INTRODUCTION {#S1}
============

The genetic basis of host preference is relevant to a number of fundamental evolutionary questions. These include evolution of specialization and generalization ([@R53]; [@R24]), sympatric speciation ([@R38]; Dieckman and Doebeli, 1999; [@R26]), host shifts ([@R25]; [@R16]), and parasite-host and plant-herbivore co-evolution ([@R11]; [@R8]). For example, the presence of host choice can drive the evolution of specialization, as organisms adapt to the hosts to which they are more frequently exposed ([@R53]; [@R22]). In speciation models, the simpler the genetic basis of host preference and performance, and the more these traits are tightly linked, the more likely sympatric speciation is to occur ([@R9]). An additional consideration is that parasitoids are widely used for biological control of insect pests of agricultural importance ([@R35]). A better understanding of the genetics of host range in parasitoids could also facilitate genetic improvement of these insects in biological pest control, by providing mechanisms for genetic manipulation of host usage.

Host selection behavior involves several phases, including habitat location, host location, host recognition and host acceptance ([@R20]; [@R48]). All these stages may be under both genetic and environmental influence ([@R13]). However, the genetic basis of these behaviors has been investigated in only a few systems. Most work on host selection behavior in arthropods has been done in phytophagous insects (reviewed by [@R20]) parasitic hymenoptera (reviewed by [@R48]), and ticks and mites ([@R30]). Genetic studies have suggested wide variation between systems in the number of loci and mode of inheritance involved in host preference (*e.g.*, [@R19]; [@R45]; [@R23]; [@R32]; [@R47]; [@R17]; [@R34]).

The most well-studied genetic system for host preference is that of *Drosophila sechellia*, a species of *Drosophila* endemic to the Seychelles Islands which feeds solely on the fruit of *Morinda citrifolia*, which is toxic to most other *Drosophila* species ([@R28]; [@R21]). It was subsequently shown that two genes encoding odorant binding proteins affect the species' responses to hexanoic and octanoic acid, and therefore their attraction to the fruit ([@R31]). Knockout of one of these genes in *Drosophila melanogaster*, *Obp56e*, caused the flies to lose much of their aversion to morinda fruit ([@R5]). In aphids, [@R17] found a complex basis to host preference, with several groups of tightly linked quantitative trait loci involved in host choice and fitness.

The present work analyzes the genetics of host preference differences in the parasitic wasp *Nasonia*. There are four species of *Nasonia*: *N. vitripennis*, *N. giraulti*, *N. longicornis*, and the newly described *N. oneida* ([@R37]). The species are interfertile once cured of *Wolbachia* ([@R1], [@R2]), allowing traits from one species to be introgressed into another ([@R51]; [@R27]). With the recent sequencing of the genomes of three species ([@R49]) and a wealth of other resources becoming available (e.g., [@R29]; [@R33]; Pannebacker *et al.*, 2009), *Nasonia* provides a powerful system for studying the genetic basis of interspecies differences ([@R50]).

*Nasonia* consists of both a generalist and specialist species. *N. vitripennis* has a holarctic distribution and is a generalist that parasitizes a wide range of calyptrate flies, including blow flies, house flies, and flesh flies. *N. giraulti* and *N. longicornis* specialize on bird blowflies (*Protocalliphora*, which *N. vitripennis* also parasitizes) and occur in northeastern and northwestern North America, respectively ([@R4]). The newly described *N. oneida* also specializes on bird blowflies and is currently known to occur only in upstate New York ([@R37]). *N. vitripennis*, *N. giraulti*, and *N. oneida* occur microsympatrically in bird nests in eastern North America, whereas *N. vitripennis* is also found associated with carrion-breeding flies.

As host choice is hypothesized to drive the evolution of specialization ([@R53]; [@R22]), the genetics of host preference is particularly relevant to the evolution of host usage in *Nasonia*. Evidence of a host preference locus was originally detected during introgression of male-specific wing-size locus *ws1~g~* from *N. giraulti* into *N. vitripennis* ([@R51]). Pure breeding of the line with *ws1~g~* in a largely *N. vitripennis* genetic background was difficult, because homozygous females did not sting the *Sarcophaga* (flesh fly) hosts that are used for maintenance of wasp strains in the laboratory and are regular hosts of *N. vitripennis* in the wild. A pilot experiment indicated that they did sting *Protocalliphora* hosts. Subsequently, *ws1~g~* was separated from these effects by recombination, allowing purebreeding of *ws1~g~* ([@R51]). Here, we backcross the region around *ws1~g~* from *N. giraulti* into *N. vitripennis* using newly available visible markers, and map a major host preference effect in the region.

