Géraldine Roux-Morabito

N E Gillette

A Roques

L Dormont

J Stein

F A H Sperling

Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA

Keywords: Dioryctria, mtDNA, COI, COII, seed orchard

Coneworms of the genus Dioryctria Zeller include a number of serious pests of conifer seeds that are notoriously difficult to distinguish as species. We surveyed mitochondrial DNA variation within the abietella species group by sequencing 451 bp of COI and 572 bp of COII genes from 64 individuals of 6 major species in the group. In addition to examining phylogenetic relationships within European members of the group, the study focused on the two most damaging species, D. abietivorella Grote from North America and D. abietella Denis and Schiffermüller from Europe and Asia, which have been considered taxonomically synonymous in the past. In order to detect different levels of divergence, we extensively sampled in seed orchards and natural forests for D. abietella on different hosts. Maximum parsimony and maximum likelihood analyses confirmed the monophyly of the abietella species group and its separation into three clades. The grouping of North American species (Clade A) received strong support in both analyses, whereas relationships between Clade A and the two other European clades were weakly supported. D. simplicella Heinemann could not be unambiguously separated from D. abietella populations. The diverse haplotypes observed in the network analysis conducted with eight populations of polyphagous D. abietella suggested the presence of two distinct lineages in France.

Introduction

The greater diversity of phytophagous insect clades compared to their nonphytophagous sister groups has lead biologists to postulate that host plants strongly influence the diversification and speciation of herbivorous insects [START_REF] Kelley | Strong differentiation in mitochondrial DNA of Dendroctonus brevicomis (Coleoptera: Scolytidae) on different subspecies of Ponderosa pine[END_REF]. Two patterns of association between phytophagous insects and their host plants exist, with some species using a diversity of host taxa (polyphagy), and others being restricted to one particular plant species (monophagy). According to numerous authors [START_REF] Bush | Modes of animal speciation[END_REF][START_REF] Mitter | Population genetic consequences of feeding habits in some forest Lepidoptera[END_REF][START_REF] Diehl | An evolutionary and applied perspective of insect biotypes[END_REF][START_REF] Tauber | Sympatric speciation in insects: perception and perspective[END_REF][START_REF] Dres | Host races in plant-feeding insects and their importance in sympatric speciation[END_REF][START_REF] Rundle | Ecological speciation[END_REF], changes in host preference can be critical to the formation of new species. Such genetic differentiation has been associated with host use in several polyphagous species, which frequently consist of locally specialized populations, races or even sibling species complexes [START_REF] Menken | Pattern and process in the evolution of insect-plant associations: Yponomeuta as an example[END_REF]. Lepidoptera include numerous examples of species complexes in which there are evolutionarily significant entities that may or may not represent species [START_REF] Sperling | Butterfly molecular systematics: from species definitions to higher level phylogenies[END_REF][START_REF] Wahlberg | Phylogenetic relationships of Phyciodes butterfly species (Lepidoptera: Nymphalidae): complex mtDNA variation and species delimitations[END_REF]. Because morphological characters of such related species or subspecies are very hard to distinguish, their autecology and host plants are often used for taxonomic identification, but the use of such labile characters raises doubts about the validity of the taxonomic status of these taxa.

Coneworms of the genus Dioryctria Zeller (Lepidoptera: Pyralidae) comprise several species groups within which numerous species have been identified mainly on the basis of their larval host-plant, but also by forewing morphology and geography [START_REF] Neunzig | Pyraloidea, Pyralidae (part), Phycitinae (part)[END_REF].

These coneworms are serious pests of conifer seed cones in the Holarctic region [START_REF] Turgeon | Insect fauna of coniferous seed cones -Diversity, host-plant interactions and management[END_REF], where over 70 species have been recorded [START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF][START_REF] Roe | Identification of Dioryctria (Lepidoptera: Pyralidae) in a seed orchard at Chico, California[END_REF]. Most of them are associated with the Pinaceae, especially with Pinus Linnaeus, Picea A. Dietrich, Abies Miller, Larix Miller, and Pseudotsuga Carrière species [START_REF] Hedlin | Cone and Seed insects of North America Conifers[END_REF][START_REF] Roques | Les insectes ravageurs des cônes et graines de conifères en France[END_REF], Cibrian-Tovar et al. 1986[START_REF] Neunzig | A new species of Dioryctria (Pyralidae: Phycitinae) from Mexico[END_REF][START_REF] Turgeon | Management of Insect Pests of Cones in Seed Orchards in Eastern Canada[END_REF], with a few Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. [START_REF] Merkel | The Baldcypress coneworm Dioryctria pygmaeella Ragonot (Lepidoptera: Pyralidae)[END_REF]. Because these species may drastically limit crops of genetically superior seeds in seed orchards, their biology has been studied extensively during the past forty years [START_REF] Lyons | Insetcs affecting seed production in red pine. II. Dioryctria disclusa Heinrich, D. abietella (D. & S.), and D. cambiicola (Dyar.) (Lepidoptera: Phycitidae)[END_REF][START_REF] Zocchi | Contributi alla conoscenza degli insetti delle piante forestali. V. Il gen. Dioryctria Zell. (Lepidoptera, Pyralidae) in Italia[END_REF][START_REF] Neunzig | Observations on the biology of four species of Dioryctria in North Carolina (Lepidoptera: Phycitidae)[END_REF][START_REF] Charles | Observations sur la biologie et l'écologie de Dioryctria mutatella Fuchs (Lepidoptera Phycitidae), ravageur des pousses et des cônes de Pin sylvestre en forêt de Fontainebleau[END_REF][START_REF] Grant | Sexpheromone identification and trapping of Dioryctria resinosella (Lepidoptera, Pyralidae)[END_REF][START_REF] Millar | 3Z, 6Z, 9Z, 12Z, 15Z)-pentacosapentaene, a key pheromone component of the fir coneworm moth Dioryctria abietivorella[END_REF].

However, a paucity of clear morphological characters for separating Dioryctria taxa creates taxonomic uncertainties that hinder the analysis of their host relationships [START_REF] Chatelain | Seasonal attack periods of cone-feeding insects of loblolly pine cones[END_REF][START_REF] Hedlin | Cone and Seed insects of North America Conifers[END_REF][START_REF] Sopow | Identification of the "grey' Dioryctria species of British Columbia (Lepidoptera: Pyralidae)[END_REF]. Using a combination of external characters and adult genitalia, [START_REF] Mutuura | American species of Dioryctria (Lepidoptera: Pyralidae) III. Grouping of species: species of the auranticella group, including the Asian species, with the description of a new species[END_REF] defined seven species groups but could not definitively place all the studied species into groups [START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF]). Among these groups, the abietella species group comprises 13 species and is defined by the absence of raised scales on the forewings, a feather-like maxillary palpus in the male, and a narrow valva in the male genitalia. According to Mutuura andMunroe (1972, 1973) and [START_REF] Neunzig | Pyraloidea, Pyralidae (part), Phycitinae (part)[END_REF], this group includes the widespread Palaearctic species D. abietella Denis and Schiffermüller, and species from Europe (D. pineae Staudinger, D. mendacella Staudinger, D. simplicella Heinemann= D. mutatella Fuchs; [START_REF] Fazekas | Systematisches und synonymisches Verzeichnis der Microlepidopteren Ungarns (Lepidoptera: Microlepidoptera)[END_REF], North Africa (D. alloi Barbey, D. peyerimhoffi Joannis), Asia (D. stenopterella Amsel, D. assamensis Mutuura, D. raoi Mutuura), North America (D. abietivorella Grote, D. ebeli Mutuura and Munroe, D. pinicolella Amsel), and Central America (D. sysstratiotes Dyar).

Dioryctria abietella and D. abietivorella are undoubtedly the most important lepidopteran pests of conifer cones in Europe and North America [START_REF] Roques | Les insectes ravageurs des cônes et graines de conifères en France[END_REF][START_REF] Hedlin | Cone and Seed insects of North America Conifers[END_REF]. They both have a wide host-range for larval development. So far, D. abietella has been recorded from western Europe and Scandinavia to the Russian Far East and northern China on a broad range of hosts, including cones of pine, spruce, larch, fir and Douglas fir, and more rarely twigs, buds, and adelgid-induced galls [START_REF] Roques | Les insectes ravageurs des cônes et graines de conifères en France[END_REF]. D. abietivorella has been reported from Alaska to central Mexico, and from California to Newfoundland, on more Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

Manuscrit d'auteur / Author manuscript

Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 5 than 20 different hosts, including pine, spruce, and Douglas fir [START_REF] Lyons | Insetcs affecting seed production in red pine. II. Dioryctria disclusa Heinrich, D. abietella (D. & S.), and D. cambiicola (Dyar.) (Lepidoptera: Phycitidae)[END_REF][START_REF] Hedlin | Cone and Seed insects of North America Conifers[END_REF], Turgeon and de Groot 1992). Because these two species are generally similar in genital characters, they have variously been considered under the names D. abietella, D. assamensis and D. raoi [START_REF] Munroe | Canadian species of Dioryctria Zeller (Lepidoptera: Pyralidae)[END_REF][START_REF] Byun | Taxonomic Notes of Dioryctria abietella and D. sylvetrella (Lepidoptera: Pyralidae) in Korea[END_REF].

