Genetic Mapping of Two Components of Reproductive Isolation Between Two Sibling Species of Moths, Ostrinia Nubilalis and O

Home , European corn borer, Ostrinia, Ostrinia scapulalis

ORIGINAL ARTICLE Genetic mapping of two components of reproductive isolation between two sibling species of moths, Ostrinia nubilalis and O. scapulalis

Re´jane Streiff1, Brigitte Courtois2, Serge Meusnier1 and Denis Bourguet1

We report the quantitative trait loci (QTL) mapping of reproductive isolation traits between Ostrinia nubilalis (the European corn borer) and its sibling species O. scapulalis (the Adzuki bean borer), focusing on two traits: mating isolation (mi) and pheromone production (Pher). Four genetic maps were generated from two backcross families, with two maps (one chromosomal map and one linkage map) per backcross. We located 165–323 AFLP markers on these four maps, resulting in the identification of 27–31 linkage groups, depending on the map considered. No-choice mating experiments with the offspring of each backcross led to the detection of at least two QTLs for mi in different linkage groups. QTLs underlying Pher were located in a third linkage group. The Z heterochromosome was identified by a specific marker (Tpi) and did not carry any of these QTLs. Finally, we considered the global divergence between the two sibling species, distortions of segregation throughout the genome, and the location and effect of mi and Pher QTLs in light of the known candidate genes for reproductive isolation within the genus Ostrinia and, more broadly, in phytophagous insects. Heredity (2014) 112, 370–381; doi:10.1038/hdy.2013.113; published online 13 November 2013

Keywords: QTL mapping; European corn borer; Adzuki bean borer; Ostrinia; mating isolation; ecological speciation

INTRODUCTION by wing color patterns in Heliconius, with variations of these patterns In animals, algae and plants, many species initially classified as being involved in both mating preference and mimicry defense generalists have proved to be complexes of different taxa—be it strategy (Jiggins, 2008; Servedio et al., 2011). Host adaptation and varieties, races or sibling species—displaying little or no phenotypic mate preference are thus both putative components of reproductive differentiation, but each specializing in a particular habitat (Leliaert isolation while it has been suggested that the genetic architecture of et al., 2009; Speybroeck et al., 2010; Keller and Seehausen, 2012). In reproductive isolation and adaptive traits is a major element in this particular, phytophagous insect species previously considered to be adaptation – isolation link (Rundle and Nosil, 2005). Indeed, the polyphagous are frequently found to be mixtures of genetically probability of speciation is increased by the existence of tight linkage differentiated, specialist taxa, each feeding on a restricted range of (or even pleiotropy) between genes conferring host adaptation and host plants (Berlocher and Feder, 2002; Dre`s and Mallet, 2002; Dyer genes involved in reproductive isolation, and by these genes being few et al., 2007; Feder and Nosil, 2010; Matsubayashi et al., 2010). These in number (Hawthorne and Via, 2001; Berlocher and Feder, 2002). specialized taxa display a recurrent pattern of host-plant associations, Matsubayashi et al. (2010) have shown that the reproductive together with partial or complete reproductive isolation in sympatry. isolation process may comprise multiple components. For instance, This has led to claim that adaptation to different host plants may be a six incomplete isolation mechanisms are known to occur in two closely major source of speciation in insects (Matsubayashi et al.,2010). related species of the phytophagous ladybird beetles, Henosepilachna For phytophagous species living in sympatry and specializing on vigintioctomaculata and H. pustulosa, resulting jointly in almost different host plants, the crucial issue is the establishment and complete reproductive isolation. Studies on multiple components of maintenance of reproductive isolation. At the postzygotic level, host reproductive isolation are rare but are required if we are to understand adaptation may cause reduced hybrid viability and directly induce a which barriers contribute to speciation and how different barriers reproductive isolation even in the absence of a mate preference. At the might interact (Coyne and Orr, 2004). In line with this complex prezygotic level, various mechanisms have been described, from direct establishment of reproductive isolation between host-affiliated species effects of host-plant phenology, leading to a shift in reproductive of phytophagous insects, we describe here the genetic architecture of season in the host-affiliated insect (Wood and Keese, 1990; Pratt, the reproductive isolation between two sibling host-affiliated moth 1994; Feder and Filchak, 1999), to ‘magic’ traits (or genes) involved in species: the European corn borer (ECB, Ostrinia nubilalis Hu¨bner) and both adaptation and mating success. A striking example is provided the Adzuki bean borer (ABB, Ostrinia scapulalis Walker).

1INRA, UMR CBGP (INRA–IRD–CIRAD–Montpellier SupAgro), Campus International de Baillarguet, Montferrier sur Lez Cedex, France and 2CIRAD, UMR AGAP, Montpellier, France Correspondence: Dr R Streiff, INRA, UMR CBGP (INRA–IRD–CIRAD–Montpellier SupAgro), Campus International de Baillarguet, CS 30016, F-34988 Montferrier sur Lez Cedex, France. E-mail: [email protected] This paper is dedicated to the memory of Serge Meusnier. Received 19 October 2012; revised 16 July 2013; accepted 6 September 2013; published online 13 November 2013 Mapping reproductive isolation in Ostrinia spp RStreiffet al 371