MATERIALS AND METHODS {#S2}
=====================

Nasonia strains and maintenance {#S3}
-------------------------------

The general biology of *Nasonia* is described by [@R52]. Cultures of *Nasonia* were maintained in the laboratory with constant light and temperature (25°C) on *Sarcophaga* pupae. Under these conditions the generation time is approximately 14 days. For laboratory experiments on host preference and acceptance, the standard reference strains of *N. vitripennis* (ASymCx) and *N. giraulti* (RV2Xu) were used ([@R49]) To introgress (backcross) the region around *ws1~g~* from *N. giraulti* into *N. vitripennis*, we used the mutant *N. vitripennis* strain peach (*pe333*). Previous studies had revealed that this eye color mutant interacts epistatically with a natural eye color allele in *N. giraulti* (*bk~g~*) that is linked to *ws1~g~*, permitting easy tracking of the region during backcrossing.

For host preference experiments we used *Sarcophaga bullata* and *Protocalliphora sialia* pupae. A *Sarcophaga* culture was maintained in the laboratory, while *Protocalliphora* were obtained as larvae from bluebird and tree swallow nests during the summer months. The larvae were separated and allowed to pupate. Two days following pupation, both *Sarcophaga* and *Protocalliphora* were placed in a refrigerator at 4°C, where they were stored for up to 4 weeks prior to their use in experiments.

Host acceptance tests of field collected wasps {#S4}
----------------------------------------------

To test for acceptance of *Sarcophaga* hosts by field-collected wasps, we collected bluebird and tree swallow nests from 8 different states in the eastern and midwestern US (New York, Ohio, Virginia, Pennsylvania, Indiana, Minnesota, Michigan, Wisconsin). Wasps were allowed to emerge from the nests in the laboratory, and we collected females from nests that contained either all *N. giraulti* (14 nests) or all *N. vitripennis* (102 nests). To assess acceptance of *Sarcophaga* hosts, the females were placed in a vial with one *Sarcophaga* pupa and allowed to parasitize until the wasp dies (\~3--6 days). Two to three weeks later, hosts were scored for the presence of adult flies or wasps (adults or diapausing larvae).

Host preference and acceptance experiments {#S5}
------------------------------------------

Observations were performed to characterize the behavioral response of *Nasonia* strains to *Sarcophaga* and *Protocalliphora* pupae. Virgin females 2--3 days old were placed in individual vials. Each female was given either one *Sarcophaga* host and one *Protocalliphora* host in a host preference (*i.e.* choice) experiment or two *Sarcophaga* in a host acceptance experiment. Vials were set horizontally so the female\'s behavior could be observed. The female\'s contact with and stinging of hosts was recorded. A 'contact' was recorded if the female was on the host but not stinging it. A 'sting' was recorded if the observer could see the ovipositor probing/stinging the host. Observations were made every five minutes for the first hour, every 10 minutes for the second hour, and subsequently every 15 minutes. Observations ceased approximately 4.5 hours after the female was first given the host. Each wasp was then scored for 1) whether or not it contacted a host at all during observation (contact), 2) how much time it spent contacting each host (time spent on host), and 3) whether or not it stung during observation (stinging). After 24 hours, each host was removed and scored two to three weeks later for the presence of adult flies or wasps (adults or diapausing larvae). Statistical comparisons were performed using contingency χ^2^ tests or Mann-Whitney U tests. Both host acceptance and preference tests were performed on *N. vitripennis* (strains ASymCx and peach), *N. giraulti* (strain RV2Xu), and heterozygous bkbw~g~/+~v~ introgression females (described below).

