PCR-based O. nubilalis population and pedigree analysis indicated female specificity of a (GAAAAT)n microsatellite, and male specificity of a CAYCARCGTCACTAA repeat unit marker. These loci were respectively named Ostrinia nubilalis W-chromosome 1 (ONW1) and O. nubilalis Z-chromosome 1 (ONZ1). Intact repeats of three, four, or five GAAAAT units are present among ONW1 alleles, and biallelic variation exists at the ONZ1 locus. Screening of 493 male at ONZ1 and 448 heterogametic females at ONZ1 and ONW1 loci from eleven North American sample sites was used to construct genotypic data. Analysis of molecular variance (AMOVA) and F-statistics indicated no female haplotype or male ONZ1 allele frequency differentiation between voltinism ecotypes. Four subpopulations from northern latitudes, Minnesota and South Dakota, showed the absence of a single female haplotype, a significant deviation of ONZ1 data from Hardy-Weinberg expectation, and low-level geographic divergence from other subpopulations. Low ONZ1 and ONW1 allele diversity could be attributed either to large repeat unit sizes, low repeat number, reduced effective population (Ne) size of sex chromosomes, or the result of recent O. nubilalis introduction and population expansion, but likely could not be due to inbreeding. These sequences have been deposited in GenBank AF442958, and AY102618 to AY102620.
Microsatellites are repetitive nucleotide elements that have a core repeat structure of 2 to 6 nucleotides (Chambers and MacAvoy, 2000). Microsatellite allele mutations predominantly occur via strand slippage during chromosome replication at a rate of 10−6 to 10−2 (Eisen, 1999), and is influenced by repeat unit length, number of repeat units in the array, microsatellite flanking sequence, and recombination (Chambers and MacAvoy, 2000).
Ostrinia nubilalis (Hübner), the European corn borer, is an invasive agricultural pest in North America introduced from Europe around 1917. The herbivorous O. nubilalis larvae are adapted to feeding on cultivated Zea mays (L.) and annually cause major economic losses (Mason et al. 1996). Phenotypic diversity in the North American population is present in two sex pheromone races, and three voltinism types (Showers 1993). Females of the two sex pheromone races are differentiated by their emission of either E- or Z-11-tetradecenyl acetate as the dominant pheromone component. Pheromone races show little allozyme marker differentiation (Harrison and Vawter 1977; Cardé et al. 1978; Glover et al. 1991). They interbreed in the laboratory and under natural conditions continual gene flow between the races apparently occurs (Roelofs et al. 1985; DuRant et al., 1995). In contrast, diapause ecotypes differ in the number of degree days required prior to adult emergence (Showers 1993), and the resultant asynchrony of adult mating cycles between voltinism types was suggested to minimize genetic exchange (Roelofs et al. 1985). Reduced gene flow between voltinism ecotypes was supported by RAPD-PCR marker data (Pornkulwat et al. 1998), but not by allozyme marker (Bourguet et al. 2000) or mitochondrial RFLP data (Marcon et al., 1999).
To date, locus specific genetic markers used for O. nubilalis population analysis have inherent limitations that restrict the levels of variation detected, which might contribute to the conflicting evidence provided between O. nubilalis ecotypes. Intraspecific diversity of allozyme markers is reduced by negative selection on nonsynonymous changes and environmental influence on post translational modification pathways (Hartel 1988). Mitochondrial DNA is also prone to fixation by genetic drift due to a reduced effective population size of the molecule compared to chromosomes (Avise et al., 1988). Given the limitations of previously used markers, unresolved questions regarding gene flow among North American ecotypes, and our desire to estimate both male- and female-based population differentiation (movement), we developed sex-specific O. nubilalis nuclear microsatellite markers. In the following allele variation of the North American population is characterized at two such markers, ONW1 (Ostrinia nubilalis W-chromosome 1) and ONZ1 (Ostrinia nubilalis Z-chromosome 1). Genotypic and haplotypic data were used to evaluate intraspecies differences, infer the presence of any population subdivision, and estimate the levels of genetic exchange (migration) among O. nubilalis voltinism ecotypes and geographically distinct North American subpopulations.
Materials and Methods
Satellite DNA Isolation
Genomic DNA from O. nubilalis was enriched for CA microsatellites by methods described by Lyall et al. (1993). Resultant PCR products were ligated into pGEM-T easy cloning vector (Promega, www.promega.com) according to manufacturer instructions. Electroporation competent E. coli SURE® cells (Stratagene, www.stratagene.com) were transformed on a MicroPulsar apparatus (BioRad, www.bio-rad.com), and clone selection and blue white screening was performed. Positive clones were propagated overnight at 37° C in 25 ml Terrific Broth that contained ampicillin. Plasmid DNA was isolated with a QIAprep spin miniprep kit (Qiagen, www.qiagen.com) according to manufacturer's directions. Template was sequenced at the DNA Sequencing and Synthesis Facility at Iowa State University, Ames, IA, USA.
