EVALUATION OF CROSS-ATTRACTION BETWEEN SYMPATRIC OCCIDENTALZS AND C. RETZNZANA (: ) POPULATIONS IN SOUTH-CENTRAL OREGON ANDREWM. LIEBHOLD,W. JANA. VOLNEY,and WILLIAME. WATERS Division of Entomology and Parasitology, University of California, Berkeley 94720

Abstract Can. Ent. 1 16: 827-840 (1984) The pheromone specificity of female Choristoneura occcidentalis Freeman and C. re- tiniana (Walsingham) from several locales, laboratory colonies, and interspecific mat- ings was determined by observing the numbers and wing maculations of maies attracted at several sites in southern Oregon. Pheromone s~ecificitvof females reared from field- collected brown (typical of c.-occidentalis) and green kypical of C. retiniana) larval morphs differed considerably but differed little among sites of origin. Field-collected females attracted more males than conspecific individuals from laboratory colonies. F, and F, interspecific hybrids most closely resembled C. occidentalis in the numbers and types of males attracted. As a group, progeny of backcrosses to C. retiniana appeared intermediate between pure lines of the species in their pheromone specificity. Females reared from intermediate-colored field-collected larvae varied considerably in attrac- tiveness. Most attracted groups of males similar to those attracted to female C. reti- niana, but others attracted males most similar to those attracted to female progeny of C. retiniana hybrid backcrosses. These findings support the conclusion that hybrid matings occur between these species at a low frequency in nature. Resume La spCcificitC de la phtromone chez des femelles de Choristoneura occidentalis Free- man et de C. retiniana (Walsingham) provenant de plusieurs sites, de colonies de laboratoire et de croisements interspecifiques a ete CtudiCe en determinant les nombres et les taches alaires des mdles attires a plusieurs endroits en Oregon. La spCcificitC de la phtromone differe considkrablement entre des femelles provenant de larves brunes (couleur typique chez C. occidentalis) et vertes (C. retiniana) prClevCes sur le terrain, mais trks peu entre les sites d'origine. Les femelles prklevCes sur le terrain ont attirC plus de miles que des conspCcifiques provenant de colonies de laboratoire. Des hy- brides intersptcifique de F, et F, ressemblaient plus a C. occidentalis quant aux nombres et aux types de miles attirks. En tant que groupe, la progCniture des rCtrocroisements avec C. retiniana est apparue intermediaire entre les lignCes pures quanta sa specificitk. Des femelles ClevCes a partir de larves de couleur intermkdiaire provenant du terrain ont montrC une variabilitC considCrable. La plupart ont attire des groupes de m2les similaires a ceux attires par les femelles de C. retiniana, mais certaines ont attire des mdles ressemblant plus ceux attires aux femelles provenant des rktro-croisements d'hybrides avec C. retiniana. Ces observations appuient la conclusion voulant que des croisements entre ces deux espbces se produisent avec une frkquence rCduite en nature. Despite several taxonomic treatments, the status of conifer-feeding Choristoneura species is uncertain (Freeman 1953, 1967; Powell 1964, 1980). Much of this uncertainty is due to the great intraspecific variation in, and interspecific overlap of, morphological characters (Harvey and Stehr 1967; Volney et al. 1983, 1984). In addition, the degree of reproductive isolation among various populations is uncertain. Postmating incompatibility is not a major factor in the reproductive isolation of conifer-feeding Choristoneura species. When interspecific pairs are confined in small cages, mating takes place and little inviability exists in their progeny (Smith 1953; Sanders et al. 1977; Volney et al. 1984). Thus, premating factors must be the major barriers to gene flow between these species. Considerable overlap exists in the seasonal and die1 periods of sexual activity between sympatric species (Smith 1954; Sanders 1971a; Lieb- hold and Volney 1984~).Therefore, temporal factors are not important isolating mecha- nisms. Males are attracted to female-released pheromones whether females are in host or 828 THE CANADIAN ENTOMOl.O(ilST June 1984 non-host trees (Liebhold and Volney 1984b). Consequently, host specificity is not a major isolating mechanism either. There are no major differences in the close range mating behavior of C. occidentalis Freeman and C. retiniana (Walsingham) (Liebhold and Volney unpub.). Thus, it appears that reproductive isolation between sympatric conifer-feeding Choristoneura is due largely to differences in female-produced attractant pheromones and responses of males to these compounds. Smith (1953, 1954) concluded that reproductive isolation between sympatric C. fu- miferana (Clemens) and C. pinus Freeman in Ontario was complete and was due mainly to ecological isolation (host selection), temporal isolation, and "sexual selection" (adult behavior and the role of pheromones were still not fully understood). Subsequent evalu- ations of cross-attraction between conifer-feeding ~horistoneuraspecies largely