The ranges of the nominal species Lycaeides idas and L. melissa are broadly overlapping in western North America, especially in California where their ranges are interdigitated and the butterflies occur in sympatry in at least one location (A. M. Shapiro and C. C. Nice, pers. obs.). These butterflies feed on papilionaceous legumes (Fabaceae) in California and Nevada. There is considerable overlap in the recorded use of hosts for each species, however local populations of each species are apparently monophagous. For example, L. melissa populations using introduced alfalfa Medicago sativa L. apparently ignore readily available native hosts. Where L. idas and L. melissa occur in close proximity, for instance in the Sierra Nevada of California, they are always found using different host plants. Lycaeides melissa at Carson Pass, California use Astragalus whitneyi A. Gray as their host and apparently ignore Lupinus polyphyllus Lindley, which is the host for the nearby population of L. idas at Leek Springs, California. Indeed, conspecific populations of L. idas in close proximity can be found using different host plants. In other words, the polyphagy at the species level appears to be the result of localised specialisation and is not the product of a generalist strategy (Fox & Morrow, 1981). This apparent localised specialisation is coupled with the mating behaviour of these butterflies. Mating occurs on or very near the host plant (Pellmyr, 1982; Scott, 1986; J. A. Fordyce and C. C. Nice, pers. obs.). Females perch on the host plant, males fly near the plants searching for females, and the courtship behaviour and mating usually occur on the host.
Vladimir Nabokov delineated these species by differences in wing pattern and the form of the male genitalia (Nabokov, 1949). A discriminant function analysis of variation in male genitalic characters clearly distinguished the two species (Nice & Shapiro, 1999). Two male genitalic measures (falx length and uncus length) accounted for 83% of the morphological variation between species. The discriminant function analysis based on the genitalic measures misclassified only 2.5% of males that were identified to species by wing phenotype. These species are indistinguishable using both nuclear markers (allozymes) and mitochondrial markers (mtDNA) (Nice & Shapiro, 1999), indicating recent divergence (Neigel & Avise, 1986). Genetic distances calculated from allozyme allele frequencies from 14 populations of L. idas and L. melissa from California and Nevada were extremely small (Nei's D = 0.002–0.078) and the two species were not monophyletic (Nice & Shapiro, 1999). Analysis of molecular variance (amova; Excoffier et al., 1992) of mtDNA cytochrome oxidase subunit I (COI) gene sequences revealed that < 3% of the total molecular variance was partitioned among species (Nice & Shapiro, 1999). This incongruence between morphological and molecular data indicates that divergence has either occurred recently or is ongoing and that selective factors may be important components of diversification. An alternative is that the morphological characters used to distinguish the species exhibit phenotypic plasticity or that the phenotype is host dependent (or host induced). This appears unlikely as individuals of both species reared to adulthood in the laboratory on the same hosts exhibit normal L. idas and L. melissa phenotypes (C. C. Nice, unpublished).
The lack of significant differences at the molecular level would be expected if the genomes of these species evolve in a mosaic fashion (Ting et al., 2000). That is, molecular evolution may proceed at different rates for different loci or groups of loci within a genome. Rates of evolution may be significantly higher for loci that contribute to reproductive isolation or are relevant in the context of ecological speciation compared with rates for presumed neutral loci (Butlin & Tregenza, 1998; Ting et al., 2000). In fact, such differences between quantitative trait loci and neutral markers may be considered evidence for divergent natural selection (Whitlock, 1999). The absence of intermediate morphologies in Sierran populations of Lycaeides indicates that divergent selection on morphological characters is quite strong and/or that gene flow among populations is low. Strong host fidelity in Lycaeides is one mechanism by which reproductive isolation may evolve and restrict gene flow. This hypothesis was tested by measuring host fidelity in populations of Lycaeides that use different larval host plants. The allozyme data of Nice and Shapiro (1999) were also re-examined to test specifically for population differentiation that may result from the evolution of host fidelity. Do females prefer the hosts they use in the field? If so, is gene flow restricted among populations using alternative hosts?