Introgression of the bkbw~g~ region into N. vitripennis {#S6}
-------------------------------------------------------

To test for host preference effects in the *ws1~g~* region, we introgressed (backcrossed) the region around *ws1* from *N. giraulti* into an *N. vitripennis* genetic background. We hereafter refer to this region as bkbw~g~ (for black eyes, big wings, naturally occurring *N. giraulti* visible markers in the region). Specifically, the bkbw~g~ region contains the visible markers *ws1~g~*, *sww~g~*, and *bk~g~* (see bottom of [Figure 1](#F1){ref-type="fig"}). These visible markers, described below in more detail, allow heterozygous bkbw~g~/+~v~ females to be visibly distinguished from homozygous bkbw~g~/bkbw~g~ females and bkbw~g~ males to be visibly distinguished from +*~v~* males.

The process of the introgression is outlined in [Figure 1](#F1){ref-type="fig"}. Initially, *N. giraulti* females were crossed to *N. vitripennis* peach males ([Figure 1](#F1){ref-type="fig"}, parental). F~1~ hybrid females were then backcrossed to *N. vitripennis* peach males ([Figure 1](#F1){ref-type="fig"}, backcross). Each backcross generation, hybrid heterozygous bkbw~g~/+~v~ females, identified using visible markers in the region ([Figure 1](#F1){ref-type="fig"}, m), were mated to *N. vitripennis* peach males. After ten generations, an attempt was made to produce a homozygous bkbw~g~ strain by crossing bkbw~g~ hybrid males to heterozygous bkbw~g~/+~v~ females ([Figure 1](#F1){ref-type="fig"}, purebreed). In the next generation, all females were mated to bkbw~g~ males. The incidence of failure to parasitize *Sarcophaga* hosts increased dramatically, and nearly all females parasitizing hosts proved to be bkbw~g~/+~v~ heterozygotes rather than bkbw~g~/bkbw~g~ homozygotes. This made production of a purebred bkbw~g~ introgression line impossible. Note that these hybrid incompatibilities are only seen in the purebreeding stage and not in the late-generation backcrosses, because hybrid incompatibilities in *Nasonia* tend to be recessive ([@R2]). The bkbw~g~ strain in therefore maintained heterozygously, by crossing heterozygous bkbw~g~/+~v~ females with bkbw~g~ males.

Visible markers in the bkbw~g~ region {#S7}
-------------------------------------

Integrity of the bkbw~g~ introgression strain is maintained using visible markers (a map of these markers is shown at the bottom of [Figure 1](#F1){ref-type="fig"}). The region maps onto linkage group IV (chromosome 4), and on one end lies the major male wing size QTL *ws1* ([@R51], [@R49]). Approximately 0.74 cM from *ws1* is the mutant body color allele *bl13* which causes a blue-colored body. This *N. vitripennis* mutation was originally generated by [@R40] but had been mapped incorrectly to linkage group III ([@R41]). On the opposite side of *bl13*, 0.09 cM away, lay the loci *bk* and *sww*. The *bk* and *sww* loci were discovered during experiments conducted to introgress additional wing size QTL, using a *N. vitripennis* strain (peach) with the R-locus mutant *pe333* on chromosome 5 which causes "peach" colored eyes (J. H. Werren and L. Enders, unpublished data). These experiments revealed an epistatic interaction between *pe333* and *N. giraulti* wild-type eye locus *bk~g~*, which creates "oyster"-colored eyes when both markers are homozygous. Epistatic effects among some eye color mutants generated in *N. vitripennis* had been previously observed ([@R39]), apparently involving mutants in the red versus brown pigment pathways.

Subsequent genetic analyses revealed that the natural *N. giraulti* eye color effect was allelic to the *N. vitripennis* locus *bk576* on linkage group IV. A mutant at *bk576* causes blackish eyes ([@R40]). Introgression males showing the oyster eye phenotype also had large wings, even larger than standard *ws1~g~* males. The wing size effects within the region are due to introgression of *N. giraulti* alleles at *ws1* and a second locus, called *shorterwiderwings* (*sww*). Analyses of *sww* will be reported elsewhere.