Genetic, ecological and biological studies in western Europe ensuring assortative mating between ECB and ABB moths is unrelated (Bourguet et al., 2000; Martel et al., 2003; Thomas et al., 2003; to the type of female pheromone produced. The nature of the trait Malausa et al., 2007a; Malausa et al., 2007b) have shown that the ECB underlying assortative mating remains elusive, but investigations of and ABB are sibling species (Frolov et al., 2007). Each is adapted to its this trait may provide new insight into the reproductive isolation own principal host plants (maize for the ECB and hop, mugwort and between these two sibling species. hemp for the ABB), as shown by host choice for oviposition and In this study, we pursued the characterization of the mating larval feeding performance in reciprocal infestation experiments isolation (mi) between ECB and ABB, by locating QTL linked to this (Bethenod et al., 2005; Calcagno et al., 2007; Malausa et al.,2008). close-range isolation mechanism on a [ABB Â ECB] genetic linkage In addition, at least in Western Europe, these two species map. On the same map, we also located QTLs linked to Pher display strong, but incomplete, reproductive isolation: the ECB and identified the heterochromosome, because of its recurrent and ABB are interfertile, but the proportion of F1 hybrids in role in various traits, some of which being related to sexual seminatural (Bethenod et al., 2005) and natural (Malausa et al., selection (including resp (Linn et al., 1997) but also other candidates 2005) conditions is very low. Much of our research focuses on such as olfactory genes (Lassance et al., 2011)). The location and whether the specialization of ECB on maize—a crop introduced into number of QTLs linked to mi and Pher are discussed in light of the native area of Ostrinia spp., that is, Europe, B500 years ago previous candidate genes thought to be involved in reproductive (Tenaillon and Charcosset, 2011)—triggered its ongoing speciation isolation. Other characteristics of the interspecific map (segregation from the ABB, as presumed in ‘ecological speciation’ theory (Rundle distortions and heterozygosity level of the F1 hybrids) are also and Nosil, 2005). discussed in light of the process of divergence between these two The pheromone communication system is a key trait involved in host-affiliated species. reproductive isolation within the genus Ostrinia (Carde´ et al., 1975; Huang et al.,1997;Huanget al.,2002;Thomaset al.,2003;Bontemps MATERIALS AND METHODS et al., 2004; Takanashi et al.,2005).Naturalpopulationsofboththe Moth stocks and rearing ECB and ABB have a polymorphic communication system with two Two strains, one for each sibling species, were used as a source of initial different blends of the female sex pheromone. Some females produce mapping families. The ECB strain was founded from B100 pupae taken from a 3:97 mixture of (E)- and (Z)-11-tetradecenyl acetate (‘Z’ females), an outbred strain reared at INRA-Le Magneraud (France). This strain whereas others produce a 99:1 E/Z blend (‘E’ females, Klun, 1975). ‘Z’ originated from wild larvae collected from maize (Zea mays L.) in the vicinity and ‘E’ males preferentially respond and fly toward a 3:97 E/Z and a of Surge`res (France, 461100N, À01750E). The ABB strain was established and 99:1 E/Z blend of female sex pheromones, respectively (Roelofs et al., reared at the CBGP-INRA laboratory (France) from B50 diapausing larvae 1987). Formal genetic studies (Roelofs et al.,1987)andgenetic collected from mugwort (Artemisia vulgaris L.) near Paris (France, 481510N, 0 0 0 mapping (Dopman et al.,2004;Dopmanet al., 2005) have indicated 2121 E) and Lille (France, 50138 N, 3103 E) during the spring of 2004. All 1 ± 1 that female pheromone production and the male behavioral response strains were reared at 22 C 2 C under a 16:8 h light/dark photoperiod. Larvae were fed on a standard artificial diet (Gahukar, 1975). to this pheromone are autosomal and sex-linked traits, respectively, each being determined by a single major gene (designated Pher and resp, respectively, on the map). Finally, a locus encoding a fatty-acyl Crossing scheme reductase (pgFar,Lassanceet al., 2010; Lassance et al., 2013) has ECB females were crossed with ABB males to obtain F1 hybrids. These hybrids recently been identified as the principal functional gene underlying were then individually backcrossed with males or females of the two parental the Pher quantitative trait loci (QTL) and, thus, the shift from an E to strains. Two backcrosses, C04 and P10, were selected from the several dozen a Z pheromone blend. produced, on the basis of their having the largest family sizes obtained at the adult stage (70 offsprings for C04 and 71 for P10). The C04 and P10 This variability of the sex pheromone blend and of the male backcrosses correspond to the following pedigrees: [ABB female Â F1 male] for response ensures a certain level of reproductive isolation between E C04 and [F1 female Â ABB male] for P10 (Figure 1). and Z individuals within natural populations of both the ECB and ABB. Indeed, in locations at which E and Z individuals coexist (for example, in the United States for the ECB (Linn et al., 1997) and in Mating assessment Japan for the ABB (Takanashi et al., 2005)), the proportions of The level of mating isolation (mi) was measured in a no-choice experiment. females producing hybrid blends are lower than what would be Virgin females and virgin males from C04 and P10 backcrosses (Figure 1), within 24 h of emergence, were paired with an ECB virgin male or virgin expected assuming random mating between E and Z moths (Linn female, as appropriate, for three consecutive nights at 22 1C±2 1C, with a et al., 1997). This sex pheromone variation may also be involved in 16:8 h light/dark photoperiod. the reproductive isolation between the ECB and ABB. Indeed, in At the end of the third night, females were killed and dissected to determine Europe, sympatric ECB and ABB females produce Z and E blends of their mating status (virgin or mated), according to the content of the bursa pheromones, respectively (Pe´lozuelo et al., 2004). One straightforward copulatrix of the genital duct. The sperm and nutritious substances transferred explanation for this would be that the reproductive isolation between by males during copulation form an easy identifiable solidified structure (the these two sibling species is a mere consequence of long-range spermatophore) that is later used by females for fertilization. assortative mating due to differences in female sex pheromones, This mating design excluded sources of isolation other than the short-range resulting in the specific attraction of males to females of the same type acceptance or rejection of mating (for example, mating competition between (Thomas et al.,2003;Pe´lozuelo et al., 2004). However, experiments in males, long-distance pheromone attraction, and so on). It thus mainly target the ‘Am’ (assortative mating) trait described by Pe´lozuelo et al. (2007). This controlled conditions (Pe´lozuelo et al., 2007) have shown that even study demonstrated that a mating barrier exists between the ABB and ECB when moths are paired in a small box, forcing short-range encounters based on quantitative genetics and comparative analysis between intra- and and, thus, mating between sexual partners, the mating success inter-specific mating trials. Yet, by contrast with the comparative analysis based of E Â ZandZÂ E crosses remains lower than that of E Â Eand on multiple crosses in the study by Pe´lozuelo et al. (2007), the present Z Â Z crosses. By crosses and backcrosses of E and Z strains, Pe´lozuelo phenotype measurements do not distinguish between the reproductive et al. (2007) have also shown that the close-range mechanism isolation between the ABB and ECB, and other intrinsic individual propensities

Heredity Mapping reproductive isolation in Ostrinia spp RStreiffet al 372

O.n O.s this fragment of Tpi from the parents and offspring of the C04 backcross, by PCR, as follows: 5 min at 95 1C, 30 cycles of 30 s at 95 1C, 30 s at 60 1Cand 1 min at 72 1C, followed by 10 min at 72 1C. The PCR products were then digested with EcoRI, according to the manufacturer’s protocol. Digestion products were run in a 2% agarose gel and stained with ethidium bromide. The F1 male of the C04 backcross had two alleles for the target SNP (mixture H O.s H O.s of cut and uncut PCR fragments). Conversely, the ABB female of this backcross appeared to be homozygous. We then examined the segregation of the male parental alleles in the offspring of the C04 backcross.

