the B. tabaci complex, such as the Med, MEAMI, T, M or S species as delineated by Dinsdale et al. (2010) are known to be present in the Mediterranean basin (Brown et al. 1995; Banks et al. 1998; Simo´n et al. 2003, 2007; Demichelis et al. 2005; Bosco et al. 2006; De la Ru´a et al. 2006; Tahiri et al. 2006; Tsagkarakou et al. 2007; Dalmon et al. 2008; Vassiliou et al. 2008; Bel-Kadhi et al. 2008). In Tunisia, all markers and approaches agree that only Med and MEAMI species are present: Med B. tabaci

123

123

8.33

South

5.44

4.89

3.56

Tomato

Aubergine

Courgette

0.388 ± 0.220

0.433 ± 0.189

0.508 ± 0.223

0.488 ± 0.225

0.492 ± 0.228

0.474 ± 0.216

0.362 ± 0.266

0.282 ± 0.194

0.378 ± 0.238

0.329 ± 0.241

0.341 ± 0.223

0.320 ± 0.244

0.023 \ FST \ 0.121

FST = 0.004

4.56

4.22

6.0

(–)

5.11

6.88

0.194 ± 0.244

0.143 ± 0.230

0.155 ± 0.236

(–)

0.150 ± 0.236

0.176 ± 0.213

0.143 ± 0.198

0.087 ± 0.132

0.090 ± 0.138

(–)

0.094 ± 0.134

0.109 ± 0.138

Observed percentages followed by different letters differ significantly (G test of independence, 1 df, P \ 0.05)

He, unbiased expected heterozygosity; Ho, observed heterozygosity; m ± SE, mean allele size ± standard error (bp); SE, standard error

The prevalent species is in bold (-) Data not given for the MEAMI B. tabaci from Lantana because it represented only 2 individuals

8.56

Lantana

Per host-plant

7.67

North

Per region

Ho (SE)

Pairwise FST

0 \ FST \ 0.059

FST = 0.015

17.9/82.1 b

N = 123

38.5/61.5 c

N = 148

20/80 b

N = 290

99.1/0.9 a

N = 222

N = 391 55/45 b

36.2/63.8 a

N = 392

Observed percentage

He (SE)

G A

G A Pairwise FST

N = 405

N = 249 Ho (SE)

Med/MEAMI species (28 samples) N = 783

MEAMI species (23 samples)

Med species (23 samples)

He (SE)

Distribution

Genetic diversity

Table 3 Patterns of genetic diversity and distribution of each species according to the region and host plant sampled

270 Popul Ecol (2012) 54:261–274

Popul Ecol (2012) 54:261–274

(specifically Q1, Gueguen et al. 2010) and MEAMI B. tabaci, on tomato, aubergine, and courgette, and on the ornamental, L. camara. In the present study, both B. tabaci species were well represented whereas previous preliminary studies based on only a few individuals mainly collected from tomato, suggested a higher prevalence of MEAMI than of non-MEAMI specimens, very probably Med (Chermiti et al. 1997; Bel-Kadhi et al. 2008). No hybrids were detected in our study, suggesting that they are genetically isolated in the field. This is consistent with (1) FST values for MEAMI and Med B. tabaci estimated at national and sampling scales in Tunisia (0.31 B FST B 0.58) and with the results of previous studies (Moya et al. 2001; Simo´n et al. 2007; Dalmon et al. 2008), (2) the percentage divergence between Med and MEAMI mtCOI haplotypes in the present study (4.9–5.4%) and in different parts of the world (Boykin et al. 2007; Dinsdale et al. 2010) which identifies them as two distinct species, and (3) the widely reported reproductive incompatibility between the two species (Elbaz et al. 2010; Xu et al. 2010; De Barro et al. 2011; Sun et al. 2011). Currently, although Med and MEAMI species are the most frequently observed species in the Mediterranean basin, they are often observed separately (Bosco et al. 2006; De la Ru´a et al. 2006; Tahiri et al. 2006; Tsagkarakou et al. 2007; Dalmon et al. 2008; Vassiliou et al. 2008). Where multiple species of the B. tabaci complex—above all Med and MEAMI—have co-occurred for some time, their distributions have often been reported to be highly dynamic in space and time. After a period of co-existence, only one species is detected in field surveys, because under specific conditions evaluated in the laboratory and using models, one species may be favoured over another (De Barro and Hart 2000; Moya et al. 2001; Pascual and Callejas 2004; Horowitz et al. 2005; De Barro et al. 2006; Liu et al. 2007; Delatte et al. 2009; Crowder et al. 2010a, b). Hence, in recent years, in Mediterranean countries such as Spain (Simo´n et al. 2007), France (Dalmon et al. 2008), Greece (Tsagkarakou et al. 2007), and Morocco (Tahiri et al. 2006), MEAMI species have been reported to have been supplanted or displaced by Med B. tabaci species. This makes Tunisia a particularly interesting case study because unlike in many Mediterranean countries, MEAMI and Med B. tabaci continue to co-exist and are well-represented at the national scale. Could this be just a question of time, since the displacement of one species by another may take a long time? (Wang et al. 2002). Patterns and factors of genetic diversity between MEAMI and Med species Med B. tabaci presented higher genetic diversity, with some novel Q1 haplotypes, than MEAMI B. tabaci in all