compared allopatric populations of these species (Sanders 1971a,b; Sanders et al. 1977). Thus, the degree of reproductive isolation between sympatric conifer-feeding Choristoneura, es- pecially western species, remains uncertain. Our observations on the relative abundance, distributions, host associations, and phenotypic variation of C. occidentalis and C. retiniana in southern Oregon have indicated that these two species are sympatric over much of this area (Volney et al. 1984). We also have found larvae of intermediate phenotypes in addition to the characteristic green larvae of C. retiniana and brown larvae of C. occidentalis. We therefore undertook this study to determine the attractive abilities of females from sympatric and allopatric populations of C. occidentalis and C. retiniana in this area. Specifically, we wished to determine if geographically separated populations of the same larval morph were similar in their abil- ities to attract males, the degree to which the two different morphs were cross-attractive in sympatric populations, and what sort of attractive abilities females reared from inter- mediate larvae possessed. With this knowledge we hoped to better evaluate whether hy- bridization between sympatric populations of these species occurs and what consequences this hybridization would have on the genetic structure of these populations. Methods Descriptions and locations of trapping sites are given in Volney et al. (1984). An additional trapping test was conducted in a predominantly young growth Douglas-fir, Pseu- dotsuga menziesii (Mirb.) Franco, stand located in the Mt. Hood National Forest near Bear Springs, Wasco Co., Oregon (BS). This area has a history of C. occidentalis out- breaks (Dolph 1980) and C. retiniana is not known to occur there (V. M. Carolin Jr., Ders. comm.: Powell 1980). All were collected as larvae either by pruning and searching branches from the entire crown of host trees (Douglas-fir and whte fir, (Gord. & Glend.) Lindl.), or by searching foliage of host trees at eye level. Larvae were returned to a field laboratory and in the sixth instar they were classified as either brown, green, or inter- mediate larval morphs using the method of Volney et al. (1984). All field-collected larvae were reared at ambient laboratory temperature in 90 x 23 mm shell vials containing foliage of the host tree species on which they were found. Upon pupation, foliage was removed, pupae were sexed, and were allowed to emerge. For convenience, females reared from field-collected green larval morphs, characteristic of C. retiniana, will be referred to as 'green', females reared from field-collected brown larval phenotypes, characteristic of C. occidentalis, will be referred to as 'brown', and females reared from field-collected larvae of intermediate phenotype will be referred to as 'intermediate'. One- to two-day-old virgin females were placed singly in 12 x 6 x 4 cm aluminum screen cages.u To Drevent desiccation of moths. a half dram water-filled vial fitted with a cotton wick was added to each cage. Females were exposed to at least 24 h of natural photoperiod before being taken to the field. Each cage was attached under the roof of a Pherocon 1C trap (Zoecon Corp., Palo Alto, CA). Control traps contained a cage with Volume 116 THE CANADIAN ENTOMOLOGIST 829 only a water vial. Traps were placed 20 m apart in a grid and hung from tree foliage approximately 1.5 m above the ground. Treatments (i.e. female types and control) were assigned at random to the trap locations within a grid. In 1979, green larval morphs were collected at Dutch Oven Flat (DOF), Riverbed Butte (RB), and Dry Lakes Flat (DLF). Females from these sites were deployed in traps at all sites except that DLF females were not deployed at RB, and RB females were not deployed at DLF. Control traps were deployed at all sites. Traps were left up for 2 nights after which trapped males were counted. If the caged female had died, data from the trap were excluded from the analysis. In 1980, green females from Mt. Ashland (MA) and RB, and brown females from Hyatt Lake (HL) and Tolman Creek (TC) were deployed at MA. At RB, green females from MA and RB, and brown females from HL were deployed. At DLF, green females from MA and DLF were deployed. At TC, green females from MA and RB, and brown females from TC were deployed. At HL, green females from MA and RB, and brown females from HL were deployed. Four control traps were deployed at all sites also. After 2 nights, the number of males per trap was recorded, excluding counts from traps in which the female had died. Pupae of C. occidentalis and C. retiniana were obtained from non-diapausing col- onies maintained at the U.S. Forest Service, Pacific Southwest Forest and Range Exper- iment Station, Berkeley, California. Females reardd from the C. occidentalis laboratory colony will be referred to as 'OCC' and females from the C. retiniana laboratory colony will be referred to as 'ret'. Hybrid crosses and backcrosses were made by mating pairs of adults in 250 mL waxed paper cups and rearing their