P10 C04 mapping population mapping population Mapping analysis No crossing over occurs during oogenesis in Lepidoptera, so that all the markers on the same chromosome are inherited as a single unit (that is, O.n O.n O.n O.n O.n O.n O.n O.n O.n O.n O.n O.n without recombination) from the female parent (Heckel et al.,1999). Conversely, crossing over does occur during spermatogenesis in males, making Figure 1 Experimental design for ECB and ABB crosses and backcrosses. it possible to carry out crosses in which recombination affects the cosegrega- Circles and squares represent females and males, respectively. Individuals tion of markers on the same chromosome as a function of the distance from parental strains are shown in black (for the ECB strain) and white (for between markers. the ABB strain). F1 hybrids are shown in gray and the offsprings of Based on this unusual feature of female recombination in these species, four backcrosses are shown by pale gray graduated shading. Lines indicate linkage maps were constructed as follows. The first two maps were based on crosses (mapping scheme), and arrows indicate the mating experiment (mi the segregation of markers (i) present in the F1 male and absent from the ABB QTL experiment). ‘O.n’ is for Ostrinia nubilalis (ECB), ‘O.s’ for O. scapulalis female of the C04 backcross, and (ii) present in the F1 female and absent from (ABB) and H for hybrid. the ABB male of the P10 backcross. These two maps show the patterns of segregation of markers present in the F1 hybrid parent. We also constructed two other maps, based on the reverse configuration: markers present in the to mate. We thus distinguish the mating isolation (mi) trait measured here ABB female and absent from the F1 male of the C04 backcross, and present in from the Am trait observed in the study of Pe´lozuelo et al. (2007). the ABB male and absent from the F1 female of the P10 backcross. These two maps provided the pattern of segregation of markers present in the ABB parent. Female sex pheromone characterization We also made use of recombination patterns in the generation of these four Before dissecting female offspring of the C04 and P10 backcrosses to determine maps: for each backcross, markers present in the female and absent from the their mating status, we extruded the pheromone glands by pressing gently on male cosegregate as a single unit if they are located on the same chromosome. m the abdomen. Individual glands were incubated for 20 min in 20 lof99% We therefore obtained what we refer to hereafter as a ‘chromosomal map’—a hexane (Prolabo, Fontenay-sous-Bois, France) for pheromone extraction. The map on which all markers from the same linkage group display the same extract was then stored at 20 1C. We analyzed 3 ml of the frozen mixture on a À pattern of segregation in a given family (with the exception of mutations and/ ´ gas chromatograph, as described by Pelozuelo et al. (2007). Pure synthetic E or typing errors). For clarity, these maps are referred to as NR_P10 and and Z isomers of the female sex pheromone were used as internal standards. NR_C04 maps, where NR means ‘non recombinant’ and C04 or P10 refer to Pheromone characterization was performed on the female offspring of the P10 the segregating backcrosses. Conversely, when markers are present in the male backcross only, for external reasons. parent and absent from the female parent, recombination occurs and classical genetic maps are obtained, showing the ordering of and distances between the AFLP genotyping protocol markers defining the pattern of recombination in the offspring. These maps are DNA was extracted with the DNeasy Kit (Qiagen, Venlo, Netherlands), referred to as the R_C04 and R_P10 maps, where ‘R’ means recombinant and according to the manufacturer’s protocol, from the heads of adults stored in C04 or P10 indicates the segregating backcross concerned. ethanol. DNA concentration was assessed by fluorimetric quantification of In summary, NR_P10 is a chromosomal map for an F1 female hybrid, dsDNA with the PicoGreen Kit (Life Technologies, Carlsbad, CA, USA). AFLP R_C04 is a linkage map for an F1 male hybrid, NR_C04 is a chromosomal analyses were conducted as described by Midamegbe et al. (2011). Briefly, we map for an ABB female parent and R_P10 is a linkage map for an ABB male digested B100 ng of genomic DNA with EcoRI and BamHI (Promega, parent (Figure 1). Madison, WI, USA) enzymes, and then carried out two successive selective Linkage analysis was performed with Joinmap4.0 software (Stam, 1993). 0 0 0 PCRs with the EcoRI (5 -GACTGCGTACCAATTC-3 )andBamHI þ A(5- Linkage maps were based on ‘informative’ AFLP markers, that is, those that 0 ATGAGTCCTGATGATCCA-3 ) primers for the first reaction and the were heterozygous in the mapped parent. Chi-squared (w2)testswere EcoRI þ 2andBamHI þ 3 primers for the second reaction. In total, 16 pairs performed on informative AFLP markers to check the goodness-of-fit for of EcoRI þ 2andBamHI þ 3 primers were used. AFLP products were subjected the expected 1:1 Mendelian segregation ratio of each marker in the offspring. to electrophoresis on an ABI 3130XL (Applied Biosystems, Foster City, CA, Loci with distorted segregation patterns deviating significantly from this ratio USA) automated sequencer. Raw data were analyzed with GENEMAPPER with Po0.001 were excluded from map construction to avoid pseudo-linkages (Applied Biosystems, Foster City, CA, USA) version 4.0 software and (that is, 22, 19, 11 and 15 AFLP markers in R_C04, NR_P10, NR_C04 and individuals were scored for the presence or absence of each AFLP marker. R_P10, respectively). Linkage groups were then identified on the basis of a logarithm of odds (LOD) score of 8 in NR_P10 and NR_C04, and 6 in R_P10 Tpi genotyping and R_C04. The haploid number of chromosomes in Ostrinia spp. is n ¼ 31 The gene encoding triose phosphate isomerase (Tpi) has been shown to be sex- (Guthrie et al., 1965). We therefore expected to obtain 31 linkage groups. linked in the ECB (Glover et al., 1990). It is located close to a QTL For R_C04 and R_P10, the ordering of the markers within linkage groups contributing to the male response to female sex pheromone blend (resp locus, was determined by maximum likelihood and default parameters, in Joinmap. Dopman et al.,2004).Inthisstudy,wemappedTpi as follows. The DNA Recombination values were converted into map distances (in centimorgans, sequences obtained by Malausa et al. (2007b) were used to identify an EcoRI cM) by applying the Kosambi mapping function (Kosambi, 1944). restriction site overlapping a SNP distinguishing between the ABB and ECB. The genus Ostrinia, like most lepidopterans (Sahara et al., 2012), has a Using primers flanking this restriction site (forward: 50-GCCCAAGACGTCCA female-heterogametic sex chromosome system: males and females are ZZ and CGCTGC-30 and reverse: 50-TCTCCGCATGATACTTGAGA-30), we amplified WZ, respectively. Hence, the Z heterochromosome was recognized in R_C04 as

Heredity Mapping reproductive isolation in Ostrinia spp RStreiffet al 373 the linkage group bearing the Tpi locus. In chromosomal maps (NR_C04 and group varied from 2 to 15 in NR_P10 and from 2 to 14 in NR_C04. NR_P10), it was recognized on the basis of the segregation pattern of Z-linked Twenty-six markers remained unlinked in NR_P10 and three in markers: in such maps, the F1 parent was female and the mapped markers NR_C04. were present in the female parent and absent from the male parent and segregated in the offspring. The segregation pattern is thus that of a (ZAW) Linkage groups and distribution of AFLP markers female Â (ZaZa) male cross, where a indicates an absence of the AFLP band, We found 31 linkage groups in the R_C04 and NR_P10 maps, and A indicates its presence. The male and female offspring would be expected corresponding to the haploid number of chromosomes in the genus to be ZAZa and ZaW, respectively. We thus introduced a virtual marker into the data set with the sex of each offspring, and simple ordering of the AFLP Ostrinia (Guthrie et al., 1965). The number of linkage groups markers made it possible to identify those that were Z-linked. identified was lower for both NR_C04 (29) and R_P10 (27). This probably reflects the lower level of saturation of these maps. Clustering of markers The distributions of AFLP markers between linkage groups did not The number of AFLP markers per linkage group in the four maps was differ significantly from random expectations in R_C04 (w2 ¼ 5.02, compared with the number expected under a Poisson distribution, to test the d.f. ¼ 4, P ¼ 0.285) and NR_C04 (w2 ¼ 6.78, d.f. ¼ 4, P ¼ 0.148), randomness of the distribution of AFLP within the genome, with the ‘goodfit’ whereas weak deviations from random expectations were observed function and ks.test of the ‘vcd’ library in R/qtl software (available from http: for R_P10 (w2 ¼ 10.73, d.f. ¼ 4, P ¼ 0.030) and NR_P10 (w2 ¼ 14.90, //www.rqtl.org/). The same tests were applied to the distribution of the d.f. ¼ 5, P ¼ 0.011). However, all the w2 values were in the same order distances between two consecutive markers within linkage groups for of magnitude. R_C04 and R_P10. The results of this second analysis are directly dependent Within linkage groups, the frequency distribution of the distances on the bin sizes used to calculate frequencies (Winter and Porter, 2009). We used a bin size of 2 cM. between consecutive markers in R_C04 and R_P10 showed the markers to be significantly clustered (R_C04, w2 ¼ 345.93, d.f. ¼ 6, À4 2 À4 QTL analysis Po10 , and R_P10, w ¼ 111.50, d.f. ¼ 6, Po10 ). QTL analysis was carried out with binary values obtained in the mi experiment, coded as 0 for non-mated individuals and 1 for mated individuals. Bridge markers between maps Pheromone type was also analyzed in the P10 offspring as a binary trait Eighty of the 937 AFLP markers were mapped on at least two of the corresponding to two classes of pheromone: E and E/Z (hybrids). Computa- four maps. No marker was mapped on both R_C04 and NR_C04 tional analysis was carried out with R/qtl software (available at http: // because, in these maps, the informative markers were in opposite www.rqtl.org/) as previously described (Broman et al., 2003). QTL detection configurations (that is, present in the F1 male and absent from the was performed by interval mapping (Dempster et al., 1977) in R_C04 female parent in R_C04, but absent from the F1 male parent and and R_P10 and by marker regression in NR_C04 and NR_P10 (because of present in the female parent in NR_C04, see Materials and Methods). the lack of recombination in these maps), with the binary model recommended for binary phenotypic traits (Broman et al., 2003). LOD significance The same reasoning holds for R_P10 and NR_P10. thresholds for QTLs were established with 1000 permutations, as described by Details of the bridge markers between maps are provided in Churchill and Doerge (1994), with a procedure adapted to the heterochromo- Supplementary Figure S1. In brief, three, two, five and one linkage somes, as recommended by Broman et al. (2006) and implemented in R/qtl group were connected by two or more markers between R_C04 and (Broman et al.,2003). NR_P10, R_P10 and NR_C04, NR_P10 and NR_C04, and NR_P10 and R_C04, respectively. Total concordance was observed for these RESULTS markers. Thus, any two markers found to belong to the same linkage Linkage maps group in one map also belonged to the same linkage group on In total, 323 and 165 AFLP markers were informative (that is, another map (provided they were present on that map). heterozygous in the F1 parents) in R_C04 and R_P10, respectively (Figures 2 and 3). The number of informative markers is directly Segregation distortion related to the level of heterozygosity in the F1 and the degree of The markers showing a strong deviation to 1:1 segregation (w2-test, divergence between the parental lines. Po0.001) were discarded prior to maps’ construction (see Materials We obtained 31 and 27 linkage groups for R_C04 and R_P10, and Methods). Weaker segregation distortion was observed for 36, 14, respectively, with a LOD score of 6. The total length of the map was 29 and 7 loci (0.001oPo0.050) in R_C04, NR_P10, NR_C04 and 997 cM for R_C04 and 595 cM for R_P10, with the length of linkage R_P10, respectively. After linkage analyses, 22, 13, 26 and 4 of them groups ranging from 1.8 to 36.2 cM in R_C04 and 1.5 to 60.1 cM in were mapped to linkage groups in R_C04, NR_P10, NR_C04 and R_P10. The difference in map length between the two families likely R_P10, respectively. In total, with a significance threshold of 0.05, reflects a lack of saturation of R_P10 (less linkage groups and less distorted loci accounted for 18, 14, 19 and 13% of the screened markers). The mean distance between two consecutive markers was of markers in R_C04, NR_P10, NR_C04 and R_P10, respectively. 3.8 cM in R_C04 and 6.1 cM in R_P10. Forty-three (17) markers In all four maps, loci with distorted segregation patterns were remained unlinked in R_C04 (R_P10). highly clustered both within and between linkage groups. In R_C04, the markers with distorted segregation were present in only 6 of the Chromosomal maps 31 linkage groups. Some linkage groups displayed segregation In total, 234 and 215 AFLPs were informative in NR_P10 and distortion in highly localized areas (e.g., linkage groups 3, 13 and NR_C04, respectively. Linkage analysis identified 31 groups of 27; Figure 2). Conversely, linkage group 19 displayed distortion over cosegregating markers in NR_P10 and 29 in NR_C04 (Figures 4 almost its entire length and linkage group 14 displayed distortion over and 5). The pattern of marker cosegregation was consistent with the half its length. In R_P10, all the distorted (and mapped) markers were lack of recombination in female lepidopterans, with the exception of present in only 1 of the 28 linkage groups identified (linkage group 5, 0.73% genotyping error and/or mutations for NR_P10 and 1.00% for Figure 3). NR_C04, as estimated by counting the unexpected genotypes in the In NR_P10 and NR_C04, in which there was no recombination pattern of cosegregation obtained. The number of markers per linkage within linkage groups, a similar pattern was observed, with three