271

regions and on all host-plant species (Tables 2, 3; Fig. 2b). This difference has already been reported and explained by the fact that Med populations may have been present before the arrival of MEAMI populations (Moya et al. 2001; Tahiri et al. 2006). Under this hypothesis, are Med the indigenous species and MEAMI the invasive species, or are both invaders, with Med being the first to arrive in Tunisia? In Tunisia, the first sighting of the B. tabaci species complex has never been directly recorded. On the other hand, the first report of Tomato yellow leaf curl disease, a B. tabaci transmitted virus disease was in the 1980s (Cherif and Russo 1983). In the invasion history of B. tabaci worldwide, the first major event reported concerned MEAMI species, which commenced in the late 1980s from its region of origin in the Middle East/Asia Minor to at least 54 countries including Tunisia (De Barro et al. 2011). In recent years, the Med species has spread from its area of origin in the western Mediterranean region to the rest of the world (De Barro et al. 2011). Whether Tunisia is part of the home range of Med or not has never been ascertained but probably not considering its geographic position in the eastern Mediterranean basin. Thus Tunisia may have been invaded by both species. Loss of genetic diversity in B. tabaci populations has also been explained by high human pressure due to cropping systems with frequent cultivation practices and insecticide treatments (Delatte et al. 2009). Interestingly, in Tunisia, MEAMI B. tabaci, which displayed the lowest genetic diversity, were mainly sampled on the three cultivated vegetable crops. On the other hand, the highest genetic diversity was observed in Med specimens mainly sampled on lantana, the ornamental which is assumed to undergo less—at least intentional—human pressure than vegetables. Even though there was a difference in genetic diversity between Med specimens sampled on vegetables and those sampled on L. camara, no or little genetic differentiation was found (Table 3). Distribution patterns: regional co-existence but local exclusion, and host-plant partitioning MEAMI and Med B. tabaci were clearly seen to co-occur at the regional scale—and even within small areas—at all periods of the sampling survey, but to mutually exclude one another at the sample scale. Spatial overlapping was very rare (Fig. 1; Table 2). MEAMI and Med B. tabaci also exhibited resource partitioning between vegetables and lantana: 99.1% of the B. tabaci sampled on lantana were Med specimens (Table 3). The distribution of MEAMI and Med B. tabaci was so distinct that it obviously cannot be due to chance. Our results clearly showed that, in the Tunisian agricultural system, each species has a