Marker Screening and Analysis
Ostrinia nubilalis samples were collected from three light traps in Iowa, and samples obtained from collaborators (Table 1). DNA extractions were performed on moth thoracic tissue as described by Marcon et al. (1999). DNA pellets were diluted to 50ng/µl with TLE (10 mM Tris, 0.1 mM EDTA, pH 7.5) and stored at −20° C prior to use. Primer pairs ONW1-F (5′-TGGAAGTTGATCGGAATAAGAAGTC –3′) with ONW1-R (5′-TGGAAGAGCGGTAACCTCCT –3′), and ONZ-M1F (5′-GGTGGGACCTCCATGCGC-3′) with ONZ-M1R (5′-GCTGGGGCGTCTTCGAGGT-3′) were designed from respective DNA sequence data using Primer3 (Rozen and Skaletsky 1998). Primers were synthesized at Integrated DNA Technologies ( www.idtdna.com), and 5 pmol of each used along with 0.425 U Taq polymerase (Promega), 1.25 µl 10X thermal polymerase buffer (Promega), 2.5 mM MgCl2, and 150 µM dNTPs to PCR amplify 150 ng of individual O. nubilalis DNA sample in a 12.5 µl reaction volume. Thermocycler reactions used 94° C for 3 min., followed by 40 cycles of 94° C for 20 sec., 53° C for 30 sec. (ONW1) or 50° C for 30 sec. (ONZ1), and 72° C for 15 sec. ONW1 locus amplification from eighteen other species used modified amplification conditions (Table 2). PCR products were separated at 150V for 10.5 hr. on a 0.1 × 20 cm 8% polyacrylamide (19:1 acrylamide:bisacrylamide) 1X TBE gel with a 25 base pair ladder (Promega) for size comparison. Bands were visualized after ethidium bromide staining, and image capture took place on a PC-FOTO/Eclipse Electronic Documentation System (Fotodyne, www.fotodyne.com). Basis of variation was determined by cloning ≥ 2 loci of each observed fragment size for ONW1 and ONZ1. Plasmid DNA isolation and DNA sequencing was carried out as previously described.
ONW1 allele and ONZ1 genotypic frequency data was used to evaluate O. nubilalis population structure, and estimate male and female migration (M) between subpopulations. Population structure assumed genetic separation of univoltine Z-pheromone subpopulations (UZ; ID 1 to 4), from bivoltine and multivoltine Z-pheromone (Table 1; ID 5 to 11). Calculations of geographic-based population subdivision assumed three groups: group 1, subpopulations with ID 1 to 4; group 2, ID 5 to 9; group 3, ID 10 and 11. A second analysis assumed north-south population subdivision assumed two subpopulation groups; north: subpopulations with ID 1 through 4, and south ID 5 to 11 (Table 1). Statistical evaluations included analysis of molecular variance (AMOVA), and F-statistic or haploid allele data modified F-statistic (Θ; Excoffier et al., 1992; Weir and Cockerham, 1984; Weir, 1996). The Hardy-Weinberg equilibrium of subpopulation ONZ1 was tested using 10,000 Markov chain steps, and significance p values set at 0.05. All calculations were performed with the Arlequin software package (Schneider et al., 1997).
Two polymorphic tandem repetitive loci, O. nubilalis Z-chromosome 1 (ONZ1) and W-chromosome 1 (ONW1), were sequenced from separate clones in a microsatellite enriched partial genomic library. The ONW1-containing clone, pGEM-OnCA09, had two repetitive regions, ten tandem GT/CA repeat units and four consecutive GAAAAT repeats (Fig. 1a; GenBank AF442958), and population analysis revealed three alleles of 141 (3 repeats; allele WA3), 147 (4 repeats; allele WA4), and 153 base pairs (5 repeats; allele WA5). Two samples from each of three observed ONW1 allelic types (electromorphs) were DNA sequenced, and full GAAAAT repeat units were identified as the basis of size variation without flanking region mutation (data not shown). Successful PCR results were obtained from the ONW1 hexanucleotide repeat only from female DNA samples (data not shown). The ONZ1-containing clone pGEM-OnCA01 had a fifteen nucleotide-long imperfect repeat element, CAYCARCGTCACTAA, (underlined nucleotides optional; Fig 1b; GenBank AY102618 to AY102620). ONZ1 was PCR amplified from male derived DNA and all attempts to amplify female DNA failed (data not shown). Six ONZ1 alleles were sequenced, three from each of two size classes. Two ONZ1 allele size range variants 95 to 97 (ZA1) and 106 to 107 base pairs (ZA2) were characterized with one insertion/deletion and two substitutions within both the repeat and the flanking region (Fig 1b). Since single base changes were not resolved by polyacrylamide gel electrophoresis methods, two alleles (ZA1 and ZA2) were scored and used for genotype data. Three pedigrees with ≥ 46 F2 progeny confirmed the male specificity of ONZ1 and female specificity of ONW1. More specifically, ONW1 and ONZ1 were hemizygous in females (single locus with no corresponding homologous pair), and males were diploid at ONZ1 and lacked the ONW1 locus.