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0.0 eTAbACG162.98 0.0 eTTbACC367.62 0.0 eATbAgA507.13R * 0.0 eTAbACG462.6R 6.3 eTAbAGT262.12R eTTbACC282.4 0.9 eTTbACC178.1R 0.0 eTCbACA112.52R 0.8 CPeTAbACG156.25R 9.6 eTAbAGT484.47R 0.3 eTTbATT367.41 3.6 CPbeATbAgA421.4 * 2.0 eTAbAGA445.39R eTTbATT282.22 3.8 CPeTTbACC406.99 2.1 eTAbAAA445.52R 10.7 CPbeTAbATC237.08 6.0 eTAbATA294.3R eTAbATA229.08R 7.2 eTAbAAA369.4 2.5 eTAbATA445.66R 12.3 eAGbAGG195 5.6 6.5 eTAbATC455.56R eTAbAGT230.15R 12.9 eTAbATA397.58 5.8 CPeTAbACG402.68 12.4 eAGbATG195.16 8.9 eTAbACG307.12R 13.8 eTAbATC397.31 eTAbACG390.79R eAGbATG495.05R 14.6 9.0 eTAbATA346.89R 18.9 CPeTAbATA249.12 9.3 eTTbACC271.53R 19.6 eTAbAAA249.16 22.3 eAGbAGG102.8R 21.0 eTAbAGA249.08 33.3 eTAbATA129.12R 4 22.5 eTAbACG282.3R 34.8 eAGbAGG170 CPeAGbATG105.85R 0.0 CPeTTbACC486.59R 32.3 eTAbAAA239.06R 25.2 eTAbACG519.05 35.1 eTAbACG472.29R eTAbACG187.48R 33.1 eTAbAGA238.96R 35.3 CPbeAGbATG169.52 38.0 eACbAgg500.5 35.4 eTAbATA239.05R 35.4 eAGbAGG198R 38.9 eTAbACG409.45 10.1 eACbAgg182.19R 36.2 eTAbATC129.48R 40.2 CPeTAbACG374.54R 42.5 eTTbACC318.44 43.7 eAGbAGG292R eTTbACC314.07R 46.9 eTCbACA145.06 52.4 eTAbACG438.22R 54.1 CPeTCbACA457.99R 28.1 CPeATbACC268.2R 56.2 eTAbATC281.62R 29.3 CPeATbAgA244.98R 61.9 eATbACC245.39 62.7 eACbAgg148.91 35.2 eATbAAA268.33R 63.2 eATbAAA245.61

7 8 10 11 12

0.0 eACbAGT271.46R 0.0 CPeTAbACG133.17R 0.0 eTAbATA401.18R 0.0 eTCbACA498.07R 0.1 CPbeAGbATG394.38 0.0 eAGbAGG516R 1.8 eTAbATA273.37R 4.7 eACbAgg440.37R 0.4 eACbAgg245.64 3.0 eACbAgg173.9R 3.9 eTAbAAA273.32R 4.8 eTCbACA152.7R eTTbATT243.21R 3.8 eAGbATG516.31R 4.0 eTAbAGA273.21R 10.0 eTTbACC243.39R 4.6 eTAbACG377.53R 5.6 eTAbACG139.67R 19.2 eTAbAGA329.62R eTTbACC360.42 10.9 eTAbAGT279.98 14.4 eTAbAAA406.11R CPbeATbAgA366.26R eTTbACC277.96R 11.7 16.3 eACbAgg419.12 eTAbACG385.2 * eACbAGT265.79R 26.2 13.5 CPeAGbAGG108.7R 22.1 CPeAGbATG354.15 eATbAAA366.41R 16.7 19.8 eTAbACG178.16 22.2 eAGbAGG354 eTAbATA329.82R 9 28.6 eTAbATA312.7R 27.8 eTAbAGA160.13R 26.3 eTAbATA328.62 25.4 eTAbAAA298.03R 28.9 eTTbACC152.13 29.9 eTAbATA160.13R eTTbATT355.48 0.0 eAGbATG221.15R eTAbAGA312.51R 36.1 eTAbAGT385.48R 27.3 CPbeTAbACG329.59R 1.4 eAGbAGG221R 33.7 eATbAAA179.53R 30.0 eTAbAAA312.64R eTAbAGA328.4 37.9 eTCbACA264.31R 28.6 34.0 eATbAgA179.45R 33.5 eAGbATG325.96R 38.0 eAGbATG313.31 38.8 eACbAGT395.26R ** 37.9 eATbAgA410.43R 34.5 eAGbAGG326R 42.4 eACbAgg326.29R 41.8 eTCbACA173.98 38.2 eACbAGT119.15 39.5 eTCbACA102.17 eATbAAA165.1R * 46.2 eATbACC127.56R 43.4 42.8 eTAbAGT405.66 eTAbATC471.64 16.4 eTAbACG514.8 47.7 50.6 eATbAAA127.49R 22.1 eAGbAGG300R eTAbAAA149.2R 54.2 eTAbATA223.88R 58.3 23.5 eAGbATG300.17R 52.4 eTAbAGT282.44R eTAbAGT368.33 24.7 eACbAgg330.19R 60.0 eTAbAGT397.26 63.2 eTAbAGT256.47R