More recently, there has been similar confusion among European Dioryctria species such as D. abietella, D. simplicella, and even species outside the abietella group such as D.

schuetzeella Fuchs [START_REF] Charles | Observations sur la biologie et l'écologie de Dioryctria mutatella Fuchs (Lepidoptera Phycitidae), ravageur des pousses et des cônes de Pin sylvestre en forêt de Fontainebleau[END_REF]Roques 1977, Roques 1983). For example, D. resiniphila Segerer and Pröse was recently identified in cones of Abies cephalonica [START_REF] Segerer | Dioryctria resiniphila sp. nov., eine neue Pyralide auf Abies cephalonica Loud. in Griechenland (Lepidoptera: Pyralidae: Phycitinae). -NachrBl. bayer[END_REF] in Greece, but in the past these coneworms had been identified as D. abietella.

Species-specific treatments such as semiochemicals [START_REF] Debarr | Synthetic pheromones disrupt male Dioryctria spp. moths in a loblolly pine seed orchard[END_REF] or pathogens [START_REF] Verma | Natural enemies of Dioryctria abietella Denis & Schiff., a pest of conifers in the north-west Himalaya[END_REF][START_REF] Perez | Pr 1-3, a new entomopathogenic isolate of Beauveria bassiana (Bals.) Vuill. in Cuba[END_REF][START_REF] Glynn | Bacillus thuringiensis variety kurstaki x aizawai applied to spruce flowers reduced Dioryctria abietella (Lepidoptera: Pyralidae) infestation without affecting seed quality[END_REF] are frequently required for the control of such cryptic pests, which are not amenable to control using pesticides without resorting to highly toxic organophosphate and carbamate insecticides [START_REF] Bhandari | Chemical control of cone worm, Dioryctria abietella, infesting cones of silver fir (Abies pindrow) by systemic insecticides[END_REF] or multiple injections of systemic insecticides [START_REF] Grossman | Systemic insecticide injections for control of cone and seed insects in Loblolly Pine seed orchards-2 year results[END_REF]. The success of such species-specific treatments relies upon correct pest species diagnoses. Thus, it is important to focus on clarifying the status of taxonomically intractable taxa like Dioryctria species groups, so that effective control measures can be tailored to each species.

In taxonomic groups where morphological identification is difficult, molecular characters, particularly mtDNA, can help in assessing species boundaries (Sperling and Hickey 1994[START_REF] Brower | Delimitation of phylogenetic species with DNA sequences: a critique of Davis and Nixon's population aggregation analysis[END_REF][START_REF] Caterino | The current state of insect molecular systematics: A thriving tower of Babel[END_REF][START_REF] Templeton | Using phylogeographic analyses of gene trees to test species status and processes[END_REF][START_REF] Kerdelhué | Le séquençage des acides nucléiques et les méthodes d'analyse des données moléculaires en phylogénie[END_REF], Hebert et al. 2003a[START_REF] Wahlberg | Phylogenetic relationships of Phyciodes butterfly species (Lepidoptera: Nymphalidae): complex mtDNA variation and species delimitations[END_REF], Roe and Sperling 2007b). Several properties make mtDNA a good marker of species limits [START_REF] Avise | Ten unorthodox perspectives on evolution prompted by comparative populations genetic findings on mitochondrial DNA[END_REF][START_REF] Sperling | Butterfly molecular systematics: from species definitions to higher level phylogenies[END_REF][START_REF] Wahlberg | Phylogenetic relationships of Phyciodes butterfly species (Lepidoptera: Nymphalidae): complex mtDNA variation and species delimitations[END_REF]. It is a non-recombining, maternally inherited genome, with a smaller effective population size that leads to shorter coalescence times [START_REF] Moore | Inferring phylogenies from mtDNA variation : mitochondrial-gene trees versus nuclear-gene trees[END_REF]. Moreover, gene trees may be more likely to reflect species trees when using mitochondrial markers [START_REF] Avise | Phylogeography: The History and Formation of Species[END_REF] Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. ). Thus, the rapid evolution of mtDNA sequences has often been used in intraspecific studies [START_REF] Avise | Phylogeography: The History and Formation of Species[END_REF] as well as in investigating relationships of closely related species in Lepidoptera [START_REF] Bogdanowicz | Asian gypsy moths (Lepidoptera: Lymantriidae) in North America: evidence from molecular data[END_REF][START_REF] Brower | Practical and theoretical considerations for choice of a DNA sequence region in insect molecular systematics, with a short review of published studies using nuclear gene regions[END_REF][START_REF] Brower | Delimitation of phylogenetic species with DNA sequences: a critique of Davis and Nixon's population aggregation analysis[END_REF][START_REF] Brown | Phylogeny of Greya (Lepidoptera: Prodoxidae) based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data[END_REF], Sperling and Hickey 1994[START_REF] Sperling | DNA-based identification of introduced ermine moth species in North America (Lepidoptera: Yponomeutidae)[END_REF][START_REF] Landry | Systematics of the Argyotaenia franciscana ( Lepidoptera: Tortricidae) species group: evidence from mitochondrial DNA[END_REF][START_REF] Kruse | Molecular phylogeny within and between species of the Archips argyrospila complex (Lepidoptera: Tortricidae)[END_REF][START_REF] Sperling | Butterfly molecular systematics: from species definitions to higher level phylogenies[END_REF][START_REF] Wahlberg | Phylogenetic relationships of Phyciodes butterfly species (Lepidoptera: Nymphalidae): complex mtDNA variation and species delimitations[END_REF][START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF], Roe and Sperling 2007a, 2007b). So far no molecular study has been performed across the widespread and economically important European D. abietella coneworm group; and genetic and biochemical analyses of Dioryctria have so far been primarily limited to North American and Chinese species [START_REF] Richmond | Genetic and biochemical similarities among four species of pine coneworms (Lepidoptera: Pyralidae)[END_REF][START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF][START_REF] Roe | Identification of Dioryctria (Lepidoptera: Pyralidae) in a seed orchard at Chico, California[END_REF].

In this paper we review species delimitations and phylogenetic relationships within the Dioryctria abietella species group based on mtDNA sequences. We give special emphasis to the taxonomic status of the two most damaging Dioryctria species of this group, namely European D. abietella and North American D. abietivorella.

Materials and methods

Coneworm collections

A total of 67 specimens were selected for this study, 61 within the Dioryctria abietella species group (29 specimens identified as D. abietella, nine D. mendacella, two D. pineae, eight D. simplicella, 11 D. abietivorella and two D. ebeli) and six additional specimens representative of three other Dioryctria species groups (two specimens of the pine stem borer D. sylvestrella Rartzeburg belonging to the sylvestrella group, two specimens of D. amatella Hulst belonging to the zimmermani group, one specimen of D. pseudotsugella Munroe and one of D. reniculelloides Mutuura and Munroe belonging to the schuetzeella group). The list of corresponding larval hosts and locations is presented in Table 1. In order to ascertain insect-host relationships, only insects reared from identified host cones were considered. We Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

Manuscrit d'auteur / Author manuscript

Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 7 carried out most sampling, but some specimens were provided by collaborators. In addition, three published sequences were incorporated in order to relate our study to previous publications (Table 1). These included one D. abietella from China [START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF], and two D. abietivorella from Chico, California [START_REF] Roe | Identification of Dioryctria (Lepidoptera: Pyralidae) in a seed orchard at Chico, California[END_REF].

Sampling of D. abietella was designed to survey different potential levels of divergence. Populations were defined according to the larval host tree and collecting localities. Thus, sympatric populations of D. abietella developing simultaneously on different hosts were sampled on three locations in France (Fig. 1): (i) in a natural forest with mixed Pinus cembra (Linnaeus) and Picea abies (Linnaeus) in the northern Alps; (ii) in a seed orchard that included the native species Picea abies and Larix decidua (Miller), and the exotic species Pseudotsuga menziesii (Mirbel) Franco at Latronquière (south-central France); and (iii) in an arboretum that included, among other species, the exotic species Picea smithiana (Wall.) and Pinus koraiensis Linnaeus) (Les Barres, northern-central France). No natural forests surrounded the Latronquière seed orchard.

Dioryctria larvae were extracted from damaged cones, and either reared until adult emergence or killed immediately in 95% ethanol and kept at -80°C until DNA extraction.