progeny on artificial diet described by Robertson (1979). In this manner, at least 10 families were obtained for each of the occ X occ (female X male), ret X ret, occ X ret, ret X occ, ret X (ref X occ), ref X (OCCX ret), and (ret X occ) X (ret x occ) crosses. In 1982, traps baited with females from these families, traps baited with females reared from field-collected brown, green, and intermediate larval morphs from HL, and control traps were deployed at HL, DOF, and BS. Traps that contained more than 40 trapped males after 1 night were supplied with a fresh liner for the second night. The total trap count was recorded as the sum of the first and second nights' catches. Because the underlying distributions of trap catches were unknown, we relied on non- parametric statistical methods in our analyses of data. We first used a Kruskal-Wallis test (Lehmann 1975) to compare trap catches between treatments at each site. When this test was significant, indicating differences among treatments, we then used a two-sided Wil- coxon rank-sum test (Lehmann 1975) to make painvise comparisons between treatments. Because we made a large number of comparisons, the probability of rejecting a true null hypothesis (type I error) may be large for this study as a whole. Since this study was exploratory, we chose a significance level of 0.01 for individual painvise comparisons as a compromise between reducing the experimentwise probability of a type I error and simultaneously reducing the probabilities of painvise type I1 errors (failure to reject a null hypothesis when it is false) (Tukey 1962). We also attempted to reduce the experimentwise probability of a type I error by limiting the number of comparisons; within each plot, field- collected females of a given morph were compared with field-collected females of other morphs and with controls, comparisons were made among the different types of laboratory- reared females and with controls, and field-collected females were compared with con- specific laboratory-reared females (e.g. green females were compared with ret x ret females). C. occidentalis and C. retiniana adult males are not positively distinguishable in these areas (Volney et al. 1983, 1984). Therefore, trapped males were removed from all traps used in 1982 tests and all traps used in 1980 tests at MA and TC. Males were then 830 THE CANADIAN EhTOMOLffiIST June 1984 degreased by soaking in xylene for 2 h and then rinsing with hexane. Males missing large patches of forewing scales were discarded. The remaining males were described using the scoring methods of Volney et al. (1983). There were insufficient trapped males scored in 1980 to develop a classification scheme as was done by Volney et al. (1983). However, at MA and TC pairwise com- parisons of banding intensity x background color cell frequencies were made between the groups of males trapped by different sources of females using chi-square tests (Sokal and Rohlf 1981) (a = 0.05). Using wing pattern frequencies from males trapped in the 1982 experiments, we examined the relationship between trapped male classification characters and the type of female to which it was attracted. We did this by applying the non-parametric procedure of Grizzle et al. (1969) to the males trapped by green and brown females at each of the three sites. At the HL site, a binomial classification rule (Hand 1981, p. 99) was developed using character states of all five forewing characters from the descriptions of males trapped by brown and green females collected at HL. Thus, males from each treatment were classified as most similar to those trapped by either brown or green females. We found the binomial classification technique (Hand 1981) to be superior to the use of a discriminant function, such as developed by Volney et al. (1983), because it more successfully clas- sified individuals in the calibration data and because it does not assume classification variables to be either normally distributed or continuous. A non-parametric simultaneous comparison of binomial populations (Miller 1966) was used to compare the proportions of males classified as 'brown' which were trapped by different female types. Only treat- ments (female types) for which there were five or more traps with 15 or more described males per trap (brown, green, intermediate, and occ x ret females) were compared in this analysis. A chi-square test of heterogeneity (Sokal and Rohlf 1981) was also performed on the classification results from each treatment to determine if the classification frequen- cies were significantly heterogeneous within treatments. Results All populations of females tested in 1979 were significantly more attractive than controls (Table I). However, there were no significant differences in the number of males trapped by females from different sites. Except for HL females tested at RB, all populations of females were significantly more attractive than controls in 1980 (Table 11). No significant differences in trap catch were observed among populations of females tested in 1980 except that at HL, local fe-