13 14 15 16 17

0.0 eTAbACG205.31 0.0 CPbeAGbAGG415 0.0 eATbAAA305.43 * 0.0 eTTbATT503.77R 0.0 eATbACC82.96R 2.0 eTCbACA304.55 1.9 eAGbATG414.97 1.5 CPeATbAAA422.59 4.2 eTTbACC465.79R * eTAbACG332.16 ** 13.2 eTAbAGA316.78R 14.4 CPeTAbAAA316.92R 15.8 CPeTAbATA316.96R 14.6 eTCbACA418.93 20.7 CPeAGbATG208.6 16.3 eTAbATA438.86 19.9 eATbAgA485.41R 16.7 CPbeTAbACG340.02R 21.1 eAGbAGG209 16.6 eTAbAGA438.62 20.8 CPeTAbACG129.62R 18.3 eATbAgA132.41 20.3 eACbAGT314.64R 23.5 eATbACC485.29R 21.7 eTAbACG317.3 eTAbACG316.46R 22.1 eACbAGT196.37R 27.4 eTGbAGT236.99R 23.5 eTTbATT164.34 29.7 eTGbAGT508.33R 31.6 CPbeATbACC282.09 30.0 CPeAGbATG141.01 30.9 eTTbACC139.58R 31.2 eTAbAGT247.49R 37.4 eTTbATT187.96R * 39.0 eTCbACA332.87R 37.8 eTTbATT154.24R 37.8 eTTbACC193.1 39.3 eACbAGT90.37 40.7 eTAbACG226.13R 18 49.6 eACbAgg346.01R ** 49.7 eATbACC285.56R 54.9 eTAbATA457.95R * 0.0 CPeACbAgg399.12 55.6 eAGbAGG308R * 54.3 eATbAAA285.74R 56.0 eAGbATG307.7R * 10.2 CPeTGbAGT276.7

19 20 21 23 24

0.0 eATbAAA335.56 0.0 eAGbATG321.96 0.0 eTAbATA161.82 0.0 eTAbAGA519.26R 0.0 eATbAAA267.69 2.3 eATbACC335.41 ** eTAbACG161.74 1.7 eATbACC267.5 3.5 CPbeTAbACG338.98R** 4.0 eTAbATC428.71 3.7 eTTbATT290.09R ** 9.2 eAGbAGG322 8.3 eAGbATG167.42R eTAbATC127.65R * 8.5 CPbeAGbAGG167R 11.7 CPeTGbAGT183.19 5.9 * eTAbAGA254.56 eTGbAGT449.11R 17.8 13.5 CPeTAbACG245.34 12.5 ** eTAbACG503.66 18.0 18.9 eAGbAGG164.6R 18.3 eTAbATA254.67 22 19.4 eTAbAAA254.66 23.7 eAGbATG164.6R 26.2 eTAbATA513.53R 0.0 eATbAgA511.1R 27.9 eTAbATC513.22R eTAbATC238.18 eACbAGT451.49 3.7 30.7 eTAbACG436.47 31.1 eACbAGT67.55 30.1 *** 33.9 eATbAgA478.15 eTCbACA501.41R 9.7 eACbAgg483.51 35.8 * 41.9 eTAbACG334.58R 42.0 eTCbACA165.37 42.3 eTGbAGT503.99R 44.9 eTAbAGT513.64 21.8 eAGbATG118.41

25 27 28 29 30

0.0 eTAbACG309.14R 0.0 eATbAgA371.86R eACbAgg371.25 0.0 eTAbACG358.01R 0.1 eATbACC372R 0.0 CPeTAbAGA435.52R 0.0 eACbAgg227.11R Tpi 3.0 eTAbAAA461.96R 0.2 eTGbAGT272.21R 1.8 eTAbATA435.78R 8.1 eATbAgA225.98R 10.0 eACbAgg468.65 31 22.7 eTGbAGT448.12R 16.5 eTAbAAA410.86R eACbAgg128.12 19.6 eTAbAAA428.36R 0.0 eTAbAGT410.38 19.8 eTCbACA157.82 ** 26.1 eATbACC220.32R CPeTGbAGT392.22 0.1 eTAbACG449.95 27.3 eTGbAGT225.36R 21.9 eTAbATA428.4R 1.8 23.9 eAGbAGG412 CPbeTAbACG416.9R 28.0 CPeACbAGT424.48R eATbACC225.11 28.7 eTAbAGA428.27R 3.4 28.4 eTAbACG168.6 6.9 eATbAgA103.52R 11.0 eATbAgA401.36 29.0 eTGbAGT194.85 26 31.0 eTAbACG191.87 0.0 eTAbAGA112.66 eTAbAAA112.72 0.1 eTAbATA112.6 6.9 eTAbACG319.52 Figure 2 R_C04 linkage map. Vertical black bars correspond to linkage groups. Linkage group IDs are given above each bar. Horizontal ﬁne bars correspond to AFLP marker positions, with the distance (in centimorgans, cM) on the left and the marker ID on the right. The Tpi marker (see Materials and Methods), identifying linkage group 27 as the Z heterochromosome, is bounded by an open black square. Black stars highlight markers displaying distorted segregation, *Po0.050, **Pp0.010 and ***Pp0.001.

Heredity Mapping reproductive isolation in Ostrinia spp RStreiffet al 375

1 2 3 4 5

0.0 eAGbATG147.06 0.0 CbPbeTAbACG310.69 0.0 CbPbeTAbACG349.89R 0.0 CbPbeTAbATA200.35R 0.0 CbPbeAGbATG508.27R 0.2 CbPbeTTbATT203.17 1.6 eATbAAA251.96 * 4.7 eTAbACG127.85 8.6 eTGbAGT214.28R 9.3 CPbeTAbACG340.07 12.8 eTCbACA195.07 11.5 CbPbeATbAGA282.07R 15.2 14.2 CPbeATbAAA282.31R 13.9 eTTbATT507.78R * 15.4 CbPbeTAbACG427.92 eTAbACG394.16R * CPbeAGbATG169.52 eAGbATG410.36 17.1 * 18.8 18.7 eTAbACG126.69 19.8 eTCbACA329.86R 19.0 eATbAGA156.54 eTTbACC428.35R eTCbACA309.69R 21.4 23.6 CbPbeAGbATG113.84R 23.8 eAGbAGG113.84R 27.7 CPbeTAbATC237.02

34.1 eATbACC354.46R 34.2 CbPbeTCbACA279.2 37.0 eAGbATG149.28 40.0 eATbAGA287.37R 41.0 eTTbATT159.93

53.2 CPbeATbAGA366.66R 53.9 eTCbACA480.06R 55.5 CbPbeTAbAGA193.29 56.8 eTGbAGT139.2R 60.1 eTAbATC425.12

6 7810 11

0.0 eAGbAGG394.13 0.0 eATbAGA431.66 0.0 eTCbACA482.56 0.0 eACbAgg300.26R 0.0 CbPbeAGbAGG293.99 eAGbAGG390.16R 1.7 eATbAAA431.95 1.6 1.7 CPbeAGbATG394.33 5.5 eTAbATC328.03 1.9 eTGbAGT390.55 6.6 CPbeAGbAGG167.64 11.9 eTAbAGA361.1R 15.2 eTAbATA92.68 12.2 eTAbACG492.69R 14.7 CPbeATbAGA421.25 15.8 eAGbATG369.59R 15.3 eTAbACG92.43 17.3 eATbACC316.81 18.5 eACbAgg279.05 16.8 eTAbACG120.6R 20.2 eTAbAGA385.33 21.8 eAGbATG100.51R 24.5 CbPbeTCbACA295.7R 9

0.0 eACbAgg406.14R 1.7 eTCbACA464.15R 42.5 eTAbACG464.02R

12 13 14 15 16

0.0 CPbeTAbATA406.08 0.0 eATbACC333.5R 0.0 eTTbATT257.23 0.0 eAGbATG471.04 0.0 eTAbAGA343.08 1.7 eATbACC338.78 3.0 eATbAAA185.66 2.0 eTCbACA401.61R 6.1 eTTbATT353.01R 4.5 eTAbACG102.48 eTAbATA136.55 6.2 CPbeTAbACG329.64 8.0 eTTbATT440.04 8.6 eTAbATA280.11 7.6 13.2 CbPbeTCbACA302.52R 13.4 eACbAgg240.87 15.2 eATbAGA413.46 19.6 eAGbATG371.44R 23.7 eTAbAAA202.71R 26.8 eATbAAA173.81R 26.8 eTAbACG442.52R 30.8 eTAbACG357.45