Adults were identified using morphological descriptions from [START_REF] Zocchi | Contributi alla conoscenza degli insetti delle piante forestali. V. Il gen. Dioryctria Zell. (Lepidoptera, Pyralidae) in Italia[END_REF] and Mutuura andMunroe (1972, 1973), or on the basis of the larval host [START_REF] Charles | Observations sur la biologie et l'écologie de Dioryctria mutatella Fuchs (Lepidoptera Phycitidae), ravageur des pousses et des cônes de Pin sylvestre en forêt de Fontainebleau[END_REF] when morphological identification was uncertain and compared with adult DNA from the same species (four larval specimens were identified as D. mendacella and five larvae identified as D. simplicella).

DNA protocols

The methods used for DNA extraction, amplification using the polymerase chain reaction (PCR), and sequencing follow Sperling et al. (1994). Genomic DNA was extracted Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. One microliter of extracted DNA was used as template for amplification of mtDNA fragments by PCR following methods primarily developed for spruce budworm (Sperling and Hickey 1994). Using Promega Taq, 30 cycles of amplification were performed as follows in 50 μl reaction volumes: denaturation step at 94°C for 1 min, annealing step at 45°C for 1 min, and extension step at 72°C for 1 min 30 sec. An initial cycle employed a 3 min denaturation at 95°C and a final cycle had an extension step of 72°C for 5 min.

Overlapping sections of a 2272-bp region of one individual of each of Dioryctria abietivorella and D. reniculelloides, and 1975-bp for two specimens of D. abietella that are respectively homologous to bases 1457-3729 and 1754-3729 in Drosophila yakuba Burla [START_REF] Clary | The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code[END_REF] were PCR-amplified using heterologous primers (list in Table 2). This region includes the gene coding for the cytochrome oxidase subunit 1 (COI), and extends through the tRNA leucine gene, and ends in the cytochrome oxidase subunit 2 gene (COII). An additional 60 samples, representing one to five specimens per population, were sequenced over 451 bp of COI (primers N°4, 7) and 572 bp of COII (primers N°10, 12).

Both strands of the PCR product were sequenced for all samples. Fragments were sequenced directly using Big Dye Terminator (Applied Biosystems) and detected with an ABI 377 automatic sequencer (Applied Biosystems, Foster City, CA).

Data analysis

DNA contigs were constructed using Sequence Navigator (Applied Biosystems) and aligned manually with published sequences of Dioryctria abietella from China and D.

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

Manuscrit d'auteur / Author manuscript

Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 9 abietivorella from USA [START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF][START_REF] Roe | Identification of Dioryctria (Lepidoptera: Pyralidae) in a seed orchard at Chico, California[END_REF]. Sequences of two species in the Phycitini, Oncocera faecela and Ceroprepes ophthalmicella [START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF] were used as outgroups to root Dioryctria.

Maximum parsimony (MP) and maximum-likelihood (ML) phylogenetic analysis were performed with PAUP*4b10 [START_REF] Swofford | Phylogenetic analysis using parsimony[END_REF]. For maximum parsimony analysis, a heuristic search was implemented with the tree bisection-reconnection (TBR) branchswapping option. Variable nucleotide positions were treated as unordered characters with one state for each nucleotide base. The relative level of support for each phylogenetic grouping was assessed with the bootstrap method [START_REF] Felsenstein | Confidence limits on phylogenies: an approach using the bootstrap[END_REF]. For analysis, MODELTEST v3.07 [START_REF] Posada | MODELTEST: testing the model of DNA substitution[END_REF] was used to determine the model of evolution across the COI and the COII genes. To test for homogeneity of our data set, we used a partition homogeneity test (ILD) for detecting incongruence caused by differences in evolutionary constraints and/or tree topologies [START_REF] Farris | Testing significance of incongruence[END_REF]. We performed the ILD test in PAUP using heuristic searches with tree bisection-reconnection (TBR) branch swapping, and 100 random taxon addition replicates.

Sequence divergences were calculated using uncorrected pairwise distances with PAUP. A statistical parsimony network was constructed with D. abietella haplotypes using TCS version 1.21 [START_REF] Clement | TCS: a computer program to estimate gene genealogies[END_REF].

Genetic structure within and among European D. abietella populations was examined by Analysis of Molecular Variance (AMOVA) [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data[END_REF] as implemented in ARLEQUIN version 3.0. Populations were grouped either by geographical location (region) or by host species or by host origin (see Fig. 1 and Table 4 for details). Levels of significance were determined through 1000 random permutation replicates.

Results

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. Ten of the variable sites showed transversions (8 in COI and 2 in COII). There was variation in amino acids at 4 locations in COI and COII. No insertion or deletion of sequence was observed between the two species. On the basis of variation observed in these longer sequences, as well as the relative effectiveness of different primer combinations, two shorter regions were chosen to survey mitochondrial sequence variation for the remainder of the study.

Sequence selection

Manuscrit

Fifteen haplotypes were found among the 29 D. abietella specimens, five haplotypes among the eleven D. abietivorella specimens, five haplotypes among the nine D. mendacella and two haplotypes among the eight D. simplicella. Divergences within and between lineages (species) and species groups of Dioryctria were compared between the 451-bp COI versus the 572-bp COII fragments and across the 2 combined gene fragments (Table 3 and Appendix for complete data of pairwise divergences). For all data sets (COI, COII, COI+COII), maximum divergence between lineages did not overlap minimum divergence between species group, and uncorrected pairwise distances were comparable to divergences already reported in the genus Dioryctria by Roe andSperling (2007a, 2007b) and [START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF]. By contrast, intraspecific divergence in D. abietella (within lineages) exceeded the interspecific divergence between sister pairs of the abietella species group for two data sets (maximum 0.035 in D. abietella versus minimum 0.014 in abietella species group and maximum 0.023 versus minimum 0.011 for COII and COI+COII respectively). Moreover, Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

Manuscrit d'auteur / Author manuscript

Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 11 when sequence divergence was compared within D. abietella, mean pairwise distances differed substantially between the two genes (0.002 in COI versus 0.018 in COII), the number of variable nucleotide sites being five times less in COI (4 transitions with 1-0-3 changes at first, second and third codon positions, respectively) than in COII (18 transitions and 2 transversions with 2-1-17 changes at first, second and third codon positions, respectively). There were three amino acid replacements within the COII gene, including one valine versus isoleucine (bp 506), one phenylalanine versus leucine (bp 518), and one phenylalanine versus serine (bp 952). No ambiguous site (double peak) was detected. When compared between species belonging to abietella species group, mean pairwise distances were similar (0.027 in COI versus 0.029 in COII). Mean pairwise comparisons between the two species D. abietivorella and D. abietella were higher in the COI fragment gene (0.041) versus in the COII gene (0.034). To minimize the great variability detected in intraspecific divergence rate between the COI fragment gene and the COII gene and to minimize the stochastic variation across taxa, we used the combined sequence because this gave a better average of overall divergence rates across COI and COII.

The ILD test between full length sequences of COI and COII genes revealed no significant conflict (P=0.13). Modeltest was applied to determine the most appropriate model of sequence evolution. The general time reversible model with the following base composition (A=0,33290; C=0,10870; G=0,12960; T=0,42880), rate of invariable sites (0.6068), and gamma distribution (0.6041) (GTR+I+G, Tavaré 1986) was the substitution model selected for the combined COI+COII data set.

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. pineae, and the two others (mend1+2+3 and mend4+5) were identified as D. mendacella, indicating uncertainty in the phylogenetic relationships of these two species.

Phylogenetic reconstruction

Manuscrit

The European pine stem borer D. sylvestrella (sylvestrella species group) was clearly separated from the abietella group and clustered with specimens of D. amatella (zimmermani species group). The specimen of the North American species D. pseudotsugella formed a well supported monophyletic group with the specimen of D. reniculelloides, supporting the placement of both species in a separate species group.

Dioryctria abietella haplotype network

Thirteen mitochondrial haplotypes were detected among the 30 D. abietella specimens analysed. Interestingly, most of the haplotype diversity was distributed in artificial stands in France (Fig. 1 and Table 1). Of the eleven specimens from natural stands in the French and Italian Alps, ten had the abt1 haplotype, regardless of host and locality. This major haplotype (abt 1) was also present in an artificial stand in the Latronquière seed orchard, on three different hosts (Picea abies, Pseudotsuga menziesii and Larix decidua). Seven other unique haplotypes (abt3, abt5-9 and abt11) were found in this locality, plus one divergent haplotype (abt12) shared with the Picea smithiana population from Les Barres Arboretum. Three remaining haplotypes were also found in Les Barres (abt4 on Picea smithiana, abt11 on Pinus koraiensis and abt10 on both species). Haplotypes from China (abt13) clustered with the major haplotype abt1.