Table I. Mean numbers of males caught in female-baited tram at study sites in southern Oregon, 1979

Trap Morph of Source of No. of Mean Coefficient location female female traps trap catch' of variation DOF Green DOF 9 42.7 a 25 Green RB 9 43.0 a 44 Green DLF 6 33.8 a 39 Blank 6 Ob 0 RB Green DOF 8 74.0 a 27 Green RB 7 80.7 a 11 Blank 12 0.1 b 346 DLF Green DOF 6 36.7 a 63 Green DLF 6 53.2 a 29 Blank 8 0 b 0

'Within a location, treatments (types of females and blanks) with means followed by the same letter are not significantly different (a = 0.01). ABBREVIATIONS:DOF, Dutch Oven Flat; RB, Riverbed Butte; DLF, Dry Lakes Flat. Volume 116 THE CANADIAN ENTOMOLOGlST 83 1 Table 11. Mean numbers of males caught in female-baited traps at study sites in southern Oregon, July 1980

Trap Morph of Source of No. of Mean Coefficient location female female traps trap catch1 of variation MA Green MA 8 12.7 a 99 Green RB 10 12.5 a 6 1 Brown TC 6 7.2 a 98 Brown HL 8 10.5 a 141 Blank 4 0 b 0 RB Green MA 8 27.9 a 50 Green RB 10 25.0 a 52 Brown HL 9 0.2 b 300 Blank 4 Ob 0 DLF Green MA 8 14.4 a 126 Green DLF 8 22.4 a 90 Blank 4 0 b 0 TC Green MA 8 9.0 a 127 Green RB 10 11.1 a 93 Brown TC 4 16.2 a 83 Blank 4 0 b 0 HL Green MA 7 4.1 b 70 Green RB 10 2.6 b 127 Brown HL 8 34.6 a 11 Blank 4 0 c 0

'Within a location, treatments (types of females and blanks) with means followed by the same letter are not significantly different (a = 0.01). ABBREVIATIONS.MA, Mt. Ashland; RB, Riverbed Butte; DLF, Dry Lakes Flat; TC, Tolman Creek; HL, Hyatt Lake.

males (collected as brown larval morphs) were more attractive than both MA and RB females (collected as green larval morphs). In 1982, all females were significantly more attractive than controls except ret X (ret x occ) at HL, ret X occ and (ret X occ) x (ret x occ) at DOF , and ret X ret at BS (Tables 111, IV). At HL, field-collected females (brown and green) were more attractive than conspecific laboratory colony females (occ x occ and ret X ret respectively). Simi- larly, at DOF, field-collected C. retiniana females (green) were more attractive than lab- oratory colony females. At DOF, where C. retiniana predominated (Volney et al. 1984), green females were more attractive than brown females and ret x ret females were more attractive than occ x occ females. At BS, where only C. occidentalis is presumed to occur, brown females were more attractive than green females and occ x occ females were more attractive than ret X ret females. At all sites, intermediate females were not significantly different from either brown or green females in trap catch. The number of males trapped by both types of Fl hybrid females (occ X ret and ret X occ) did not differ significantly from the number trapped by occ x occ females at all sites. However, at BS both types of Fl hybrids caught significantly more males than did ret X ret females. At DOF, a similar, but not significant difference was observed between catches by both F, hybrids and those by ret x ret females. At no site was attraction to the two types of F, hybrids significantly different. Similarly, F, hybrids, (ret x occ) x (ret x occ), tended to attract numbers of males similar to those trapped by occ X occ females. In contrast, the hybrid backcrosses, ret x (occ x ret) and ret X (ret x occ), were more similar to pure C. retiniana than to C. occidentalis in the number of males trapped. However, only at BS did backcrosses trap a significantly different number of males than did pure C. occidentalis females. There was little or no difference in coloration (combination of forewing banding intensity and ground color) between male moths trapped by green females from MA and those trapped by green females from RB (Fig. 1). No significant difference was observed 832 THE CANADIAN ENTOMOLOGIST June 1984 Table 111. Trap catches of female-baited traps at study sites in southern Oregon, 1982