ONZ1 and ONW1 were PCR amplified from 448 female, and ONZ1 from 493 male, DNA samples from 11 North American subpopulations of O. nubilalis. Male ONZ1 genotypic and ONZ1 and female ONW1 haplotype frequencies were calculated (Table 1). Male genotypic and female haplotype data were separately evaluated by AMOVA and fixation indices, and independently showed stronger evidence for geographic-compared to voltinism-based population structuring (Table 2).
Low allele size diversity was present at O. nubilalis W chromosome (ONW1) and Z-chromosome (ONZ1) microsatellite loci. The ONW1 allele with four repeat units [(GAAAAT)4; WA4), was present among 76% of North American O. nubilalis females, and described from 87.1 % of 41 female O. furnicalis (Gunée) and a single O. pentitalis (Grote) sample, indicating that the allele likely is ancestral to the genus. The molecular basis of low ONW1 allele size variation might lie in the repetition of a hexanucleotide repeat that is less prone to strand slippage during recombination, the short array of 3 to 5 GAAAAT repeats, and the potential location of ONW1 in the nonrecombining region of the W chromosome that eliminates unequal crossover as a mechanism generating allele diversity. ONW1 WA3 alleles outnumbered WA5 alleles, 19.3% to 4.7% (Table 1), indicating a preponderance of microsatellite repeat unit loss, as opposed to gain that occurs typically at microsatellite loci (Rubinsztein et al., 1995, Weber and Wong, 1993). Rose and Falush (1998) determined that a minimum of eight repeats was required for evolution of hypervariable microsatellites, and array lengths such as ONW1 that are below this threshold suggest insensitivity toward repeat number expansion bias. The ONZ1 locus is biallelic, with the dominant ZA1 allele frequency at 95% (937 of 986) in males and 99% (445 of 448) in females. Near fixation of ZA1 in female O. nubilalis and not in males might result from differences in effective population sizes (Ne) of the W chromosome compared to Z chromosomes. Assuming a 1:1 sex-ratio, lepidopteran W chromosomes have a three- and fourfold reduction in effective population size compared to Z and autosomal chromosomes respectively (Charlesworth et al., 1987; Hartel, 1988), whereby associated loci are be more prone to fixation and interpopulation divergence (Charlesworth et al., 1987). Despite being less prone to molecular fixation, ONZ1 locus allele diversity was lower than ONW1 that might result from its longer imperfect repeat, or be a result of population effects (see below).
No genetic divergence was detected between female ONW1 and ONZ haplotypes based on voltinism ecotype, as indicated by – 6.44% (0.0%) of total population variation among groups. Similarly at the ONZ1 locus, grouping samples by voltinism showed an FST = 0.028, and 0.83% of total population variance among ecotypes (remaining data not shown). Results indicated no evidence of O. nubilalis voltinism-based ecotype structure and are in agreement with mitochondrial PCR-RFLP (Marcon et al. 1999) and allozyme data (Bourget et al. 2000), but in conflict with voltinism ecotype variation detected by RAPD-PCR (Pornkulwat et al. 1998). Additionally, a single E-pheromone subpopulation from Newark, Delaware did not show significant ONZ1 allele or genotype variance with Z-pheromone race subpopulations. A triose phosphate isomerase (TPI) marker was previously mapped to the Z chromosome and linked to inter-pheromone race behavioral or response differences in males (Glover et al. 1991), suggesting a Z chromosome location of ONW1 distant from TPI and male pheromone response genes. Lack of both voltinism ecotype-based population differentiation or a correlation of ONZ1 with pheromone race might be an artifact of recent O. nubilalis range expansion across North America (Showers 1993), suggesting slowed ONW1 and ONZ1 lineage sorting (Avise et al. 1984) and allele extinction by genetic drift (Takahata 1983) between ecotypes.