The hostplant association and phylogenetic relationships of all haplotypes in D. abietella are summarized in a network (Fig. 3

Genetic structure of D. abietella populations

The results of the AMOVA analyses performed in D. abietella are presented in Table 4. When populations were grouped by geographic region (Alps, Central France and South Western France) (see Fig. 1), genetic variation was partitioned half between regions (51.08%) and half within each population (48.92%), this result being significant. When populations were grouped by host plant, 58% of the genetic variation was found within populations, 40% between populations within hosts and 2% between hosts. Only genetic variation within populations was significant. When populations were grouped by type of stand, 55.6% of the genetic variation was significantly found between groups, i.e. natural forest versus artificial stands, variation within population being also significant. (Roe and Sperling 2007a) examined patterns of evolution of these two mitochondrial genes and ramifications for delineating species boundaries in Lepidoptera and Diptera. They demonstrated that DNA substitution patterns can vary between independent lineages and change as taxa become increasingly diverged (see also [START_REF] Galtier | Mutation hot spots in mammalian mitochondrial DNA[END_REF]. Nevertheless, studies of intraspecific patterns of divergence are performed with a limited number of conspecific populations (Wahlberg et al. 2003, Roe andSperling 2007a).

Discussion

Better sampling throughout the geographic range of the species should maximize sampling of mtDNA haplotype diversity and consequently minimize the effect of localized stochastic mutational anomalies.

Our study provides an opportunity to evaluate the variability of divergence rates between COI and COII in a species group with sequence available at different taxonomic levels (populations, sister species and species groups). The high variability observed in intraspecific divergence rate (five times more in COII than in COI within Dioryctria abietella) was unexpected and contrasts with the interspecific divergence rates in Dioryctria demonstrated by Roe and Sperling (2007a). We ruled out technical artifacts due to DNA contamination or PCR-sequencing errors. However, extreme sequence divergence in COII could reflect the presence of nuclear copies of mitochondrial DNA (numts) that had contaminated sequences of D. abietella. Nevertheless, there were neither ambiguous polymorphic sites, nor unexpected stop codons in any of the sequences analysed, nor elevated numbers of amino acid changes. Numts have been more commonly found in plants than in animals and few studies have been reported in insects (Bensasson et al.2001[START_REF] Keller | Transition-transversion bias is not universal: a counter example from grasshopper pseudogenes[END_REF].

The presence of numts was also inferred but not proven in tropical Lepidoptera by [START_REF] Hajibabaei | DNA barcodes distinguish species of tropical Lepidoptera[END_REF]. Similar divergence variability between COI and COII was also observed within the longhorn beetle Monochamus galloprovincialis, in which numerous numts have been detected [START_REF] Koutroumpa | Incorporation of mitochondrial fragments in the nuclear genome (Numts) of the longhorned beetle Monochamus galloprovincialis (Coleoptera Cerambycidae)[END_REF].

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

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Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 16 Roe and Sperling (2007a) found that maximum intraspecific diversity in Lepidoptera, including Dioryctria pentictonella,was usually found in COI. Although such patterns of sequence variation may be due to random stochastic variability, they may also suggest that the common assumption of neutral molecular evolution in mtDNA is not justified. Non random regional variation has previously been shown to occur in mtDNA [START_REF] Broughton | Spatial covariation of mutation and nonsynonymous substitution rates in vertebrate mitochondrial genomes[END_REF]Reneau 2006, Galtier et al. 2006) and several factors such as functional constraints, mutation hot spots, or adaptive substitutions could explain heterogeneous evolutionary rates observed in Dioryctria [START_REF] Lunt | The insect cytochrome oxidase I gene: evolutionary patterns and conserved primers for phylogenetic studies[END_REF][START_REF] Stoneking | Hypervariable sites in the mtDNA control region are mutational hotspots[END_REF][START_REF] Innan | Recombination or mutational hot spots in human mtDNA?[END_REF].

Our study highlights the importance of considering other genes than COI, such as COII, as well as independent markers as nuclear markers, when studying phylogenetic relationships between closely related species, especially if they display high genetic diversity or low interspecific divergence. In this context, it is reasonable to ask whether the COII gene is optimally informative by itself for reconstructing phylogenetic relationships of closely related species. Short fragments have commonly been used to identify sister species, especially for DNA barcoding (Hebert et al. 2003a). However, such reliance on short fragments has been controversial [START_REF] Wahlberg | Phylogenetic relationships of Phyciodes butterfly species (Lepidoptera: Nymphalidae): complex mtDNA variation and species delimitations[END_REF][START_REF] Roe | Identification of Dioryctria (Lepidoptera: Pyralidae) in a seed orchard at Chico, California[END_REF], especially when only a single specimen is used to define a lineage. Roe and Sperling (2007a), argued that it may be advantageous to focus on regions that give accurate and consistent estimates of divergences relative to longer mtDNA regions. They identified a 600-bp fragment as the best indicator of total COI-COII divergence (mean percent divergence of 100.7% relative to total COI-COII divergence) for sister species in Lepidoptera and Diptera. The COI gene fragment used in our study partially spanned this region, and showed overall interspecific and intraspecific pairwise divergences similar to other Lepidoptera [START_REF] Wahlberg | Phylogenetic relationships of Phyciodes butterfly species (Lepidoptera: Nymphalidae): complex mtDNA variation and species delimitations[END_REF][START_REF] Blum | A molecular phylogeny of the neotropical butterfly genus Anartia (Lepidoptera: Nymphalidae)[END_REF][START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF]) (0-0.011). In contrast, the COII gene showed lower minimum interspecific divergence (0.14) and higher maximum intraspecific divergence (0.35)

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

compared to distances previously recorded in Dioryctria species (Du et al. 2006[START_REF] Roe | Identification of Dioryctria (Lepidoptera: Pyralidae) in a seed orchard at Chico, California[END_REF]. Incongruences between species trees and mtDNA trees have often been reported in closely related taxa [START_REF] Avise | Ten unorthodox perspectives on evolution prompted by comparative populations genetic findings on mitochondrial DNA[END_REF][START_REF] Funk | Species-level paraphyly and polyphyly: frequency, causes and consequences, with insights from animal mitochondrial DNA[END_REF][START_REF] Ballard | The incomplete natural history of mitochondria[END_REF]. Thus reliance on a single DNA region can be misleading, in part due to underestimates or overestimates of sequence divergence between taxa, particularly between sister pairs.

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Intraspecific variability of the polyphagous European Dioryctria abietella:

Although short fragments of COI have commonly been used to identify closely related species in Lepidoptera [START_REF] Caterino | The current state of insect molecular systematics: A thriving tower of Babel[END_REF] and for DNA barcoding (Hebert et al. 2003b), the combination of more than one DNA region (or longer DNA fragments) to identify closely related species or to distinguish populations is strongly supported [START_REF] Wahlberg | Phylogenetic relationships of Phyciodes butterfly species (Lepidoptera: Nymphalidae): complex mtDNA variation and species delimitations[END_REF][START_REF] Roe | Identification of Dioryctria (Lepidoptera: Pyralidae) in a seed orchard at Chico, California[END_REF]). In addition to the fact that most D. abietella populations in our study contained at least three specimens, the combination of two mtDNA fragments showing contrasting evolutionary rates, targeting a region of maximum divergence (COII gene which was more divergent than COI in this species), should improved resolution considerably in intraspecific analysis.

Analysis of molecular variance within D. abietella did not show any clear genetic differentiation among hosts (Table 4), even if most variation in the plains haplotypes was restricted to single host species (see Fig. 1 and Table 1). Dioryctria abietella is unusual among the European members of its group in that it is relatively polyphagous, feeding on Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

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Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 18 hostplant species from a number of unrelated coniferous genera. According to numerous authors [START_REF] Johnson | Conditions for sympatric speciation: a diploid model incorporating habitat fidelity and nan-habitat assortative mating[END_REF][START_REF] Funk | Species-level paraphyly and polyphyly: frequency, causes and consequences, with insights from animal mitochondrial DNA[END_REF][START_REF] Rundle | Ecological speciation[END_REF], divergent habitat preferences are more likely to cause prezygotic isolation when mating occurs in or near the preferred habitat, for example between herbivorous insect populations that mate on the plant on which they feed. But according to [START_REF] Emelianov | Host-induced assortative mating in host races of the larch budmoth[END_REF], mating behavior in many plant-feeding insects does not depend directly on host cues. Hence, in most Lepidoptera, the host plant is not required for mating, and females call for males using longrange pheromones. Because of its polyphagy, D. abietella populations are less likely to experience disruptive selection following shifts to novel host plants [START_REF] Mopper | Genetic structure and local adaptation in natural insect populations. Effects of ecology, life history and behavior[END_REF][START_REF] Berlocher | Sympatric speciation in phytophagous insects: moving beyond controversy?[END_REF], Funk and Omland 2002). Furthermore, our sampling was performed on numerous exotic conifer trees that may not represent of the natural host range of the species. Additional studies of the ecology and genetics of this species are needed to investigate further insect-host relationships.