40.2 eTAbATA220.95 41.2 eTAbAAA220.93

47.6 eTAbATA416.84R 47.7 CPbeTAbACG416.72R

17 18 19 20 21

0.0 CPbeAGbAGG415.57 0.0 eATbACC370.3R 0.0 CPbeTGbAGT429.4R 0.0 eAGbATG246.37R 0.0 CPbeTAbACG338.24R 3.5 eTAbACG362.54R 5.3 eAGbATG506.36 7.0 eAGbAGG506.15 10.5 CbPbeATbAGA281.18R eTAbACG367.04 10.8 eATbAAA281.39R 12.3 12.4 CbPbeTAbACG506.55R

19.9 CbPbeAGbAGG248.02R 21.1 eATbACC282.13 22.8 CbPbeACbAgg207.97 26.9 eATbAAA241.04 27.2 eATbAGA240.89 30.3 eTAbACG228.26

39.5 eTCbACA375.12R 41.5 eTAbACG89.88R

22 25 26 27

0.0 eTGbAGT219.61 0.0 eTAbAGT131.14 0.0 eAGbATG331.13 eAGbATG268.28 eATbAAA379.48 0.0 eAGbAGG268.14 1.5 1.5 eTAbACG103.43

15.1 eTAbACG483.87 23 16.6 eTAbACG325.51 28 eTTbACC322.37 eAGbATG303.92R 19.0 0.0 eATbAAA467.92R 0.0 CbPbeTAbACG269.66 2.9 eATbACC473.5 1.6 eTAbAAA514.27R Figure 3 R_P10 linkage map. Vertical black bars correspond to linkage groups. Linkage group IDs are given above each bar. Horizontal ﬁne bars correspond to AFLP marker positions, with the distance (in centimorgans, cM) on the left and the marker ID on the right. Black stars highlight markers displaying distorted segregation, *Po0.050, **Pp0.010 and ***Pp0.001. The initial coding of linkage group names was retained throughout the entire mapping process, leading, by chance, to the omission of linkage group 24. We retained the initial IDs to prevent errors in subsequent analyses.

groups displaying high levels of segregation distortion in both linkage group affecting all the markers from that group, as they are NR_P10 and NR_C04 (Figures 4 and 5). In NR_P10 and NR_C04, fully linked. However, as noted before, some genotyping errors (or this pattern should be ‘perfect’, with any distortion within a given mutations) and missing data may account for these discrepancies.

Heredity Mapping reproductive isolation in Ostrinia spp RStreiffet al 376

CbPeAGbAGG262.4 CbPeATbAAA375.29R CbPeTAbAGA132.88R CPeTTbACC400.96R CbPeAGbATG262.49 eAGbAGG318.74 CbPeTTbATT319.22R eACbAgg251.67 CPeATbAAA422.6R eAGbATG318.89 CPeATbAAA481.62 eAGbAGG155.17 4 eAGbAGG119.79R eAGbATG481.47R3 eACbAgg481.13 eAGbATG155.1 * eATbAGA459.62 eATbACC104.54R eAGbAGG102.14R eTAbACG95.98 1 eTAbACG194.29 eTAbACG429.64R eTAbAAA132.91R eTTbATT486.52R eTAbACG446.83R eTAbACG86.4 eTCbACA344.77 eTAbATA472.85 eTCbACA468.9R eTGbAGT179.19R eTCbACA172.61 eTTbACC249.84R5 eTGbAGT181.02 eTCbACA405.64 eTGbAGT261.85 eTTbACC370.86R

CbPeAGbATG498.43R CbPeATbAAA120.81R eACbAGT110.06 eACbAgg175.12 CbPeTTbATT345.79R CPeTAbATA249.18R eAGbAGG298.59R eACbAGT480.69R CPeTAbACG374.57R eACbAgg219.08 eAGbAGG342.41R eAGbATG476.52R CPeTTbATT339.19R eAGbATG189.07 eAGbATG298.78R eTAbAAA519.1R eACbAgg264.68R eATbAAA141.2R eAGbATG342.51R eTAbACG308R 9 eAGbAGG291.74R eATbACC345.38 eATbAAA216.21R eTAbAGA470.77 7 eAGbATG291.96R eTAbACG142.1R eATbAGA216.07R eTAbATA470.91 6 eTAbACG343.43R eTAbACG324.41R eATbAGA224.47 eTAbATA519.4R eTAbACG407.91 eTAbATA324.57R eTAbACG443.73R eTAbATC395.08R eTAbATC310.72R eTAbATC206.95 eTAbACG84.06R eTAbATC398.12 eTTbACC346.02R eTAbATC399.51R eTTbATT267.01 eTTbACC361.47R eTCbACA223.5 eTTbACC381.06R eTTbATT380.89R eTTbATT518.91R

CbPeATbACC296.31R eACbAGT160.8 CPeATbAGA503.48R eTGbAGT356.99R 13 CbPeATbAGA296.21R eATbACC367.52R ** eAGbAGG124.33R eTGbAGT367.69 *** 11 10 CbPeTAbACG327.01 eATbAGA266.52R * eAGbAGG197.05 12 eACbAgg250.42R eTTbACC232.03 * eAGbAGG457.19 eTAbATA110.49R eTCbACA298.32R eTCbACA314.65

CPeAGbATG141 CPeAGbATG105.95 CbPeTAbATC415.02R CbPeAGbAGG246.86R CPeAGbATG208.59 CPeTAbACG135.25 CPeTAbAAA425.75 eACbAgg136.49 CPeATbACC268.58R CPeTAbACG402.72R CPeTAbATA425.89 eTAbACG200.95 eAGbAGG140.9515 CPeTCbACA457.9816 eATbACC423.26R17 eTAbACG433.37R 14 eAGbAGG179.51 eAGbAGG105.77 eTAbATC329.75 eTAbACG451.48 eAGbAGG208.56 eTAbACG221.6 eTAbATC363.96 eTCbACA320.36 eAGbATG179.54 eTAbATC136.45 eTCbACA316.51R eTCbACA324.51R eAGbATG437.4R eTAbACG112.49

CbPeAGbAGG250.78 CPeTAbAAA316.79R eACbAgg364.99 eACbAgg184.08R CPeAGbATG354.29R CPeTAbATA316.93R eATbACC289.08R eATbAAA332.99 * eAGbATG250.87 eTAbACG336.72 eTAbACG232.2821 eATbACC453.03R 18 19 20 eATbAAA381.66R eTAbAGA218.09 eTAbACG234.89R eATbAGA463.52R eTAbATA402.75R eTCbACA250.2 eTAbACG480.77R eTCbACA129.76 eTAbATC243.18R eTTbATT150.83R eTTbACC124.91R

CbPeTAbATC233.85R ** CPeTAbACG129.76 CbPeTAbAAA433.67R eACbAgg109.31 CPeTAbAGA503.86R ** eACbAgg162.94R eTAbACG244.15R eACbAGT270.78R 25 22 eAGbATG357.22R * 23 eAGbAGG461.824 eTAbATC411.96R eAGbATG460.09 eATbACC416.15R *** eATbACC210.24R eTCbACA102.22R eTAbACG273.61 eTAbATC266.64R eTCbACA196.52 eTTbATT405.07R

CPeAGbATG89.03R CPeTAbACG245.38 eACbAgg510.04 CPeTGbAGT392.08R 29 eACbAgg179.02R27 eTAbACG457.9428 eAGbAGG284.28R eTGbAGT451.57R 26 eTAbACG452.48 eTCbACA333.46 eTAbATA150.17 eTAbAGA252.83R

eAGbAGG378.15R ** CPeACbAGT424.46R 30 31 eATbACC200.04R *** eAGbATG272.45 Figure 4 NR_P10 chromosomal map. Vertical black bars correspond to linkage groups, with group ID shown in white. As no recombination occurs in the female parent, all markers within a linkage group are inherited without recombination, so it was not possible to calculate genetic distances in this pedigree. Black stars indicate markers with distorted segregation, *Po0.050, **Pp0.010 and ***Pp0.001. The Z heterochromosome was identiﬁed here as linkage group 2 (framed in black), on the basis of the particular pattern of segregation expected and observed for Z-linked markers (see Materials and Methods).