Trap No. of Mean trap Coefficient location Type of female traps catch of variation HL Field- Brown 11 29.5 15 collected Green 9 37.4 40 Intermediate 12 30.1 43 Lab. occ X occ 9 9.7 30 colony ref X ref 9 7.3 148 occ X ref 9 14.0 59 ref X occ 10 13.7 68 ref X (occ X ref) 7 9.4 110 ref X (ret X occ) 5 4.2 149 (ret X occ) x (ref x occ) 10 12.5 126 Blank 7 0.1 316 DOF Field- Brown 8 7.1 89 collected Green 7 53.5 57 Intermediate 8 43.6 101 Lab. OCC x occ 5 1.2 37 colony ref X ret 7 17.2 94 occ X ref 8 2.1 73 ref X occ 8 3.3 95 ret x (occ x ret) 6 18.0 90 ref x (ret x occ) 8 28.5 125 (ret x occ) x (ref x occ) 6 3.8 220 Blank 8 0.1 283 Field- Brown 6 111.8 4 1 collected Green 5 27.8 63 Intermediate 7 45.9 56 Lab. occ x OCC 7 95.7 30 colony ref x ret 6 4.8 167 occ X ret 7 56.9 65 rer X occ 6 66.8 41 ref x (occ x ref) 8 14.4 109 ref x (ref X occ) 7 31.2 97 (ret X occ) X (ref X occ) 7 66.1 92 Blank 10 0.1 316

ABBREV~ATIONS:HL, Hyatt Lake; DOF, Dutch Oven Flat; BS, Bear Springs. at either MA (the probability of obtaining a larger x2by chance was 0.690; we denote this: {P > x2} = 0.690) or at TC ({P > x2} = 0.340). TOOfew moths were scored to compare males trapped by brown females from TC with males trapped by brown females from HL. However, when all males trapped by brown females (HL and TC) and all males trapped by green females (MA and RB) were compared, there was a significant difference in coloration at both MA ({P > x2) = 0.001) and at TC ({P > x2) = 0.001). Analysis of the contribution of trapped male forewing characters in predicting the larval morph of the bait female at DOF and BS indicated that none of the characters had a significant value (Table V). We were unable to formulate rules for classifying trapped males at DOF or at BS because these relationships between bait female type and trapped male characters were weak, and because there were relatively few males trapped by brown females at DOF and few males trapped by green females at BS. However, at HL ground color, banding intensity, and flecking intensity made a significant contribution to the pre- diction of the larval morph of the female to which males were attracted (Table V). Thus, at this site it was possible to generate a rule for classifying trapped males as being most similar to those trapped by green females or most similar to those trapped by brown females. Table IV. Pairwise comparisonsof numbers of males trapped by different typesof females: probabilityof a greater W < I: Inter- ret x ret x (refx occ) x B Green mediate occ X occ retX ret occ X re1 refX occ (occ X ref) (re1X occ) (refx occ) Blank + u.- Hyatt Lake Brown 0.254 0.999 0.002* Green 0.227 0.002" Intermediate OCI'X OC'C' rerX TPI OCI'X re1 re?x occ rtJt X (~~t.c.X re!) re? X (rctXocc) (re!Xoct,l X (m X occ) Dutch Oven Flat Brown 0.005* 0.104 0.004' Green 0.651 0.032 Intermediate OC'C x ucv rcr X ref occ X ref ref x wc rer X (OCCX rer) ref X (re?X mc) (re!X occlX (w!X OCC) Bear Springs Brown 0.010* 0.015 0.685 Green 0.291 0.045 intermediate ore X err ref X re! DCC X ret ref x occ re1 x (o~cX rrr) ref x (rrrxnrc) Ire: X or.^.) X (re1X occ)

*Valuesfor which P < 0.01. 834 THE CANADIAN ENTOMOLOGIST June 1984

TRAP green larvae brown larvae CHARACTER LOCATION MA RB TC HL LEGEND

MA 01

ground color rn 2 = 4 TC 5

MA 0 2 banding intensity 3 - 4 TC

MA 0 2 flecking color rn 3 -4 TC

MA 0 2 flecking intensity m 3 = 4 TC

MA discal fleck = 2 TC

MA 2 8 44 16 19 NO. INDIVIDUALS SCORED TC 33 42 23 0

FIG. 1. Forewing character frequencies of males trapped by virgin females at two sites in south-central Oregon, 1980. Pies in one column represent character frequencies of males trapped by females originating from a single locality (site abbreviations are: MA, Mt. Ashland; RB, Riverbed Butte; TC, Tolman Creek; HL, Hyatt Lake). See Volney et al. (1983) for descriptions of character states. Volume 116 THE CANADIAN ENTOMOLOGIST 835 Table V. Contribution of trapped male wing characters to the diagnosis of bait female larval morph; probabilities of obtaining a greater chi-square