Results of the AMOVA revealed the presence of significant population structure, showing that 51% of the variation was due to the subdivision of populations by geographic origin (Table 4). This result was mainly due to the strong divergence of populations from the plains, fixed for a number of unique singleton haplotypes, compared to populations from the Alps that displayed the widespread haplotype abt1 and only one singleton (abt2).

Furthermore, when populations are grouped by origin of stand (native forest vs. artificial plantation), the percentage of variance accounted for is higher (56%), which is more indicative of geographic distribution of the stand than of the type of the stand (i.e. native forests in Alps versus artificial plantations in plains localities, and Latronquière seed orchard versus Les Barres Arboretum).

Distinct selection pressures could play a role in the difference between genetic diversity in populations from the Alps and those in the plains. For most insects that are Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. governing their population dynamics [START_REF] Turgeon | Insect fauna of coniferous seed cones -Diversity, host-plant interactions and management[END_REF]. In the natural forests of the Alps, it is likely that coneworm populations have evolved together with the hosts and adapted to masting; i.e., substantial annual fluctuations in cone abundance. In contrast, the plain populations, and especially those developing within the Latronquière seed orchard, faced only limited fluctuations in annual cone crop because the orchard trees were submitted to treatments to promote annual flowering. So diet breadth might be an important parameter in the observed genetic patterns, as can be observed in other forest insects (Kerdelué et al. 2002), and the low genetic variability observed in the Alps could be due to more episodic cone production than populations from the plains. However, we cannot rule out the hypothesis that past climatic oscillations during the Quaternary period may have affected the patterns of genetic diversity of D. abietella [START_REF] Hewitt | Some genetic consequences of ice ages, and their role in divergence and speciation[END_REF]. The low genetic diversity in present-day populations from the natural forest in the Alps could be attributable to a single mountain refugium, whereas the high genetic diversity in introduced areas could result from multiple origins from different refugial sources during the ice ages or, more likely, from movements of insects due to transportation of cones into these areas. It would be useful to conduct a more comprehensive phylogeographic study on this palearctic species.

The unexpected discovery of two distinct groups of D. abietella haplotypes, which was particularly evident in the haplotype network (Fig. 3), may indicate the presence of at least two diverged lineages within the species D. abietella. The widespread abt1 haplotype found mainly in the Alps and in some plains localities is very close to the Chinese haplotype, and is therefore most likely to represent the specimens originally described as D. abietella from the vicinity of Vienna (Wienergegend) in Austria [START_REF] Denis | Systematische Werzeichniss der Schmetterlinge der Wienergegend[END_REF]. The second haplotype group appeared to be much more difficult to define without further exploration. However, even species placed in different species groups may be difficult to Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

Manuscrit d'auteur / Author manuscript

Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 20 distinguish. For example damage from the trunk borer D. splendidella [START_REF] Jactel | Infestation dynamics of Dioryctria sylvestrella (Ratz.) (Lepidoptera: Pyralidae) in pruned maritime pine (Pinus pinaster Ait.)[END_REF] has long been confused with that of D. abietella in France or D. schuetzeella, since it is also known to attack cones of Picea abies in Europe [START_REF] Schwenke | Die Forstschiidlinge Europas. Bd. 4. Hautflugler und Zweifliigler[END_REF]. Nonetheless, we did not observe specimens that were morphologically diagnosable to D. splendidella or D. schuetzeella in our surveys, nor did we find any haplotypes similar to the D. schuetzeella mtDNA [START_REF] Knölke | A procedure for combined genitalia dissection and DNA extraction in Lepidoptera[END_REF]. The recently identified D. resiniphila [START_REF] Segerer | Dioryctria resiniphila sp. nov., eine neue Pyralide auf Abies cephalonica Loud. in Griechenland (Lepidoptera: Pyralidae: Phycitinae). -NachrBl. bayer[END_REF] will also need to be considered in further analyses.

Molecular systematics of the abietella species group

Our results from mtDNA sequence variation among species are congruent with the findings of [START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF] and [START_REF] Roe | Identification of Dioryctria (Lepidoptera: Pyralidae) in a seed orchard at Chico, California[END_REF], with D. abietivorella and D. abietella supported as a separate species. The results confirm the original diagnosis of [START_REF] Munroe | Canadian species of Dioryctria Zeller (Lepidoptera: Pyralidae)[END_REF] who separated D. abietella from D. abietivorella using external and internal morphological criteria, including larger size, darker hind wings, more conspicuous pale markings, more transverse dark lines on forewings, and a different configuration of the male valva in D. abietella. Numerous other studies have advocated the use of mtDNA sequences as a valuable marker for identifying closely related species, especially where morphological differences are subtle, in some cases confirming and in others refuting previous interpretations (e.g. Sperling and Hickey 1994[START_REF] Sperling | DNA-based identification of introduced ermine moth species in North America (Lepidoptera: Yponomeutidae)[END_REF][START_REF] Caterino | Papilio phylogeny based on mitochondrial cytochrome oxidase I and II genes[END_REF][START_REF] Cognato | Incomplete barriers to mitochondrial gene flow between pheromone races of the North American pine engraver, Ips pini (Say) (Coleoptera: Scolytidae)[END_REF], Kerdelhué et al. 2002[START_REF] Kruse | Molecular phylogeny within and between species of the Archips argyrospila complex (Lepidoptera: Tortricidae)[END_REF][START_REF] Wahlberg | Phylogenetic relationships of Phyciodes butterfly species (Lepidoptera: Nymphalidae): complex mtDNA variation and species delimitations[END_REF][START_REF] Damgaard | Phylogeny and reclassification of species groups in Aquarius Schellenberg, Limnoporus Stal and Gerris Fabricius (Insecta: Hemiptera-Heteroptera, Gerridae)[END_REF].

The topology of the phylogenetic reconstruction shown in Fig. 2 reveals the monophyly of the abietella group and its separation from members of the three other Dioryctria groups (sylvestrella, schuetzella and zimmermani) as defined by [START_REF] Mutuura | American species of Dioryctria (Lepidoptera: Pyralidae) III. Grouping of species: species of the auranticella group, including the Asian species, with the description of a new species[END_REF] and [START_REF] Neunzig | Pyraloidea, Pyralidae (part), Phycitinae (part)[END_REF]. When considering the mitochondrial data set, within-Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

Manuscrit d'auteur / Author manuscript

Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 21 species group genetic distances (1.1-4.5%) were always lower than between-species group pairwise distances (5.7-8.6%). A recent study of the same genus showed similar results but more overlap, with sequence divergence ranging from 0.3% to 5.6% among species within groups and 3.3% to 9.2% among species in different groups [START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF]. Low rates of overlap in mtDNA genetic distances within and between species have been found in other Lepidoptera. In neotropical Nymphalidae for example, distances ranged from 3 to 5% between sister species and from 5 to 8% between species in the three separate phyletic lineages defined in the genus Anartia [START_REF] Blum | A molecular phylogeny of the neotropical butterfly genus Anartia (Lepidoptera: Nymphalidae)[END_REF].

Both MP and ML analyses strongly supported clade A, which comprised the two North American species (D. abietivorella and D. ebeli). MtDNA genetic distances between D. abietivorella and D. abietella ranged between 3.3% and 4.2% (mean sequence divergence of 0.037, whatever the fragment length considered, i.e. 1975, 451 or 572bp). This result was similar to divergence observed within Tortricidae for the Argyrotaenia franciscana species group [START_REF] Landry | Systematics of the Argyotaenia franciscana ( Lepidoptera: Tortricidae) species group: evidence from mitochondrial DNA[END_REF], although these pairwise divergences were higher than these found between most closely related species or within species complexes in Lepidoptera. For example, within the spruce budworm species complex, divergences ranged from 2.7%-2.9%

between Choristoneura fumiferana and the other members of the group, divergences between these other members were all <1% (Sperling and Hickey 1994). Sequence divergence within the Archips argyrospila complex ranged from 1.47% to 2.53% between populations of A. argyrospila and A. goyerana [START_REF] Kruse | Molecular phylogeny within and between species of the Archips argyrospila complex (Lepidoptera: Tortricidae)[END_REF]. Less than 1% divergence was observed among three species of ermine moths (Yponomeutidae) [START_REF] Sperling | DNA-based identification of introduced ermine moth species in North America (Lepidoptera: Yponomeutidae)[END_REF]. In contrast, some swallowtail butterfly species groups showed higher sequence divergences, ranging from 2.6 to 5.4% in the Papilio machaon group, 1.3 to 3.7% in the P. glaucus group, and 7.3 to 9.4% in the P. dardanus group [START_REF] Caterino | Papilio phylogeny based on mitochondrial cytochrome oxidase I and II genes[END_REF].