Geographic specialisation in oviposition preference by a phytophagous insect may occur in two ways (Thompson, 1993): phytophagous insects evolve heritable differences in preference for a particular host species or their host choice may be affected by ecological variables such as relative host species abundance, host phenology, or larval conditioning. The phenomenon of larval conditioning, also known as Hopkins host selection principle (Hopkins, 1917), posits that females prefer to lay eggs on the plant species that they ate as larvae; however there is little or no experimental evidence that supports this hypothesis (Szentesi & Jermy, 1990), especially in the Lepidoptera (Wiklund, 1974; Stanton, 1979; Tabashnik et al., 1981; Thompson & Pellmyr, 1991; Bossart & Scriber, 1995; Kuussaari et al., 2000).
If L. idas and L. melissa populations have developed differences in female oviposition preference, high levels of host fidelity and differences in preference hierarchies (i.e. the ranking of potential host species) are expected. If, on the other hand, host use is determined by some other mechanism(s), broader host ranges with low levels of fidelity for the natal host are expected (Thompson, 1993). To assess host fidelity in these butterflies, oviposition preference tests were conducted with wild-collected females from four populations (Fig. 1). Populations using either native host plants (Carson Pass, Mt Rose, Leek Springs, Yuba Gap) or a recently introduced host plant (Verdi) were chosen to test the hypotheses that females specifically prefer their natal host and that host fidelity is lower for butterflies with a shorter evolutionary history with a host plant than for those with a longer association.
For 10 experiments, three wild-caught females (individual caged females do not lay eggs; three females interact and stimulate oviposition) from the same population were enclosed in an arena (≈ 2000 cm3) with three plants: their natal host plant, a negative control, Melilotus alba Medikus, which is a legume that is not recorded as a host of either L. melissa or L. idas, and a test plant (the natal host of an adjacent population). Each experiment consisted of nine to 11 replicates (number of replicates was dependent on capture success and mortality of females in transit). Sample sizes are shown in Table 1. Females were collected from Carson Pass, California (38°58′N, 119°83′W), Mt Rose, Nevada (39°32′N, 119°96′W), Yuba Gap, California (39°31′N, 120°63′W), and Verdi, Nevada (39°51′N, 119°99′W). The Carson Pass and Mt Rose (L. melissa) sites are above the treeline in the Sierra Nevada. The Yuba Gap (L. idas) site is at mid-elevation on the west slope of the Sierra Nevada. The Verdi (L. melissa) site is located at relatively low elevation, east of the Sierra Nevada. Natal hosts are: Carson Pass, Astragalus whitneyi; Mt Rose, A. whitneyi; Yuba Gap, Lotus nevadensis (S. Watson) E. Greene; Verdi, M. sativa. Medicago sativa was introduced into North America less than 150 years ago (Bolton et al., 1972; Michaud et al., 1988). Populations and test plants were: Carson Pass (three experiments), L. polyphyllus, M. sativa, L. nevadensis; Mt Rose (one experiment), L. nevadensis; Yuba Gap (two experiments), M. sativa, A. whitneyi; Verdi (three experiments), L. nevadensis, A. whitneyi, L. polyphyllus. The Mt Rose population was included for comparison with the ecologically and morphologically similar Carson Pass population.
Table 1. Results of natal host oviposition preference experiments. Results of multiple comparisons are given in Fig. 1 .
|Species/population/ natal host||Test plants||n||T3||P|
|Yuba Gap, California||M. alba , M. sativa||10||7.95||<0.005|
|L. nevadensis||M. alba , A. whitneyi||10||9.12||<0.005|
|L. melissa||M. alba , L. nevadensis||8||0.28||>0.250|
|Verdi, Nevada||M. alba , A. whitneyi||9||5.49||<0.025|
|M. sativa||M. alba , L. polyphyllus||9||3.23||<0.100|
|L. melissa||M. alba , L. polyphyllus||11||22.83||<0.001|
|Carson Pass, California||M. alba , L. nevadensis||11||19.41||<0.001|
|A. whitneyi||M. alba , M. sativa||10||17.05||<0.001|
|Mt. Rose, Nevada|
|A. whitneyi||M. alba , L. nevadensis||10||31.42||<0.001|
|Leek Springs, California|
|L. polyphyllus||A. whitneyi , L. nevadensis||13||9.68||<0.005|
In another experiment of similar design, L. idas females were collected from Leek Springs, California (38°71′N, 120°25′W) and given three choices that did not include the negative control. The Leek Springs site is a wet meadow at mid-elevation on the west slope of the Sierra Nevada (Fig. 1). For this experiment, the choices included Lupinus polyphyllus (the natal host of the Leek Springs population), L. nevadensis (host of the Yuba Gap L. idas population), and A. whitneyi (host of the alpine L. melissa populations at Mt Rose and Carson Pass). This experiment consisted of 13 replicates.