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272

different habitat, which raises further questions regarding the factors involved in this outcome. Insecticide resistance has often been assumed to play a major role in the distribution of B. tabaci in agricultural systems. Thus, the Med species is reported to be prevalent over MEAMI species in agricultural environments, and this is usually explained by the reduced susceptibility of Med species to insecticides (Horowitz et al. 2005; Crowder et al. 2010a, b). In contrast, our study demonstrated that MEAMI was prevalent over Med on the vegetable crops, and not found at all on the ornamental outside the context of cropping systems. Since the most susceptible B. tabaci were found in environments that receive fewer insecticide treatments, the most susceptible species would logically be Med populations on lantana, clearly contradicting observations usually reported in the literature. Tunisian Med populations displayed low frequencies of resistance alleles for Kdr and Ace genes (N. Gauthier and A. Tsagkarakou, unpublished data), but to date, nothing is known about the Tunisian MEAMI populations’ resistance to insecticides. Consequently, if differential resistance to insecticides interferes in the distribution of B. tabaci in Tunisia, it may be in a different way from that usually described. Concerning plant resources, some B. tabaci populations are known to have a restricted host-plant range (De Barro et al. 2011) but, to the best of our knowledge, host-plant ‘‘specialization’’ or host plant preference has never been reported in the highly polyphagous MEAMI and Med species. Interestingly in our study, within Med B. tabaci, no genetic differentiation was found to be correlated with the use of lantana versus vegetables as host plants, suggesting that (1) ‘‘specialization’’, if it exists, is probably still in progress or is too recent to be genetically significant among the different populations and (2) mixing of Med B. tabaci even on different host plants and in different Tunisian regions still exists, mainly due to human activities, as no isolation by distance was revealed by our analyses. Finally, host-plant species could influence the population dynamics or distribution on whitefly resources (Mun˜iz 2000; De Barro et al. 2006, 2011; Brown 2007; Naveed et al. 2007; Delatte et al. 2009). Weeds have widely been considered to be alternative hosts to vegetable crops and as shelter for insects, and in whitefly management schemes, some weed species are intentionally included as trap crops for parasitoids (Bel-Kadhi and Onillon 2006; Naveed et al. 2007). Lantana is known to be an alternative host for several agricultural insect pests but also has insecticidal or/ and repellency effects on other insect species (Dua et al. 1996; Day et al. 2003). As a result, if lantana plays the role of a shelter, it is only for Med B. tabaci. The reasons (fitness in particular) for the absence of MEAMI specimens on lantana remain to be clarified.

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Habitat partitioning and mechanisms behind the coexistence of Med and MEAMI species Habitat partitioning among seemingly similar species which interact for resources and space has been proposed as a mechanism that limits interference and potential competition at small spatial scales, and thus favours the coexistence of species at a larger spatial scale (Chesson 2000; Gilbert et al. 2008; Debout et al. 2009). In our study, the distribution pattern of Med and MEAMI, two species that have been shown to strongly interfere with one another under laboratory conditions, is consistent with the general expectations of the competitive exclusion principle. In particular, asymmetric reproductive interferences have been suggested as a key to the displacement and population structure of B. tabaci species in nature (Pascual and Callejas 2004; Liu et al. 2007; Crowder et al. 2010a, b; De Barro et al. 2011). More recently, a B. tabaci species, the stronger competitor, was shown to preferentially use a resource that was unacceptable to its competitor and to avoid a host that was mutually acceptable (De Barro et al. 2010; De Barro and Bourne 2010). Such behaviour enables a species to take refuge in a competition-free space, and has been suggested to foster long-term co-existence. Our results suggest that no interference occurs on lantana, as only Med specimens were collected there. By contrast, both Med and MEAMI were found on the other three vegetable species, meaning these were surely acceptable for both of them and hence potential sources of interference. Is our situation the same as that described by De Barro et al. (2010) and De Barro and Bourne (2010) in which Med species apparently chose a host plant (lantana) which is unacceptable for MEAMI, in an attempt to avoid interfering with its competitor? In the end, competition and/or its limitation could be the driving force behind the distribution pattern observed in Tunisia, even though this is difficult to demonstrate unequivocally under natural conditions.

Concluding remarks and future outlook To the best of our knowledge, our study is one of the rare field studies to report that the MEAMI and Med B. tabaci species, probably both invaders in Tunisia, co-occur with comparable prevalence in a single country. The distribution pattern demonstrated in the field on four different host plants is consistent with the general expectations of competition theory suggesting that competition and its limitation could be the driving force here. For the first time, L. camara clearly emerged as an important factor in mediating the overall population structure of the B. tabaci species complex as it plays a leading role in the distribution of Med and MEAMI species on their resources.

Popul Ecol (2012) 54:261–274

Based on our case study, some interesting questions arise regarding the factors and events which may have influenced the outcome in many Mediterranean countries (i.e., an initial period of co-existence followed by the quasi exclusion of MEAMI by Med species). Is Tunisia representative of what previously prevailed in other Mediterranean countries? If so, we would expect one species to displace the other in the future. If not, to what extent do Tunisian populations differ from the other Mediterranean populations? Many exciting new hypotheses emerged from this research and should be the subject of laboratory testing and future field investigations, including specialization on L. camara, and the role of other ornamentals in the distribution of B. tabaci. Acknowledgments This study was mainly funded by the ‘‘Institut de Recherche pour le De´veloppement’’ through a grant for the Master student D. Saleh and some financial support for our associate researcher A. Laarif from the Tunisian ‘‘J.E.A.’’, and by a French National Research Agency Program (BemisiaRisk project 06-PADD04). The authors thank the reviewers and the editor for their useful comments which helped us improve the manuscript.

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