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. The high variability of sequence divergence between closely related species of Lepidoptera implies that it is not a good predictor of whether two unknown populations constitute reproductively isolated species [START_REF] Landry | Systematics of the Argyotaenia franciscana ( Lepidoptera: Tortricidae) species group: evidence from mitochondrial DNA[END_REF][START_REF] Sperling | Butterfly molecular systematics: from species definitions to higher level phylogenies[END_REF][START_REF] Cognato | Standard percent DNA sequence difference for insects does not predict species boundaries[END_REF]). Nevertheless, sequence divergence observed within the Dioryctria abietella species group most likely reflects relatively recent separation of the mitochondrial lineages, during the Quaternary ice ages, according to the mtDNA clock of [START_REF] Brower | Practical and theoretical considerations for choice of a DNA sequence region in insect molecular systematics, with a short review of published studies using nuclear gene regions[END_REF]. This pattern has been observed in other sibling species of forest insects [START_REF] Emelianov | Genetic differentiation in Zeiraphera diniana (Lepidoptera: Tortricidae, the larch budmoth): polymorphism, host races or sibling species?[END_REF][START_REF] Boato | High genetic variability despite haplodiploidy in primitive sawflies of the genus Cephalcia (Hymenoptera, Pamphiliidae)[END_REF][START_REF] Cognato | Incomplete barriers to mitochondrial gene flow between pheromone races of the North American pine engraver, Ips pini (Say) (Coleoptera: Scolytidae)[END_REF][START_REF] Stauffer | Phylogeography and postglacial colonization routes of Ips typographus L. (Coleoptera, Scolytidae)[END_REF][START_REF] Nice | Population genetic evidence of restricted gene flow between host races in the butterfly genus Mitoura (Lepidoptera: Lycaenidae)[END_REF]. Although D. abietella and D. abietivorella have very close morphological characters and similar degrees of polyphagy and broad geographic ranges, they differ strongly in their distributions, the first one being palearctic and the second one nearctic. According to [START_REF] Hewitt | Some genetic consequences of ice ages, and their role in divergence and speciation[END_REF], most species are confined to continents and closely related species often occupy different parts of a continent.

European Dioryctria species of the abietella group fall into two major groups, one comprising species that develop on Mediterranean pine cones (D. mendacella and D. pinae) and the other comprising species that develop on cones of more northern conifers (D. abietella and D. simplicella). The delimitation between D. mendacella and D. pineae is not clear. The fact that specimens of D. mendacella collected on the same host as D. pineae fell into two distinct haplotype lineages could indicate a complex of three cryptic species instead of two, as the two currently recognized species can be found in sympatry on the same hosts (Pinus pinaster, P. halepensis and P. pinea: Roques 1983; Pinus brutia, [START_REF] Karanicola | Biology, ecology and damage of cone and seed insects on Pinus brutia[END_REF]).

Nevertheless, further investigation with more extensive sampling of their ecology and genetic variation, especially with nuclear markers, is needed to clarify this result.

The clade represented by D. abietella and D. simplicella appeared even more problematic. The separation between the two species was not well supported, and three D.

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. These three specimens were collected in different localities from D. simplicella and, to date, D. simplicella has not been found on Pseudotsuga menziesii or on Picea smithiana. This species is recorded from cones as well as shoots of diverse coniferous species including Pinus sylvestris [START_REF] Charles | Observations sur la biologie et l'écologie de Dioryctria mutatella Fuchs (Lepidoptera Phycitidae), ravageur des pousses et des cônes de Pin sylvestre en forêt de Fontainebleau[END_REF]. The morphological characters of adults were clear and no differences in genitalia were noted between these specimens, whereas the genitalia of D. simplicella differ greatly from those of D. abietella [START_REF] Zocchi | Contributi alla conoscenza degli insetti delle piante forestali. V. Il gen. Dioryctria Zell. (Lepidoptera, Pyralidae) in Italia[END_REF]).

Nevertheless, it seems plausible that abt12 specimens were simplicella specimens. Further sampling is clearly needed to clarify the incongruence between morphological and molecular data.

For the North American species, the identity of the two specimens labeled as

Dioryctria ebeli is open to question, since the D. ebeli mtDNA haplotype showed only 0.1 to 0.6% divergence from D. abietivorella haplotypes. Other Dioryctria species also display low sequence divergence (less than 1.8% separate D. zimmermani, D. tumicolella and D. taedivorella), but morphological characters were not effective in confirming the distinctness of these lineages [START_REF] Du | Phylogenetic framework for Dioryctria (Lepidoptera: Pyralidae) based on combined analysis of mitochondrial DNA and morphology[END_REF]. As for the European species of the abietella group, additional sampling is clearly needed in order to resolve the specific status of D. ebeli.

Our study confirmed that nucleotide diversity within and between taxa was quite variable across both COI and COII genes. As divergences are low between sister species of Dioryctria, it is crucial to target regions with maximum divergence to ensure the greatest probability of consistently delimiting species boundaries by sequencing regions with the most informative nucleotide variation (Roe and Sperling 2007a). Mitochondrial DNA data may Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

Manuscrit d'auteur / Author manuscript

Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 24 compensate for insufficient information from morphological characters, especially at the species and species group levels, but it also shows that currently recognized taxonomic relationships based on morphological similarities and host plant origin need to be reevaluated in the European D. abietella species complex. As an integrative approach is essential to testing species delimitations (Roe et al. 2007b), multiple independent markers such as independent molecular loci (nuclear markers), morphology, larval host plant and geographic range should be also considered to be confident of species delineations.

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. * following [START_REF] Simon | Evolution, weighting, and phylogenetic A.utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers[END_REF] Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. 0-0,011 (0,002) 0-0,035 (0,018) 0-0,023 (0,010) Between lineages 0,011-0,049 (0,027) 0,014-0,044 (0,029) 0,011-0,045 (0,027) Between species groups 0,051-0,086 (0,068) 0,049-0,096 (0,074) 0,057-0,086 (0,069)

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. 1, with different pattern codes for host tree species.

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

  Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 3

  Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 8 from the thorax of both larvae and adults. The remaining body parts, including head, legs, wings and abdomen were stored at -80° and retained as vouchers at the University of Orleans. Specimens were vacuum-dried to remove ethanol before extraction. DNA was purified using a phenol/chloroform based extraction and eluted in 200 μl of LTE buffer.

  d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 10 Sequence was obtained for the full 2272 bp region (including the COI+tRNAleu+COII genes) in one D. abietivorella specimen (GenBank accession number EU407773). For two specimens of D. abietella a fragment of 1975 bp (start of COI missing) was obtained (GenBank number EU407772). Overall sequence divergence between these two species was estimated at 3,7% (73 substitutions, 51 in COI and 22 in COII), based on the 1975 bp region.

  d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 12 One sequence for each combination of haplotype, host and locality was retained for phylogenetic reconstruction. The consensus of 3 most parsimonious (MP) trees (CI=0.572; RI=0.863; excluding uninformative characters) and the maximum likelihood (ML) tree, using 451 bp in COI and 572 bp in COII, are shown in Fig. 2. The monophyly of the abietella species group relative to species in the three other Dioryctria groups is strongly supported by MP analysis but weakly by ML analysis (100% and 56% bootstrap values respectively). The monophyly of the genus Dioryctria relative to the two outgroup species in other Phycitini genera was supported by 79% in MP and 72% in ML analysis. When the MP and ML phylogenetic reconstructions were compared, there were some differences at the ingroup level. The most conspicuous difference was the position of the North American species relative to European ones. The Dioryctria abietella species group was separated into three major clades (A, B and C). Clade A contained all specimens from North America, D. abietivorella and D. ebeli. According to both MP and ML analyses this first clade was clearly separated from European and Chinese specimens (100% and 96% bootstrap support). Clades B and C were weakly supported by both analyses (bootstrap values comprised between 52 to 75%). Clade B mainly consisted of D. abietella haplotypes plus the D. simplicella haplotypes. The abt12 haplotype, corresponding to three D. abietella specimens collected at Latronquière on Douglas fir and at Les Barres Arboretum on Picea smithiana, formed a clade with specimens of D. simplicella collected in Fontainebleau and in Poland on Pinus sylvestris. The pairwise distance between abt12 and D. simplicella was 1.2%, whereas distances between abt12 and other D. abietella haplotypes ranged between 1.6 to 3.8% for haplotypes from same localities. Clade C grouped together specimens of D. mendacella and D pineae, all of which were collected on the Mediterranean pines P. halepensis and P. pinae. This clade had a basal trichotomy of three haplotype lineages that Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 13 were separated by 1.4-1.5% from each other. One of the lineages (pin1) represented D.