All host plants used were collected on the same day as the butterflies and from localities where the test populations were present (except M. alba, which does not occur in sympatry with all Lycaeides populations). Each arena consisted of an air-spun polyester mesh bag (Kleen Test Products, Brown Deer, Wisconsin) attached to a circular cardboard base, with the stems of each plant projecting through the base into a common water reservoir. Females remained enclosed in the arena and were watered and fed three times a day with a saturated sucrose solution sprayed directly onto the bag. Each experiment lasted 48 h, after which the females were removed and the total number of eggs on each plant was counted. Each arena was considered a block and the number of eggs laid on each available plant per arena was analysed using the Quade test, a rank-based randomised blocked anova (Potvin & Roff, 1993; Conover, 1999). Thus each plant was ranked one, two, or three in each arena. All host-preference experiments were carried out in a greenhouse at the University of California, Davis. These tests of oviposition preference occurred in experimental arenas of small volume where available plants were in very close proximity and often interdigitated. This arrangement only allowed an assessment of short-distance cues, not long-distance cues, such as plant architecture. Females in these experiments were using short-range cues, probably relying on leaf chemistry for making their oviposition decisions. In the field as well as in the experiments, females are observed walking on the plants after alighting, apparently inspecting the plant before laying eggs (J. A. Fordyce and C. C. Nice, pers. obs.). Electrophysiological investigations have shown that lepidopteran species commonly use sensillae located on the tarsi for chemoreception (Blaney & Simmonds, 1990; Roessingh et al., 1991) and that these cues are often important in oviposition choice (Stanton, 1979; Wiklund, 1982; Papaj, 1986).
Tests of population differentiation using allozymes
To test the hypothesis that strong host fidelity may restrict gene flow, the data from 10 allozyme loci reported by Nice and Shapiro (1999) were re-examined. The GENEPOP version 3.2a software (Raymond & Rousset, 1995a) was used to test for population differentiation among the focal populations in this study using the test of Raymond and Rousset (1995b). This procedure uses a Markov chain method to obtain an unbiased estimation of the exact test of Fisher (1935). The Genetic Data Analysis version 1.0 software (Lewis & Zaykin, 1997) was also used to calculate pairwise FST estimators (θST values) according to the formulae of Weir and Cockerham (1984) and Weir (1996). Statistical significance of θST values was assessed by 1000 bootstrap replicates.
One of the focal populations, the Yuba Gap population, was not included in the original allozyme survey because it was discovered only after that investigation was completed. The Trap Creek population (39°36′N, 120°64′W) examined in the initial allozyme survey could not be assessed for oviposition preference due to logistical constraints, however the Trap Creek and Yuba Gap populations of L. idas are similar morphologically (see Results, Fig. 2) and share identical mtDNA haplotypes (Fig. 3). Both populations use L. nevadensis as the larval host in the field and these populations are located within 7 km of each other. For these reasons, it was assumed that allozyme allele frequencies in the Trap Creek and Yuba Gap samples were similar and the allozyme data from Trap Creek were used as an approximation of the allozyme allele frequencies that would be found in the Yuba Gap location.
Figure 3. Neighbour-joining tree of mtDNA cytochrome oxidase sub-unit I haplotypes. The topology of the maximum-likelihood tree was the same as that presented in this figure. Haplotypes present in the four populations measured for oviposition preference are indicated in bold. Bootstrap values are indicated above branches.
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