QTL detection For the mating isolation (mi) trait, we found two significant QTLs For the pheromone production (Pher) trait, one significant QTL was in the NR_P10 map (linkage group 3, LOD ¼ 2.51, P ¼ 0.028, a ¼ 37% found in linkage group 1 in NR_P10 (LOD ¼ 4.86, Po10 À4), of the phenotypic variation; linkage group 4, LOD ¼ 2.65, P ¼ 0.023, accounting for 75% (a, additive effect) of the phenotypic variation a ¼ 38% of the phenotypic variation, Figure 7) and one QTL in the (Figure 6). In the same pedigree, but on the R_P10 map, a significant NR_C04 map (linkage group 8, LOD ¼ 2.32, P ¼ 0.035, a ¼ 37%). As QTL was also identified for Pher in linkage group 9 (LOD ¼ 2.57, no recombination occurred in the F1 female parent for NR_P10 and P ¼ 0.048, a ¼ 58%, Figure 6). NR_C04, no genetic distance could be inferred between markers, and

Heredity Mapping reproductive isolation in Ostrinia spp RStreiffet al 377

CbPbeTCbACA302.53 eAGbAGG205.75 CbPbeACbAgg207.98 CbPbeAGbAGG339.43R eTAbAAA221.08R eAGbATG205.79 CbPeTGbAGT319.02 CbPbeAGbATG339.63R eTAbACG460.81R eAGbATG248.33R CbPeTTbATT319.25R eACbAgg317.19 eTAbACG466.81R eTAbACG285.27 eACbAGT76.57 eATbAAA172.94R 2 3 1 eTAbAGT104.8R eTAbAGT215.79R eATbACC372.774 eATbAgA172.89R eTAbATA221.04R eTAbATA300.39R eTAbAAA196.45 eTAbACG249.5R eTGbAGT154.54R eTAbATC175.62R eTAbACG465.88R eTAbAGT110.19 eTTbAAC453.86R eTAbATC260.96R eTCbACA415.63 eTTbAAC173.68 eTTbATT295.74R eTTbATT320.55R

CbPeATbACC296.34R CbPeATbAAA120.78R ** eACbAgg271.4 CbPbeTAbAGA193.29R *** CbPeATbAgA296.47R eACbAGT261.91R ** eATbAAA215.44 CbPbeTTbATT203.18R ** CbPeTAbACG327.03 eACbAGT268.23 ** 7 eATbAgA215.32 CbPeTAbATC415.01 ** eACbAGT297.32 eAGbAGG386.35 eATbAgA216.3 eTAbAGT360.46 ** 5 6 10 eAGbAGG501.66R eTAbAAA154.91R * eTAbAGA458.42 eTAbATA193.3R ** eATbAAA495.65R eTAbAGA154.84R ** eTAbATA388.02 ** eTAbACG257.2 eTAbATA154.86R CbPbeTAbACG427.04R eTAbATC366.12R ** eTAbAGT88.95R * eTAbATA320.68R ** eACbAgg352.78 eTCbACA328.11 *** 8 eTAbATC340.64 ** eTAbACG109.89 eTCbACA297.92 * eTCbACA459.94R *

CbPeATbAAA375.22 CbPbeAGbATG113.86R CbPbeTAbAGA340.5 CbPbeATbAgA281.46 CbPeTGbAGT474.84 CbPbeAGbATG508.25R CbPbeTCbACA295.69 CbPbeTAbACG506.53 11 eACbAgg199.08 *** CbPeTAbATA297.22 eACbAGT319.58R eACbAgg407.88R eTAbACG114.64 eAGbATG95.83R eAGbAGG387.97R eACbAGT254.17 eTTbAAC249.8 eATbAAA181.99 eAGbATG202.23R eAGbAGG199.67 eATbACC298.56 eAGbATG388.21R eTAbACG121.74 eATbAgA181.93 eTAbAAA376.91R15 eTAbACG487.88R 13 14 eTAbACG118.99 eTAbACG120.33R eTAbAGA459.56R eAGbATG265.4R eTGbAGT299.71R eTAbACG153.7R eTAbAGT212.92R 12 eATbACC253.59 eTTbAAC239.82 eTAbAGT88.25 eTAbATA459.85R eTTbAAC428.27R eTAbATA279.83R eTAbATC422.71 eTTbATT239.63 eTAbATA377.01R eTGbAGT325.48R eTTbATT261.17 eTGbAGT279.44R eTTbATT165.8R eTTbATT507.67R eTGbAGT368.61

CbPbeATbAgA282.19 CbPbeTAbACG269.79R CbPbeTAbACG310.67 eAGbAGG426.14 CbPbeTAbACG349.88 CbPeAGbATG498.43R CbPeAGbAGG250.03 eAGbATG426.33 CbPbeTCbACA279.19 eTAbACG176.49 CbPeTAbATC233.87 eTAbAGT345.35 CbPeAGbAGG262.42R eTAbAGA131.17R eAGbATG250.0919 eTAbAGT497.42 18 CbPeAGbATG262.53R17 eTAbATA131.14R eTAbAAA139.43 eTCbACA382.04 16 eACbAgg480.84 eTAbATA291.64R eTAbAGA139.37 eTTbAAC417.73R eAGbAGG255.42R eTTbAAC400.23R * eTAbATA139.33 eTTbATT147.35 eAGbAGG494 eTTbAAC455.68 eTAbATC270.7 eAGbATG255.56R eTTbATT391.22 eATbAAA471.64 eATbAgA471.48

CbPbeAGbAGG293.93 CbPeTAbAAA433.74 CbPbeACbAgg263.2R CbPbeAGbATG316.76R eACbAGT69.86R eAGbAGG160.22 eAGbAGG313.83 CbPeAGbAGG246.89 eAGbAGG278.36R eAGbATG160.17 eATbAAA404.45 ** 23 eACbAgg244.55R 20 21 22 eATbACC235.69R eATbAgA345.74 eATbACC159.65 ** eAGbAGG216.67 eTAbACG254.86R eATbAgA369.42 eATbAgA462.52R * eAGbAGG316.59R eTAbATA496.02R eTAbATC232.56 eTTbATT469.64 *

eACbAgg224.71 CbPbeTAbATA200.35 eAGbAGG217.59 CbPeTTbATT345.16 eACbAgg259.94 eACbAGT63.93R eTAbACG30627 eTCbACA299.27 26 24 eATbAAA167.33R25 eTAbAGT275.29 eTAbAGT106.95R eTTbATT375.2 eATbAgA167.28R eTAbATA451.58 eTAbATA102.34R eTTbAAC472.28R eTAbATC199.61

eACbAGT476.74 eATbACC230.63 eACbAGT435.63 * 28 eTAbAGT153.5729 eTAbAGT430.06R30 eTAbACG147.57 eTGbAGT493.33 eTCbACA424.74 eTAbATA411.91 ** Figure 5 NR_C04 chromosomal map. Vertical black bars correspond to linkage groups, with group ID shown in white. As no recombination occurs in the female parent, all markers within a linkage group are inherited without recombination, so it was not possible to calculate genetic distances in this pedigree. Black stars indicate markers with distorted segregation, *Po0.050, **Pp0.010 and ***Pp0.001. The Z heterochromosome was identified here as linkage group 11 (framed in black), on the basis of the particular pattern of segregation expected and observed for Z-linked markers (see Materials and Methods). The initial coding of linkage group names was retained throughout the mapping process, leading, by chance, to the omission of linkage group 9. We retained these IDs to prevent errors in subsequent analyses. the QTLs identified on these maps are attributable solely to linkage of a sex pheromone, Pher) involved in reproductive isolation groups, without further information on the location within these and previously mapped in the ECB (Dopman et al.,2004). groups. All the linkage groups bearing QTLs involved in Pher and mi We produced genetic maps based on [ABB Â ECB] Â ABB were autosomal. backcrosses, for QTL detection and localization. These interspecific pedigrees provided information about the degree of heterozygosity DISCUSSION of the F1 hybrid parents (estimated by determining the number The goals of this study were (i) to characterize the genetic architecture of informative markers, see Results) and segregation distortion of a trait (mating isolation, mi) involved in the reproductive isolation in the offspring. Both these parameters can be considered in between two sibling species of the genus Ostrinia and (ii) to confirm light of the divergence between the parental species (the ECB and its independence from another trait (the production, by females, ABB).