Male forewing characters Banding Ground Flecking Flecking Discal Location intensitv color intensitv color fleck -- DOF 0.1824 0.0908 0.721 1 0.4868 0.5265 BS 0.4061 0.2171 0.1230 0.3505 0.4747 HL 0.0001 0.0001 0.0060 0.4026 0.6373

ABBREVIATIONS:DOF, Dutch Oven Flat; BS, Bear Springs; HL, Hyatt Lake

Using this classification rule on the calibration data set (males trapped by brown females and males trapped by green females), 85% of males trapped by brown females and 82% of males trapped by green females were successfully classified as being trapped by the correct female type. Applying this rule to the other males trapped at HL, occ X occ, occ x ret, ret X occ and (ret X occ) X (ret X occ) females attracted males that were mostly classified as 'brown'; intermediate and ret x ret females attracted males that were mostly classified as 'green', though intermediate females appeared to attract slightly more 'brown' males; ret X (ret X occ) and ret x (occ x ret) females attracted nearly equal numbers of 'green' and 'brown' males, though ret x (ret x occ) females appeared to attract slightly more 'brown' males (Table VI). The classification of males caught by individual green, brown, and occ x ret females revealed little variation within each treatment in the proportion of 'brown' males caught (Table VII). Chi-square tests of heterogeneity indicated that the classification frequencies of trapped males were not significantly heterogeneous for green females ({P > x2 829) = 0.81) or occ x ret females ({P >.x2 829) = 0.27) but frequencies were heterogeneous for brown females ({P > x2 829) = 0.04) and highly heterogeneous for intermediate females ({P > x2) = 0.001). Brown and hybrid females were not significantly different in the percentage of trapped males classified as 'brown'. However, brown, green, and intermediate females trapped groups of males that were each significantly different from each other in the percent classified 'brown'. Discussion The results of the 1979 and 1980 tests (Tables I, 11) indicated that females of the green larval morph (typical of C. retiniana) from different locations in southern Oregon possessed similar attractant pheromones. Thus, green larvae collected at MA, which is west of the previously described range of C. retiniana (Freeman 1967; Powell 1980; Dolph 1980) may be correctly classified as conspecific with C. retiniana occumng to the east in the Gearhart Mountains (DOF and RB) and on the Chiloquin Ridge (DLF). The lack of attraction of brown females from HL when tested at RB (Table II), where only green larvae had been previously collected (Volney et al. 1984), suggests consid- erable specificity in the attractiveness of brown (typical of C. occidentalis) females. This is somewhat in contrast to the findings of Sanders et al. (1977) who found that C. occi- dentalis females were ca. 8% as attractive to C. retiniana males as conspecific females. However, at BS, where only C. occidentalis is known to occur, there was significant attraction to green females (Tables 111, IV), indicating at least some cross-attraction of C. occidentalis males to C. retiniana females. This attraction to green females was con- siderably less than that to brown females. The lack of any association between trapped male character states and the type of female it was attracted to at BS (Table V) suggests that males attracted to green females here represent the same population as those attracted to brown females. However, caution must be exercised in interpreting these types of stud- 836 THE CANADIAN ENTOMOLOGIST June 1984 Table VI. Binomial classification of males trapped by various types of virgin females (pooled over traps) near Hyatt Lake, Oregon, 1982

Classification No. of No. males Percent Percent Type of female traps scored brown ereen Brown Green Intermediate OCC X OCC ref X ref occ X ref ret X occ ref X (occ x ret) ret X (ret X occ) (ref X occ) X (ret X occ)