  ), based on COI and COII sequences. This network revealed two haplotype groups separated by seven mutational steps. One comprised all 11 individuals from natural forests in the Alps (abt1, abt2) plus Chinese sequence, Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 14 separated by five mutational steps from three individuals from the Latronquière seed orchard and Les Barres Arboretum (abt3, abt4). The second group, more polymorphic, comprised only individuals from artificial stands (arboretum and seed orchard, abt5, 6, 8 to 11), with the exception of one haplotype (abt7) displayed by one individual on Larix decidua from the Latronquière seed orchard. The three individuals that had the abt12 haplotype were well separated from the other Dioryctria haplotypes and was not be included in the haplotype network produced by TCS.

  Sequence divergence in COI versus COII Although mtDNA genes have long dominated the field of molecular systematics, gene choice and fragment length are crucial when inferring phylogenetic relationship between species. The COI+COII gene region has frequently been sequenced in Lepidoptera (Sperling Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 15 2003) and a recent review

  Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 17 When separate data sets (COI or COII) for phylogenetic reconstructions of Dioryctria species group were compared, COI gave a more accurate indication of species boundaries than COII (data not shown), all haplotypes being grouped according to their respective taxon relationship in COI, whereas the COII gene separated D. abietella into two clades.

  Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 19 specific to cones, annual fluctuations in resource availability are a major driving force

  Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 22

  Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 23 abietella specimens collected on P. menziesii and P. smithiana and showing the abt12 haplotype clustered together with specimens of D. simplicella collected on Pinus sylvestris.

Table 2 .

 2 List of primers used for PCR amplification and sequencing. Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit det al. (1994) CATTTCAAGYTGTGTAAGCATC C2-J-3138a 10 Sperling et al. (1995) AGAGCCTCTCCTTTAATAGAACA C2-N-3389b 11 Du et al. (2005) TCATAWCTTCARTATCATTG TK-N-3775 12 Bogdanowicz et al. (1993) GAGACCATTACTTGCTTTCAGTCATCT

Fig. 1 .Fig. 2 .

 12 Fig. 1. Sampling sites and haplotype distributions for 12 mtDNA haplotypes detected in European populations of Dioryctria abietella (abt1 to abt12), plus the French haplotype of D.simplicella (sim1). Each haplotype is defined as in Table1.

Fig. 3 .

 3 Fig.3. Haplotype network for 13 mtDNA haplotypes detected in populations of Dioryctria abietella. Each line between circles represents one mutational change. Small empty circles represent inferred, undetected interior haplotypes. Haplotype frequencies are approximated by the area of the circle. Each haplotype is defined as in Table1, with different pattern codes for host tree species.

  Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

Table 1 . Collection and sequence data for specimens used in this study.

 1 Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859. Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

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Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J.,

Sperling, F. (2008)

.

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (

2008

). c one specimen sequenced over 1975bp of COI, tRNA leu and COII. d one specimen sequenced over 2272bp of COI, tRNA leu and COII.

e Natural forest (nat) and artificial plantations (art) as in Table

4

(AMOVA analysis)

Table 3 . Comparison of uncorrected sequence divergences (mean pairwise divergences) within lineage (within D. abietella), between lineages (within D. abietella species group) and between species groups for Dioryctria haplotypes defined from 451bp of COI, 572bp of COII and across both COI+COII.

 3

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Acknowledgements

Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 25

We thank G. Grant, G. DeBarr, J. Powell, P. Karamicola and H. Jactel for specimen sampling. We are also grateful to A. Cognato, D. Kain, B. Landry and Anna Engberg for their advice and helpful assistance in molecular work. We also thank C. Kerdelhué and A.

Roe for their insighful comments on drafts of the manuscript and an anonymous reviewer for review of the submitted manuscript. This work was supported by California Hatch funds and an NSERC grant to FAHS.

Version postprint

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.

Comment citer ce document : Roux-Morabito, G., Gillette, N., Roques, A., Dormont, L., Stein, J., Sperling, F. (2008). Systematics of the Dioryctria abietella species group (Lepidoptera: Pyralidae) based on mitochondrial DNA. Annals of the Entomological Society of America, 101 (5), 845-859.


Michael Atlan

Michel Gross

Emilie Absil

Accurate phase-shifting digital interferometry

come

Phase shifting interferometry [START_REF] Creath | Phase measurement interferometry techniques[END_REF] and holography [START_REF] Yamaguchi | Phase-shifting digital holography[END_REF] are powerful tools which have been in use for many years. In such systems, the phase of the reference arm of an interferometer is shifted by a mirror mounted on a piezoelectric transducer (PZT) and the interference pattern is recorded by a CCD camera. Since the PZT is not perfectly linear, nor calibrated, phase shift errors are introduced [START_REF] Kinnstaetter | Accuracy of phase shifting interferometry[END_REF]. This yields measurement errors and twin image aliases [START_REF] Cuche | Spatial filtering for zero-order and twin-image elimination in digital off-axis holography[END_REF]. To suppress or compensate these errors many techniques and algorithms have been proposed [START_REF] Schwider | Digital wave-front measuring interferometry: some systematic error sources[END_REF][START_REF] Hariharan | Digital phaseshifting interferometry: a simple error-compensating phase calculation algorithm[END_REF][START_REF] Lai | Generalized phase-shifting interferometry[END_REF][START_REF] Larkin | Design and assessment of symmetrical phase-shifting algorithms[END_REF][START_REF] Taylor | Multiple fringe analysis with unknown phase shifts[END_REF][START_REF] Schmit | Extended averaging technique for derivation of error-compensating algorithms in phaseshifting interferometry[END_REF][START_REF] Gutman | Phase-shifter calibration and error detection in phase-shifting applications: a new method[END_REF][START_REF] Huntley | Suppression of phase errors from vibration in phase-shifting interferometry[END_REF][START_REF] Guo | Phase-shifting error and its elimination in phase-shifting digital holography[END_REF][START_REF] Cai | Generalized phaseshifting interferometry with arbitrary unknown phase steps for diffraction objects[END_REF][START_REF] Cai | Experimental demonstrations of the digital correction of complex wave errors caused by arbitrary phaseshift errors in phase-shifting interferometry[END_REF][START_REF] Xu | Fast blind extraction of arbitrary unknown phase shifts by an iterative tangent approach in generalized phase-shifting interferometry[END_REF]. Some of these techniques [START_REF] Cai | Phase-shift extraction and wave-front reconstruction in phase-shifting interferometry with arbitrary phase steps[END_REF] yield discussion and controversy [START_REF] Ferrari | Comment on "phaseshift extraction and wave-front reconstruction in phaseshifting interferometry with arbitrary phase steps[END_REF][START_REF] Cai | Reply to comment on "phase-shift extraction and wave-front reconstruction in phase-shifting interferometry with arbitrary phase steps[END_REF].

Here, we propose to suppress most of the phase error by replacing the phase shift ∆ϕ by a frequency shift ∆ω induced by Bragg cells (Acousto Optic modulator) driven by RF (Radio Frequency) signals. We will show that the phase, which is driven by sine wave electronic signals, is very accurate. This technique has been introduced in the past in the context of holography [START_REF] Leclerc | Numerical heterodyne holography using 2d photo-detector arrays[END_REF][START_REF] Leclerc | Synthetic-aperture experiment in visible with on-axis digital heterodyne holography[END_REF]), but it can be adapted to interferometry. In this letter, we will describe the technique, and evaluate the phase shift errors by measuring the relative weight of the twin image component in an off-axis Mach-Zehnder configuration.

The setup is shown on Fig. 1. It is similar to the one used in [START_REF] Leclerc | Numerical heterodyne holography using 2d photo-detector arrays[END_REF]. The main laser beam (complex field E L , frequency ω L ) is provided by a Sanyo DL-7140-201 diode laser (λ = 780 nm, 50 mW for 95 mA of current). It is split into an illumination (E I , ω I ) and a local oscillator (LO) beam (E LO , ω LO ). A set of optical attenuators A1, A2 (grey neutral filters) allows to adjust the illumination and the LO beam intensities. The object is an USAF target in transmission (back illuminated). The CCD camera (PCO Pixelfly digital camera: 12 bit, frame rate ω CCD ≃ 10 Hz, acquisition time T CCD = 1/ω CCD ≃ 100 ms, with 1280 × 1024 pixels of 6.7 × 6.7µm) records the interference pattern of the object field E (whose frequency ω is unshifted with respect to illumination: ω = ω I ) with the LO field E LO . A set of two acousto optic modulators AOM1 and AOM2 (Crystal Technology: ω AOM 1,2 ≃ 80 MHz) shift the frequency of the illumination and LO beams (ω

I = ω = ω L + ω AOM 1 and ω LO = ω L + ω AOM 2 )
, so that the heterodyne beat frequency can be freely adjusted:

∆ω ≡ ω LO -ω I = ω AOM 1 -ω AOM 2 (1)
The relative phase shift ∆ϕ of 2 consecutive images is:

∆ϕ(∆ω) = 2π∆ω/ω CCD (2)
In the following, we will measure the signal field E by using the 4 phases method: ∆ϕ = mπ/2 where m is an integer. The complex signal field amplitude E is then obtained from a sequence I 0 , I 1 ...I 3 of 4 consecutive CCD intensity images by [START_REF] Leclerc | Numerical heterodyne holography using 2d photo-detector arrays[END_REF]: where A is a normalization factor which depends on E LO , and j 2 = -1.