Mapping of the bkbw region using a genotyping microarray {#S8}
--------------------------------------------------------

To ascertain the size and content of the bkbw~g~ region, we used a genotyping microarray which has been developed to genotype hybrids between *N. vitripennis* and *N. giraulti*. Recent sequencing of the genome of *N. vitripennis* and partial sequencing of *N. giraulti* ([@R49]) has identified an abundance of interspecies polymorphisms. *N. giraulti* reads were aligned to the *N. vitripennis* genome sequence, and single nucleotide polymorphisms and small insertions and deletions were used to design oligonucleotide probes to discriminate between *N. vitripennis* and *N. giraulti* DNA ([@R49]). Probes for \> 19,000 loci, covering 929 scaffolds and 86% of the assembled genome, were printed on custom NimbleGen microarrays (Madison, Wisconsin, USA). The details of the microarray will be described elsewhere (Desjardins *et al*., unpublished).

DNA was prepared from a single bkbw~g~ introgression male using a Puregene Gentra DNA extraction kit (Qiagen, Valencia, California, USA) using the protocol for a single *Drosophila* (<http://www1.qiagen.com/Products/GenomicDnaStabilizationPurification/GentraPuregeneCellKit.aspx>). This DNA was subsequently amplified via multiple displacement amplification using an Illustra Genomiphi V2 kit (GE Healthcare, Piscataway, New Jersey, USA). This DNA was labeled and hybridized to the array according to described protocols ([@R49]). Following hybridization, each locus on the array was identified as *N. vitripennis*, *N. giraulti*, or ambiguous. We then examined the loci in all major (\>350 Kb) scaffolds (contiguous DNA sequences) on chromosome 4 to determine the genotypes of the scaffolds.

RESULTS {#S9}
=======

N. vitripennis and N. giraulti differ in host preference {#S10}
--------------------------------------------------------

In order to assess differences in host acceptance between *Nasonia* species, *N. giraulti* and *N. vitripennis* wasps that had emerged from field collected birds nests were tested for acceptance rates of *Sarcophaga* pupae. Whereas 83% (2260 of 2739) of *N. vitripennis* females stung *Sarcophaga* hosts, only 45% (184 of 410) of *N. giraulti* females did so (χ^2^~1~=291, p\<0.001). Therefore, field-caught *N. vitripennis* females are significantly more accepting of *Sarcophaga* hosts than are *N. giraulti*.

We next tested standard *N. giraulti* and *N. vitripennis* laboratory strains for host preference and acceptance. In preference tests where wasps were provided with one *Sarcophaga* and one *Protocalliphora* host, *N. giraulti* showed a clear preference for *Protocalliphora* while *N. vitripennis* showed a preference for *Sarcophaga* (see [Figure 2](#F2){ref-type="fig"}). *N. vitripennis* was significantly more likely to both contact and sting *Sarcophaga* than *Protocalliphora* (contact: χ^2^~1~=9.5, p\<0.01; stinging: χ^2^~1~=10.1, p\<0.01), while *N. giraulti* had a significantly greater probability of contacting and stinging *Protocalliphora* hosts than *Sarcophaga* (contact: χ^2^~1~=14.4, p\<0.001; stinging: χ^2^~1~=17.0, p\<0.001). As can been seen in [Figure 3](#F3){ref-type="fig"}, *N. vitripennis* also spent significantly more time on the *Sarcophaga* host than *N. giraulti* (Mann-Whitney U test, z=8.0, p\<0.0001) while *N. giraulti* spent significantly more time on the *Protocalliphora* host than *N. vitripennis* (Mann-Whitney U test, z=6.2, p\<0.0001).

However, when the laboratory strains of both species were presented with two *Sarcophaga* hosts in acceptance experiments, both strains were highly accepting of *Sarcophaga* (84%, N=89 for *N. vitripennis* and 78%, N=79 for *N. giraulti*). There was no significant difference between the two strains relative to whether they contacted or stung the *Sarcophaga* hosts (contact: χ^2^~1~=0.20, p=0.65; stinging: χ^2^~1~=0.66, p=0.42), although *N. giraulti* spent significantly less time on the *Sarcophaga* host than did *N. vitripennis* (Mann-Whitney U test, z=2.8, p\<0.01; see [Figure 3](#F3){ref-type="fig"}).