ies at sites with "allopatric" distributions of these species since it is difficult to verify that a given species is completely absent at any one site. For example, from 1978 to 1980 at DOF, which is ca. 80 km from the nearest Douglas-fir stand (the distribution of this host approximately describes the distribution of C. occidentalis in this area (Volney et al. 1984)), we collected over 1000 larvae without finding any brown larval morphs (C. oc- cidentalis). Yet in 1982, out of ca. 300 larvae collected, one brown larval morph (male) was collected. Thus, it is difficult to determine if the males attracted to C. occidentalis at DOF in 1982 were C. retiniana or C. occidentalis. We believe similar problems exist when studying cross-attraction between other "allopatric" populations. The significant difference observed in 1980 in banding and background color between males attracted to green and brown females at MA and TC indicated at least some spec- ificity in the attraction of males to females at these sites. Thus, it was possible to develop a classification rule for distinguishing groups of males attracted to different female types where both C. occidentalis and C. retiniana occur sympatrically. Such a classification rule was developed at HL from the descriptions of males trapped in 1982. Classification frequencies for males trapped by brown females and males trapped by occ x occ females were very similar (Table VI). Similarly, green females and ret X ret females trapped very similar types of males. However, at several sites, field-collected C. occidentalis and C. retiniana were significantly more attractive than conspecific fe- males from laboratory colonies (Tables 111, IV). Thus, it appears that field-collected and laboratory colony females released pheromones of similar specificity (as measured by the types of males attracted) but differed in the magnitude of their attractiveness. It is unclear why females from laboratory colonies (reared on artificial diet) are less attractive, though this phenomenon has been observed in at least one other species of Lepidoptera (Richerson and Cameron 1974). The observed differences could be due to genetic differences (the colony has been maintained under laboratory conditions for ca. 100 generations) or due to different larval diets. Progeny of both types of F, hybrid crosses, ret x occ and occ x ret, attracted male types that were classified as most similar to those attracted to brown females (Tables VI, VII). In addition, trap catches (numbers) of both types of F, hybrids were similar to those of pure C. occidentalis (Tables 111, IV). Furthermore, at all sites there was no significant difference between the number of males trapped by the two different types of F, hybrids (occ X ret and ret X occ). These results tend to support the theory proposed by Sanders et al. (1977) that pheromone production in conifer-feeding Choristoneura is controlled by an autosomal locus (loci) and that the allele(s) for release of (E)- and (Z)-1 I-tetradecenals (principal components of the C. occidentalis pheromone system (Cory et al. 1982; Silk THE CANADIAN ENTOMOLOGIST

Table VII. Binomial classification of males trapped by individual females near Hyatt Lake, Oregon, 1982

Classification No. Female males Percent Percent Type of female id scored brown green Brown 1 36 83 17 2 29 90 10 4 27 92 8 5 2 1 90 10 6 30 83 17 7 44 93 7 8 25 60 40 10 24 75 25 12 23 82 18 mean - 83 17 a Green 1 64 14 86 2 21 14 86 3 36 17 83 4 43 19 81 6 16 13 87 7 7 1 18 82 8 27 7 93 mean - 15 85 c Intermediate 1 38 42 58 2 30 43 57 3 32 13 87 4 38 2 1 79 5 31 26 74 6 78 37 63 7 20 25 75 8 51 16 84 9 88 26 74 10 19 79 2 1 mean - 33 67 b OCC X re1 1 15 60 40 2 15 67 33 3 16 81 19 4 36 72 28 5 29 66 34 6 16 44 56 7 16 81 19 mean - 67 33 a