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same genotyping effort, more polymorphic markers (that is, ± in the mapped parents) were observed in R_C04 (323) and NR_P10 (234) than in NR_C04 (215) and R_P10 (165). According to Winter and Porter (2009), the ratio of heterozygous markers in F1 hybrid parents versus species-specific parents provides an estimate of the proportion of genome that differs between the species. This proportion was estimated at 33% in C04 (1-215/323, where 215 and 323 are the number of polymorphic markers in NR_C04 and R_C04, respectively) and 29% in P10 (1–165/234, with 165 markers in R_P10 and 234 in NR_P10). This level of differentiation is higher than that between Papilio glaucus and P. canadensis (16%, according to Winter and Porter 2009). However, too few data for mapping comparisons between interspecific and intraspecific pedigrees are available to draw any firm conclusions concerning the level of divergence, as estimated here, between the ECB and ABB.

Segregation distortion and species divergence The crossing experiments between the ECB and ABB performed here and in former studies (Pe´lozuelo et al.,2007)revealednohybrid sterility or lack of viability, although this was not formally tested. In the laboratory (Pe´lozuelo et al., 2007), seminatural (Bethenod et al., 2005) and natural (Malausa et al., 2005) conditions, reproductive isolation seems to be principally prezygotic. In the four genetic maps produced here, 13–18% of the polymorphic markers displayed significant segregation distortion. The degree of distortion was similar in ‘hybrid’ F1 maps (18 and 14% in R_C04 and NR_P10) and in maps of the ABB parents (10 and 13% in NR_C04 and R_P10). Segregation distortion is commonly detected in insect mapping (Solignac et al., 2004; Orr and Irving, 2005; Phadnis and Orr, 2009; Tatsuta and Takano-Shimizu, 2009; Winter and Porter, 2009). It may result, in part, from a sampling effect when offspring sizes are small, or from homoplasy in AFLP markers, leading to segregation at multiple loci being considered as segregation at a single locus (Gort et al., 2006). However, the clustering of distorted markers in localized regions of genomes, as reported here (Figures 2 and 4), probably reveals incompatibilities between divergent parental species, from a distortion of oogenesis to postzygotic selection (Hall and Willis, 2005; Rogers and Bernatchez, 2006). These findings pave the way for studies of the divergent genomic histories of these two Ostrinia species based on new candidate genomic regions other than those including mi and Pher and the Z heterochromosome (at least in NR_P10, Figure 4). In particular, postzygotic selection may have been neglected in our species Figure 6 QTL scan for the pheromone production (Pher) trait. QTL for all complex, but both pre- and postzygotic barriers may be involved autosomal and Z heterosomal (labeled ‘X’, shifted in the last position) linkage groups in R_P10, NR_P10. In NR_P10, the LOD score is plotted in the strengthening of reproductive isolation between the ABB and against each linkage group, represented by a single point, due to the ECB (see mutiple components of sexual isolation in phytophagous absence of recombination in the female parent (genotyping/mutation errors insects in the study by Matsubayashi et al., 2010). were manually corrected in the corresponding files). Genetic architecture of mating isolation Our study provides clues to the genetic mechanism underlying the Species differentiation and genome mating isolation (mi) trait, a prezygotic barrier between two sibling Two of the four maps (NR_P10 and R_C04) provided information species, the ECB and ABB. The fine phenotypic nature of mi is about segregation in the F1 hybrid parents, whereas the other two unknown, while the no-choice mating design used in the present (R_P10 and NR_C04) provided information about segregation in the study focused on short-range isolation including mechanisms of ABB parents. We expected hybrid F1 parents to have a larger number rejection or acceptance of a sexual partner with a different species of heterozygous markers (referred to as ‘informative’, see Results) than background (in line with the Am trait of Pe´lozuelo et al., 2007) and of species-specific parents. Indeed, the F1s should display the diagnostic individual propensity to mate regardless of the genetic background of differences between the two Ostrinia species in addition to the the partner. Our results attest that mi is independent of Pher,atleast standing variation occurring within species (Winter and Porter, as far as the major genes contributing to these two traits are 2009). Our results were consistent with this hypothesis, as for the concerned. Indeed, the QTLs involved in mi and Pher were never

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Figure 7 QTL scan for the mating isolation (mi) trait. QTL for all autosomal and Z heterosomal (labeled ‘X’, shifted in the last position) linkage groups on the NR_P10, R_C04, R_P10 and NR_C04 maps. In NR_P10 and NR_C04, the LOD score is plotted against each linkage group, represented by a single point, due to the absence of recombination in the female parent (genotyping errors and/or mutations were manually corrected in the corresponding files). assigned to the same linkage groups. In addition, all QTLs involved in empirical studies carried out on various insect species (but mostly mi were autosomal. This location excludes resp, the heterosomal QTL Drosophila) over the last few decades have provided strong responsible for the behavioral response (and preference) of males to evidence that components of reproductive isolation are female sex pheromone polymorphism, and the Z-linked pheromone controlled by a limited number of loci. According to the review receptor genes recently described in the ECB (Lassance et al., 2011; by Arbuthnott (2009), the genetic architecture of most Yasukochi et al., 2011). These findings are consistent with those of of the courtship traits involved in premating isolation involves a Pe´lozuelo et al. (2007), who suggested a complete disconnection few loci of major effect. Moreover, the percentage of phenotypic between an assortative mating trait involved in ECB/ABB isolation variance explained is large enough for natural selection to and the E/Z communication system. exert an effect through changes at a single locus. This review, It has recently been suggested that, in addition to the reproductive based on the many experiments performed on insects from isolation generated by the male response to female sex pheromone, a various orders highlighted (i) the importance of premating second prezygotic barrier based on a male pheromone (Royer and isolation and its rapid evolution, constituting a key barrier to gene McNeil, 1992) to which females respond (Lassance and Lo¨fstedt, flow in the early stages of speciation (Coyne and Orr, 1998), and 2009) may occur in the genus Ostrinia. The genetic architecture of (ii) the congruence between empirical data and theoretical expec- male pheromone production and of the female response remains tations concerning the genetic control of courtship. Courtship unknown and the mi QTLs identified here are potential candidates for traits are often controlled by small numbers of loci, which may further colocalization experiments. favor their rapid divergence, in turn driving rapid speciation Finally, our findings suggest that the mi trait is controlled by a through premating isolation. small number of major genes (two QTLs in NR_P10 and one in NR_C04). However, our experimental design relies on relatively small CONCLUSION family sample sizes. This limited sample sizes may induce an The present study allowed detecting major QTLs involved in mating overestimation of the additive variance (effect) and an underestima- isolation in the ABB and ECB. We confirmed that some components tion of the number of QTLs, due to sampling effects (Beavis, 1998). of the prezygotic isolation between these two sibling species are Our results hence may not represent a comprehensive view of independent from the pheromone communication system because the genetic architecture of the trait, but rather a truncated mating isolation and pheromone QTLs were located on distinct view towards the principal effects while additional undetected linkage groups. By design, the present QTL mapping focused on factors may be indeed implicated to a lesser extent. Nevertheless, short-range mating isolation in no-choice experiments. Additional

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