Introgression of bkbw~g~ region into N. vitripennis shows giraulti-like preference {#S11}
----------------------------------------------------------------------------------

Next, we tested host preference and acceptance of the bkbw~g~ introgression strain. Preliminary tests of bkbw~g~/bkbw~g~ homozygous females showed a complete failure to successfully parasitize *Sarcophaga* and *Protocalliphora* hosts in both the *Sarcophaga* and *Protocalliphora* host choice experiments and the two *Sarcophaga* acceptance experiments (data not shown). We therefore focused our host choice and acceptance experiments on bkbw~g~/+~v~ heterozygous females, and as an additional control, we tested the preference of mutant *N. vitripennis* strain peach (the genetic background of the bkbw~g~ introgression line).

The peach *N. vitripennis* strain shows the same host preferences as the standard *N. vitripennis* strain, as it was not significantly different from non-mutant *N. vitripennis* strain ASymCx for any behaviors (contact: χ^2^~1~=0.64, p=0.42; stinging: χ^2^~1~=1.3, p=0.25; see [Figure 2](#F2){ref-type="fig"}). Peach was significantly different from *N. giraulti* for all behaviors (contact: χ^2^~1~=21.8, p\<0.001; stinging: χ^2^~1~=23.2, p\<0.001; host stung first: χ^2^~1~=28.4, p\<0.001). Therefore, it appears that the peach mutation (*pe333*) does not impact host preference in any significant way.

In contrast, heterozygous bkbw~g~/+~v~ females showed a *N. giraulti*-like host preference in all behaviors (contact: χ^2^~1~=0.31, p=0.58; stinging: χ^2^~1~=0.49, p=0.48; see [Figure 2](#F2){ref-type="fig"}). Heterozygous bkbw~g~/+~v~ females were significantly more likely to contact and sting *Protocalliphora* than *Sarcophaga* in the 4.5 observation period (contact: χ^2^~1~=13.1, p\<0.001; stinging: χ^2^~1~=27.6, p\<0.001). Also, they spent similar amounts of time contacting each host as *N. giraulti* did (Mann-Whitney U test, time on *Protocalliphora*, z=0.9 p=0.18, time on *Sarcophaga*, z=0.7, p=0.25; see [Figure 3](#F3){ref-type="fig"}). Heterozygous bkbw~g~/+~v~ females show a *N. giraulti*-like preference for *Protocalliphora* in a *N. vitripennis*-like genetic background, suggesting that the *N. giraulti* preference for *Protocalliphora* is dominant. The trait segregates in a Mendelian fashion.

In the two *Sarcophaga* acceptance experiments, bkbw~g~/+~v~ heterozygous females did not show a significantly reduced contact rate relative to *N. giraulti* (χ^2^~1~=0.45, p=0.42) but they did show a significantly reduced stinging rate (χ^2^~1~=4.1, p\<0.05). However, the stinging rate of the bkbw~g~/+~v~ heterozygous females was still relatively high (65%) and they did not spend significantly less time contacting hosts than *N. giraulti* (Mann-Whitney U test, z=1.4, p=0.08; see [Figure 3](#F3){ref-type="fig"}), suggesting that only minor genetic incompatibility effects occur in bkbw~g~/+~v~ heterozygous females.

The bkbw~g~ region maps to the centromeric portion of chromosome 4 {#S12}
------------------------------------------------------------------

We utilized the *Nasonia* genotyping microarray to genotype all scaffolds (contiguous DNA sequences) on chromosome 4 in a bkbw~g~ introgression male ([Table 1](#T1){ref-type="table"}). For the majority of scaffolds, loci within a single scaffold were either scored as almost all *N. vitripennis* or almost all *N. giraulti*, allowing easy assignment of genotype to the scaffold as a whole. The exceptions to this were four scaffolds (23, 29, 40, 52), which each contain a region scored mostly as *N. vitripennis* adjacent to a region scored mostly as *N. giraulti*. Scaffolds 23 and 40 represent the outer bounds of the introgressed region. A few additional scaffolds, namely 26 and 133, were scored mostly as *N. giraulti* but with a small number of internal consecutive loci scored as having a *N. vitripennis* genotype.

The bkbw~g~ region maps to the central portion of chromosome 4 (markers 4.18--4.25 in Niehuis *et al.*, in press), encompassing 13 complete and 4 incomplete major (\>350 Kb) scaffolds totalling 11 megabases. The region also contains \~29 smaller scaffolds, bringing the total number of scaffolds to 46 and the total size of the introgressed region to \~16 Mb, and is a region of low recombination. Included within the region appears to be a 4.5 megabase stretch of *N. vitripennis* DNA ([Table 1](#T1){ref-type="table"}). Various lines of evidence indicate that the bkbw~g~ region spans the centromere ([@R49]). While large, this region contains a wealth of visible and molecular markers which can be used to fine-scale map the host preference allele.