NOTE:Treatments with means followed by the same letter are not significantly dif- ferent (a = 0.05). et al. 1982)) is dominant over the allele(s) for (E)- and (a-11-tetradecenyl acetates pro- duction (principal components of the C. retiniana pheromone system (Daterman et al. in press)). The backcrosses, ret x (ret x occ) and ret x (occ x ret), were less specific in the type of males that they attracted (Table VI). Both attracted nearly equal numbers of males classified as brown and green. Furthermore, these females were most similar to pure C. retiniana females in the numbers of males trapped though substantial variability oc- curred (Tables 111, IV). These results indicate that the ret x (ret X occ) and ret X (occ x ret) backcrosses possess an attractant system most similar to, but not identical with, that of ret x ret females. 838 THE CANADIAN ENTOMOLOGIST June 1984 We tested only two of the eight possible F, backcrosses. Using the genetic mechanism proposed by Sanders et al. (1977), we expect that (occ x ret) X ret and (ret X occ) X ret females would have pheromone specificities similar to ret X ret females and that (ret X occ) x OCC,(OCC x ret) x occ, occ x (occ x ret), and occ x (ret x occ) females would be most similar to occ X occ. The pheromone specificity of F2 hybrids, (ret x occ) x (ret x occ), was most similar to that of occ x occ and F, hybrids. At both DOF and at BS, the F2 hybrid trap catch was very similar to that of pure C. occidentalis and F, hybrids (Tables 111, IV). Furthermore, most males trapped by F2 females were classified as being like those trapped by brown females (Table VI). When traps at HL were pooled within treatments (Table VI), phenotypes of males trapped by intermediate females were most similar to phenotypes of males trapped by green females, though there were a substantial number of males (30%) that were classified as being attracted to brown females. However, when the classification of trapped males was summarized by. trap- (Table VII), it was evident that some intermediate females at- tracted males that were mostly like those trapped by green females, while some inter- mediate females (nos. 1, 2 & 6, Table VII) attracted males of nearly equal numbers clas- sified green and brown, and one intermediate female (no. 10) attracted males mostly classified as brown. Classification frequencies of males trapped by occ x ret and brown females were more homogeneous with the exception of brown female no. 8 which attracted noticeably more green males. Green females attracted very homogeneous types of males. These results have several implications concerning the structure of C. retiniana and C. occidentalis populations in southern Oregon. ~~ecificall~,if hybrids occur between sympatric populations, these individuals are capable of attracting mates. Other studies demonstrate that hybrid offspring are viable (Sanders et al. 1977; Volney et al. 1984). The ability of hybrid offspring to survive under natural conditions is unknown. The finding that hybrid females are more attractive to C. occidentalis than to C. retiniana males in- dicates that hybrid females will most likely mate with C. occidentalis males in nature. The mating preference of hybrid males is unknown however. The observation that some in- termediate females attracted nearly equal numbers of males classified as brown and green, indicates that these females are genetically neither pure C. occidentalis nor pure C. reti- niana. We believe that these females are the result of one or more consecutive backcrosses of hybrids to C. retiniana. Volney et al. (1984) found that 64% of the progeny of hybrid matings were brown larval morphs. Thus, we suspect that most true hybrids in nature appear as brown larval morphs and that most intermediate larvae observed in the field are the result of backcrosses to C. retiniana. This conclusion is in agreement with the finding that many of the intermediate females attracted the same type of males as green females, while others attracted the same types of males as C. retiniana backcrosses. It seems likely that the one anomalous brown female (no. 8, Table VII) was actually an Fl hybrid, thereby explaining the aberrant frequency of trapped male types. It is unclear at what rate hybrid matings occur in south-central Oregon. Certainly they are more common than hybrid matings between C. fumiferana and C. pinus reported by Smith (1954). Theoretical population genetics models indicate that hybridization between two populations rapidly counteracts the results of different selection pressures (Crow and Kimura 1970). Thus, because these two species are still at least partially distinct, we suspect that they are under strong differential selection pressures. It is unclear why a more complete isolating mechanism between these two species has not evolved as a result of this selection. One explanation is that these are incipient species and that isolating mech- anisms are still evolving. Another explanation is that there is a selective advantage to the maintenance of a low level of gene flow between these two species. Perhaps infrequent interspecific matings function as a source of genetic diversity to these species during pop- ulation bottlenecks which occur during periods of low population densities. Volume 116 THE CANADIAN ENTOMOLOGIST 839 Acknowledgments We are grateful to J. A. DeBenedictis, J. A. Powell and C. J. Sanders for reviewing this manuscript. We thank S. Omi, K. M. Daane and L. D. Merrill for technical assistance, K. Q. Lindahl Jr. for statistical advice, and J. Robertson of the USDA, Forest Service Pacific Southwest Forest and Range Experiment Station for providing insects from labo- ratory colonies. We thank the USDA Forest Service, Fremont, Rogue River, and Winema National Forests, U.S. Department of the Interior, Bureau of Land Management and the Weyerhauser Corp. for allowing us to conduct experiments on their land. Work leading to this publication was funded, in part, by a grant from the USDA Forest Service Canada1 United States Spruce Budworms Program-West to W. E. Waters and W. J. A. Volney. Grant numbers 904- 14-15 and 804- 15-23 were sponsored by USDA Science and Education Administration and grant numbers PNW-81-126 and PNW-82-210 were sponsored by USDA Forest Service, Pacific Northwest Forest and Range Experiment Station. This work was also funded, in part, by California Agricultural Experiment Station Project #3689H, entitled "Biology and Dynamics of Forest Populations".

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