Low but significant differentiation was previously shown between O. nubilalis ecotypes (Harrison and Vawter 1977; Cardé et al. 1978), but not between geographically distant samples (Bourget et al. 2000). Both sex-linked genetic markers, ONW1 and ONZ1, indicated greater contribution of geography to total genetic variance compared to voltinism. AMOVA analysis indicated that 3.78% of total population variance was between two groups; a “northern” group consisting of Minnesota and South Dakota subpopulations (ID 1 to 4), and all other subpopulations classified as “southern” (ID 5 to 11; Table 1). Combined ONZ1 and ONW1 female haplotype data indicated that 4.02% of total population variance was accounted for among “northern” and “southern” groups. Low-level geographic divergence of four subpopulations from northern latitudes might reside in their omission of the ZA1WA5 female haplotype (Table 1), and significant deviation of ONZ1 data from Hardy-Weinberg expectation at three of the four sites. In the “northern” geographic region, chi-square (χ2) tests detected significant departure of ONZ1 genotypes from Hardy-Weinberg equilibrium within Lamberton, Minnesota bivoltine Z-pheromone (p < 0.001), Lamberton, Minnesota univoltine Z-pheromone (p = 0.007), and South Shore, South Dakota univoltine Z-pheromone subpopulations (p = 0.016). Deviation for HWE was not exhibited from any other subpopulation. Except for the Brookings, South Dakota sample, the basis for Hardy-Weinberg deviations among northern subpopulations likely reside in their decreased the average heterozygosity (HS= 2 ÷ 81 ≅ 0.025) compared to the average among the remaining subpopulations (HT = 19 ÷ 317 ≅ 0.060). Reduced heterozygosity might be attributed to the presence of null alleles, or a stronger effect of the 1/4 reduction in Z chromosome effective population size in smaller fringe populations. Alternatively, recent or recurrent population size effects might disproportionately disrupt O. nubilalis population Hardy-Weinberg equilibrium in northern regions. Increased severity of cyclical genetic bottlenecks due to larval over-wintering mortality in northern regions (Hudson and LeRoux 1986) might disturb mutation-drift equilibrium since Ne likely does not remain stable for 2Ne to 4Ne generations (Nei and Li 1976). Additionally, entry of univoltine O. nubilalis moths into Minnesota in the early 1940s (Chiang, 1961), South Dakota in 1948, and North Dakota in 1950 (Chiang 1972), followed by the northern migration of bivoltine subpopulations starting in the early 1950s (Chiang 1965), suggest recent range expansion might have caused a deviation from migration-drift equilibrium (Takahata 1983).
The level of migration was inferred from F-statistics derived from male genotype and female haplotype data (Table 2). Female haplotype analysis indicated substantial effects of inbreeding and genetic drift (ΘST = 0.227; ΘIS = 0.274), whereas male ONZ1 genotypic analysis suggested little evidence of either (FST = 0.028; FIS = 0.020; Table 2). Inbreeding prevalence among female O. nubilalis might be used to infer lower migration rates as compared to males. When considered in conjunction with increased levels of genetic drift among females (ΘST = 0.227) in relation to males (FIS = 0.020), the reduced effective population size of the W chromosome and lack of homologous recombination during female gametogenesis might result in a strong influence of nucleotide fixation in female lineages. Thus differences between loci used in this study might reflect a background of chromosome position effects, and not truly be representative of population-based influences. Corroborating biological estimations of male and female flight distances are required to verify these results.
Low ONZ1 and ONW1 allele diversity was attributed to large repeat unit sizes, low repeat number, reduced effective population (Ne) size of sex chromosomes, the result of a genetic bottleneck that occurred when O. nubilalis was introduced, or genetic drift caused by continued population expansion. Analysis of male ONZ1 genotypes and female ONW1 and ONZ1 haplotypes showed no evidence for voltinism-based population structure. The similarity of west-central Minnesota and eastern South Dakota samples, compared to all other subpopulations, for both ONW1 and ONZ1 provided evidence of geographic population structure. Recent O. nubilalis population expansion into northern regions of the central United States and recurrent seasonal bottlenecks likely explain the maintenance of HW disequilibrium, and form the basis of geographic population subdivision instead of inbreeding.
North American O. nubilalis male ONZ1 genotypic, and heterogametic female ONW1 and ONZ1 haplotype frequencies in twelve subpopulations. WA3, WA4, and WA5 represent ONW1 allele with three, four, and five GAAAAT repeats, respectively. ZA1 = ONZ1 allele with one CAYCARCGTCACTAA consensus repeat, and ZA3 = ONZ1 allele with three CAYCARCGTCACTAA consensus repeats.
Male AMOVA and FST, and female AMOVA and ΘST values from test of O. nubilalis geographic variation assuming four South Dakota and Minnesota (north latitude) subpopulations are subdivided from the remainder of the North American O. nubilalis population.