DISCUSSION {#S13}
==========

*N. giraulti* shows a clear preference for *Protocalliphora*, the host genus it parasitizes in nature, over *Sarcophaga. N. vitripennis*, known to be a generalist from field studies, shows a preference for *Sarcophaga* in choice experiments. The host preference behavior of *N. giraulti* was introgressed into the genome of its sibling species *N. vitripennis*, along with chromosomal regions linked to the bkbw~g~ loci. Our genetic analysis indicates one or more genes linked to the bkbw~g~ region strongly influence host preference and that this effect segregates in a Mendelian fashion. Females heterozygous for the bkbw~g~ region show strong preference for *Protocalliphora* hosts with only minimal signs of reduced vigor, suggesting that a host preference effect is present in the region independent of any hybrid viability effects. The preference is all the more remarkable given that *Sarcophaga* hosts are much larger than *Protocalliphora* hosts (see [Figure 3](#F3){ref-type="fig"} for relative sizes), and therefore would be more likely to be encountered in the experiment. To our knowledge, this is the first report of the introgression of host preference from one parasitoid species into another one.

The preference for *Protocalliphora* seen in bkbw~g~/+~v~ heterozygous females also indicates the dominance of the *N. giraulti* allele. Based on the results, we posit a host preference locus (*hp1*) within this region with preference for *Protocalliphora* dominant over non-preference. A pattern of dominance in the inheritance of oviposition preference has been found in several phytophagous insects (*e.g.*, [@R18]; [@R19]; [@R23]), although all of these examples represent specialist versus specialist comparisons rather than the specialist versus generalist comparison done here. Additive genetic variance for oviposition preference has also been reported (*e.g.*, [@R42]; [@R43]; [@R47]; [@R32]). If specialization in *Nasonia* is derived, a dominant allele could have rapidly swept through a diverging population.

As the bkbw~g~/+~v~ heterozygous females show host preference similar to *N. giraulti*, i is possible that host preference in *Nasonia* is controlled by a small number of loci or clusters of tightly linked loci. A moderate number of loci have been found controlling host preference in other insects; [@R21] found an oligogenic basis (intermediate genetic complexity) to preference of *Drosophila sechellia* for a chemical attractant (*Morinda* fruit toxin) present in their preferred host plant. Additionally, [@R17] found 4 separate quantitative trace loci affecting host plant choice in host races of the aphid *Acyrthosiphon pisum*. This is directly relevant to some speciation models in which speciation is more likely when there are fewer loci controlling host preference ([@R9]).

For the evolution of specialization, differential performance on hosts is an important element in addition to differential preference. While field-caught *N. giraulti* showed highly reduced acceptance of *Sarcophaga* hosts, laboratory strain RV2Xu did not. This is not unexpected, as *N. giraulti* does not appear to parasitize *Sarcophaga* in the wild, but the standard strain has been reared in the laboratory on *Sarcophaga* for several years (*Protocalliphora* cannot be reared in the laboratory). Therefore it is possible that *N. giraulti* laboratory strains such as RV2Xu have developed an increased acceptance of *Sarcophaga* hosts. Clearly, however, this increased acceptance has not resulted in a preference for *Sarcophaga* over *Protocalliphora* in *N. giraulti* laboratory strain RV2Xu. This suggests that loci controlling preference and acceptance (i.e. performance) may be unlinked, a requirement for some speciation models ([@R3]; [@R9]). However, a change in host acceptance could be due to genetic or environmental causes, and further studies are needed to determine the genetic basis of host acceptance differences in *Nasonia* species.