E = A 3 m=0 (j) m I m = A [(I 0 -I 2 ) ± j (I 1 -I 3 )] (3)
In comparison to a standard phase-shifting experiment, we have replaced the phase shifts ∆ϕ of the LO induced by the displacement of a mirror mounted on a PZT, by a frequency shift ∆ω generated through the dynamic Bragg gratings. The AOMs are driven by two numeric synthesizers (Agilent 33250A), which are in coherence, the first synthesizer (master) providing the 10 MHz reference of the second synthesizer (slave). Since the dynamic phase shift we apply is accurately controlled, the twin image alias [START_REF] Schnars | Direct phase determination in hologram interferometry with use of digitally recorded holograms[END_REF] can be cancelled efficiently. To verify this point quantitatively, we have measured the field E diffracted by the USAF target, for different values of the heterodyne beat frequency ∆ω. A map of the field E is computed from a sequence of 4 recorded frames, according to Eq.3. To separate the "true" image signal from the "twin" image alias, the USAF target is angularly shifted (angle θ ∼ 1 • ) with respect to the LO beam direction. From the field E(x, y) where x, y are the CCD pixels coordinates, we calculate by 2D (fast) Fourier transformation (F T 2D) the k-space field Ẽ(k x , k y ) = F T 2D E(x, y), in which the "true" and "twin" images are well separated when the object is off-axis. Fig. 2 shows the k-space field intensity distribution (| Ẽ(k x , k y )| 2 ) in logarithmic scale for ∆ω = +2.5 Hz (a) and -2.5 Hz (b). Here, the k-space field is computed by Fast Fourier Transform (FFT) from the 1280 × 1024 CCD data that are truncated into a 1024 × 1024 calculation grid. Since the illumination beam is a plane wave, and since the USAF target transmits a large fraction of the illumination beam, the object field E remains roughly a plane wave. Thus, the USAF true image corresponds, in k-space, to a narrow bright spot visible in the center of the white circle (region 1) in Fig. 2a. The twin image (center of circle 2) is the symmetric of true image with respect to the center of the k-space. On Fig. 2a, with ∆ω = +2.5 Hz, the heterodyne detection allows one to select the true image, and reject the twin image. The true image is bright, and the twin image is dark. On Fig. 2b, on the contrary, ∆ω = -2.5 Hz so that the true image ( 1) is dark, while the twin image (2) is bright. We have measured the weight of the true (W +1 ) and twin (W -1 ) image peak defined by:

W ±1 = C±1 | Ẽkx,ky | 2 (4)
where C±1 is the sum over a 10 × 10 pixels region centered on the true image (C +1 ) and twin image peak (C -1 ) respectively. By sweeping the AOM1,2 synthesizer frequency, we have studied how W ±1 vary with the heterodyne frequency ∆ω. Fig. 3 shows W ±1 as a function of ∆ω. When the 4phases condition is fulfilled, i.e. when ∆ω = +2.5 Hz, the weight of true image is maximum, (W +1 ≃ 0.3) while the twin image is nearly zero (W -1 ≃ 10 -4 ). Contrarily, when ∆ω = -2.5 Hz, W +1 is ≃ 10 -5 while W -1 ≃ 0.3). The relative weight of the parasitic component is thus W +1 /W -1 ≃ 3 × 10 -4 when one selects the true image (+2.5 Hz), and W -1 /W +1 ≃ 3 × 10 -5 when one selects the twin image (-2.5 Hz). The parasitic twin image component can thus be neglected. We have computed the theoretical shape of W ±1 (solid gray lines). Since the E.E * LO heterodyne term is simply proportional to e jm∆ϕ where m is the index of the image I m within the sequence. We get from Eq.3:

W ±1 (∆ω) = B η 2 (∆ω) 3 m=0 j m e ∓jm∆ϕ(∆ω) 2 (5)
Here B is a proportionality factor and ∆ϕ(∆ω) is given by Eq.2. η is the factor that describes the reduction of the detection efficiency (0 < η < 1), because of the phase drift during the acquisition time of an image (i.e. T CCD = 1/ω CCD ). Since the phase drift is constant in time, this efficiency factor takes the form :

η(∆ω) = | ∆ϕ(∆ω) ϕ=0
e jϕ dϕ| ∆ϕ(∆ω) = |1 -e j∆ϕ(∆ω) | ∆ϕ(∆ω) [START_REF] Hariharan | Digital phaseshifting interferometry: a simple error-compensating phase calculation algorithm[END_REF] As depicted in Fig. 3, the experimental results for W ±1 (∆ω) (points) agree with Eq.5 (solid grey lines).

To illustrate the ability to reconstruct an image and to cancel the twin image alias, we have calculated the image of the USAF target from the complex field E(x, y) (i.e. from the complex hologram recorded in the CCD plane). The image is reconstructed by the standard convolution method [START_REF] Schnars | Direct phase determination in hologram interferometry with use of digitally recorded holograms[END_REF][START_REF] Kreis | Digital holography for metrologic applications[END_REF] that yields a calculation grid equal to the pixel size [START_REF] Zhang | Algorithm for reconstruction of digital holograms with adjustable magnification[END_REF]. To calculate the convolution product, we have used the Fourier method, like in [START_REF] Leclerc | Numerical heterodyne holography using 2d photo-detector arrays[END_REF]. To obtain a proper off-axis image, we have enlarged the 1280 × 1024 calculation grid that corresponds to the dimensions of the CCD by padding the data into a 4096 × 4096 null matrix [START_REF] Kreis | Methods of digital holography: a comparison[END_REF][START_REF] Yamaguchi | Image formation in phase-shifting digital holography and applications to microscopy[END_REF] (zero padding method).

Fig. 4a shows the USAF image obtained for a reconstruction distance z = 194.5 mm; it corresponds to the true image revealed at the modulation frequency ∆ω = +2.5 Hz. We can notice that the zero order and twin image aliases are not visible. In conclusion, we have proposed to replace, in phaseshifting interferometry (or holography), the PZT driven mechanical motion of an optical component (mirror), by AOMs driven by numeric synthesizers. The mechanical phase shift is thus replaced by an electronic frequency shift, which is much more accurate. Most of the phase errors are thus suppressed by this technique, which is simple to manage. This result is confirmed by experiment. The proposed technique is expected to greatly simplify data analysis, improving the measurement accuracy.

FIG. 1 :

 1 FIG. 1: phase-shifting digital holography setup. L: main laser; BS: Beam splitter; AOM1 and AOM2: acousto-optic modulators; BE: beam expander; M: mirror; A1/2: light attenuator. USAF: transmission USAF 1951 test pattern. CCD : CCD camera. EL, EI , ELO and E : laser, illumination, LO and object fields.

FIG. 2 :

 2 FIG. 2: USAF target k-space 1024×1024 holograms Ẽ(kx, ky) displayed in logarithmic scale for | Ẽ(kx, ky)| 2 at ∆ω = +2.5 Hz (a) and ∆ω = -2.5 Hz (b). The center of the white circles correspond to the true (1) and twin (2) images.

FIG. 3 :

 3 FIG.3: a) Weight W+1 of the true image peak as a function of the heterodyne frequency ∆ω in Hz. Vertical axis is in logarithmic scale arbitrary units. The points correspond to experimental data: ∆ω = -5 to +5 Hz with 0.1 Hz increment. The solid grey line is the theory (Eq.5) . b) Weight W -1 of the twin image.

FIG. 4

 4 FIG. 4: a,b) USAF target reconstructed image in linear scale for the field intensity |Ex,y,z| 2 with z = 194.5 mm (4096×4096 pixels): a) ∆ω = +2.5 Hz (true image); b) ∆ω = -2.5 Hz (twin image). c) Zoom of the center of the true image a (512 × 512 pixels). d) Zoom of the center of the twin image b: 512 × 512 pixels. Pixel size is 6.7 × 6.7µm.

  Fig.4b shows the twin image obtained at ∆ω = -2.5 Hz. With the chosen reconstruction distance z = 194.5 mm, the twin image is blurred. The true image and zero-order contributions are not visible. If the image reconstruction is made with z = -194.5 mm, the reconstructed twin image becomes identical to the Fig.4a true image. Fig.4c is a zoomed part of Fig.4a true image. Fig.4d is a zoomed part of Fig.4b twin image.

The authors acknowledge support from the French National Research Agency (ANR).