The results presented here indicate that a host preference gene is linked to the bkbw~g~ locus, encompassed by 16 megabases of *N. giraulti* DNA around the centromere of chromosome 4. Within the introgressed bkbw~g~ region appears to be a 4.5 megabase stretch of *N. vitripennis* DNA ([Table 1](#T1){ref-type="table"}). This may be the result of double recombination moving *N. vitripennis* DNA back into the introgressed region, possibly due to a gene in the region having a strong hybrid incompatibility effect. For example, the failure of bkbw~g~/bkbw~g~ homozygous females to parasitize any tested hosts may be indicative of *N. giraulti* alleles linked to the larger bkbw~g~ region that cause genetic incompatibilities when homozygous in an *N. vitripennis* genetic background. It is also possible that the bkbw~g~ region is contiguous and the region in question actually belongs in a different region of the genome but has been placed here by a combination of assembly and mapping errors. A few internal scaffolds also contained small stretches of *N. vitripennis* DNA. Given the low recombination rate in the region, it is unlikely that these regions are the result of double recombination moving *N. vitripennis* DNA back into the bkbw~g~ region and more likely that they are misassembled and actually belong in other parts of the genome, as only a single assembly error is required to explain each of these regions. It is now necessary to partition the region by recombination to produce more targeted introgressions of the host preference allele, and this work is underway. This is being accomplished using mapping resources available for *Nasonia* ([@R33]; [@R49]) and several visible markers present within the region ([Figure 1](#F1){ref-type="fig"}).

The presence of a *N. giraulti* preference allele allows us to make some inferences on the evolution of host preference in *Nasonia*. It suggests that the transition between generalist and specialist strategies in *Nasonia* was not only an expansion or contraction of host range, but also included an actual change in preference for the *Protocalliphora* hosts parasitized by both specialists and generalists. It is unknown whether either changes in host range or changes in host preference occurred first or if they occurred simultaneously. Given that *Trichomalopsis*, close relatives of *Nasonia*, are largely generalists ([@R14]), it is likely that generalism was the ancestral state for *Nasonia*, with subsequent evolution of specialization on *Protocalliphora* in the common ancestor of *N. giraulti*, *N. longicornis*, and *N. oneida*.

The evolution of oviposition preference is considered one of the driving forces in the divergence of phytophagous insect populations ([@R10]; [@R46]). Similar views have been presented for the divergence of parasitic Hymenoptera ([@R15]). In parasitoids, the differential usage of hosts may produce assortative mating as a pleiotropic consequence of female oviposition behavior. As *Nasonia* species mate locally on patchily distributed hosts and routinely inbreed ([@R7]), host preference differences might quickly lead to assortative mating. Interestingly, *N. giraulti* shows a high propensity to mate within the host ([@R7]), producing a strong coupling of host preference and assortative mating. Therefore it is possible that a shift in host preference was coupled with speciation events and perhaps with genetic bottlenecks due to the host shift. The low levels of genetic variation observed within *Nasonia* species is consistent with this scenario ([@R37]).

CONCLUSIONS {#S14}
===========

Results clearly indicate a major host preference locus (or set of tightly linked loci) in the region around bkbw~g~. The *N. giraulti* allele segregates in a Mendelian fashion and imparts a dominant preference for *Protocalliphora* hosts in an otherwise *N. vitripennis* genetic background. Utilizing the *Nasonia* genotyping microarray, we have mapped the host preference effect to 16 megabases of chromosome 4. Fine-scale mapping of the host preference locus can now proceed, utilizing the wealth of mapping and molecular resources becoming available for *Nasonia* ([@R49]). To our knowledge, this is the first introgression of a host preference locus from one parasitoid species into another. Furthermore, this work represents one of the few examples of introgression of behavioral genes between species.

We thank R. Edwards, C. Kennedy, and S. Patel for *Nasonia* maintenance, M. Drapeau and S. Bordenstein for assistance in data collection, A. Avery and J. Traggis for mapping visible markers in the bkbw~g~ region, J. Colbourne and J. Lopez for conducting the mapping array hybridization experiments, M. Clark, D. Loehlin, J. Jaenike, and three anonymous reviewers for helpful comments on the manuscript, and members of the North American Bluebird Society, particularly P. Conklin, J. Rogers, and R. Wells, for providing assistance in field work. Support for this research was provided by a Postdoctoral fellowship from the Spanish Ministry of Education and Culture to FP and by the National Science Foundation (IBN-9876356) and National Institute of Health (5R01GM070026) to JHW.

![Generation of the bkbw~g~ introgression line and map of visible markers in the region. In the parental cross, *N. giraulti* females were mated to *N. vitripennis* peach males. In each backcross generation, heterozygous females, identified by visible markers (m) in the bkbw~g~ region, were mated to *N. vitripennis* peach males to further reduce the size of the introgression. To purebreed the line, heterozygous bkbw~g~/+~v~ females were mated to bkbw~g~ introgression males, and their homozygous bkbw~g~/ bkbw~g~ introgression female offspring were again mated to bkbw~g~ introgression males in an attempt to produce an isogenic line. Inset is a map of the bkbw~g~ region, including the location of visible markers *ws1* (*wing size 1*), *bl13* (*blue 13*), *bk576* (*black 576*), and sww (*shorter wider wings*). Markers indicated with asterisks were used to track the bkbw~g~ introgression. Distances between visible markers are shown in centiMorgans (cM).](nihms147004f1){#F1}

![Behavior of wasps in host preference experiments. *N. vitripennis* strains ASymCx and peach, *N. giraulti* strain RV2Xu, and heterozygous bkbw~g~/+~v~ introgression females were tested. Genetic content of the wasps is shown in the chromosomes to the right, with white representing *N. vitripennis* DNA and black representing *N. giraulti* DNA. As can be seen, heterozygous bkbw~g~/+~v~ females contain a small region of *N. giraulti* DNA in a largely *N. vitripennis* genetic background. Wasps were given one *Sarcophaga* and one *Protocalliphora* host and observed for 4.5 hours. The percent which contacted and stung each host is shown, and error bars indicate standard error of proportions ([@R44]). Sample size for each strain is given to the right of the strain names.](nihms147004f2){#F2}

![Time spent on hosts by wasps in host acceptance and preference experiments. *N. vitripennis* strains ASymCx and peach, *N. giraulti* strain RV2Xu, and heterozygous bkbw~g~/+~v~ introgression females were tested. Percent time is based on 28 observations over a 4.5 hour period. Sample sizes for each strain in each experiment are shown to the right of the strain names. The relative sizes of *Sarcophaga* and *Protocalliphora* hosts are also shown.](nihms147004f3){#F3}

###### 

Genotype of major scaffolds on chromosome 4 in a bkbw~g~ introgression male. Locations of scaffolds are given as recombination-rate-based map markers from Niehuis *et al.* (in press), followed by size of the scaffolds or scaffold regions. The number of loci on the *Nasonia* genotyping microarray scored as *N. vitripennis*, *N. giraulti*, or ambiguous is also given, as is the predicted genotype of the scaffold or scaffold region (*N. vitripennis* = V, *N. giraulti* = G). The bkbw~g~ line contains two regions of *N. giraulti* DNA separated by a 4.5 megabase stretch of *N. vitripennis* DNA, suggesting double recombination has moved *N. vitripennis* DNA back into the bkbw ~g~ region.

        map                   \# of scored loci   predicted        
  ----- ------------ -------- ------------------- ----------- ---- ---
  4     4.01--4.15   5246     448                 2           20   V
  23    4.16--4.17   \~519    31                  0           7    V
        4.18--4.20   \~1900   0                   171         3    G
  29    4.20         \~200    0                   13          0    G
        4.20         \~1835   155                 0           2    V
  35    4.20         1595     142                 2           6    V
  108   4.20         481      23                  2           1    V
  52    4.20         \~832    68                  1           6    V
        4.20         \~90     0                   5           0    G
  34    4.20--4.21   1326     0                   127         0    G
  26    4.21         1596     3                   160         2    G
  123   4.21         416      1                   56          3    G
  43    4.22         879      0                   84          2    G
  51    4.22         1217     2                   124         3    G
  66    4.22         619      0                   51          0    G
  82    4.22         492      1                   30          2    G
  109   4.22         483      0                   33          1    G
  130   4.22         385      0                   42          0    G
  133   4.22         387      4                   38          0    G
  143   4.22         399      0                   38          2    G
  77    4.23         479      0                   32          0    G
  40    4.25         \~117    1                   6           0    G
        4.25--4.29   \~1760   183                 0           10   V
  9     4.29--4.41   4554     515                 5           22   V

[^1]: current address: Departamento de Genetica, Universidad de Granada, 18071 Granada, Spain

[^2]: current address: Department of Biology, University of California at Riverside, Riverside, CA 92521 USA
