Journal of Chemical Ecology, Vol. 17, No. 7, 1991

INTERSPECIFIC PAIRING BETWEEN TWO SIBLING SPECIES (COLEOPTERA: SCOLYTIDAE)

J.W.

F O X , I'* D . L .

WOOD, 1 and J.H.

Ips

CANE 2

~Department of Entomological Sciences University of California Berkeley, California 94720 2Department of Entomology Auburn University Auburn, Alabama 36849-5413 (Received September 24, 1990; accepted March 18, 1991) Abstract--Host and conspecific discrimination were tested as reproductive isolating mechanisms between Ips paraconfusus Lanier infesting Pinus coulteri (Torrey) and L confusus (Le Conte) infesting P. monophylla (Fremont). In two areas (one area largely Coulter pine and the other largely pinyon pine) where these bark beetles and hosts cooccur in southern California, we induced pheromone production in host and nonhost logs using males of each species. lps paraconfusus females joined both heterospecific and conspecific males tunneling in both tree species in each area. Ips confusus females failed to join L paraconfusus males in Coulter pine, but joined L paraconfusus in pinyon pine. Sympatry was demonstrated when females of both sibling species joined conspecific males in their respective hosts. Males attacked all four beetlehost treatment combinations in both areas. Laboratory tests confirmed these results. Males did not displace heterospecific males from nuptial chambers in hosts, and they did not occupy a gallery in which heterospecific males produced frass. Females that left conspecific males in a host were readily accepted by heterospecific males and oviposited in a nonhost. Key Words--Interspecific mating, lps confusus, lps paraconfusus, Coleoptera, Scolytidae, reproduction, reproductive isolation.

INTRODUCTION

The engraver bark beetle genus Ips contains about 60 recognized species in N o r t h a n d C e n t r a l A m e r i c a , E u r o p e , a n d A s i a ( H o p p i n g , 1963, 1965; S. L. *To whom correspondence should be addressed. 1421 0098-0331/91/0700-1421506.50/0 9 1991 Plenum Publishing Corporation

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Wood, 1982). Three sibling species in species group grandicollis Wood (1982), L confusus (Le Conte), L hoppingi Lanier, and L paraconfusus Lanier are virtually identical morphologically and electrophoretically (Cane et al., 1990b), which suggests a recent origin from a common ancestor. Their species status is based primarily upon their host association, geographic distribution, failure to hybridize, karyology, and differences in the striation density on the pars stridens, a cephalic stridulatory apparatus (Lanier, 1966, 1970, 1971, 1972). In the San Bernardino National Forest there are areas of nearly pure stands o f Coulter pine, P. coulteri (Torrey), a host of L paraconfusus, and single-leaf pinyon, P. monophylla (Fremont), a host of L confusus, as well as narrow zones of host sympatry (Critchfield and Little, 1966). One potential postmating barrier, failure of heterospecific larvae to successfully develop in a nonhost, does not occur (Cane and Wood, unpublished). Another postmating barrier, failure to hybridize, demonstrated by Lanier (1971), was recently confirmed by Merrill (1991) when she reared beetles in the laboratory and paired them with virgin females of known identity. The study reported here continues our investigation (Cane et al., 1990a, b) of possible premating barriers to hybridization between L paraconfusus and L confusus. Premating barriers likely involve discrimination of host from nonhost, conspecific from heterospecific pheromones, and mate recognition or proximal behavioral cues. Host discrimination by Ips species occurs prior to or after landing, or after penetration of the bark. Host discrimination by L paraconfusus may be influenced by the presence of feeding stimulants and the absence of feeding deterrents in the host phloem (Elkinton and Wood, 1980; Elkinton et al., 1980). However, Cane et al. (1990a) concluded that host discrimination was not absolute for these populations because they demonstrated that: (1) both male and female L confusus and L paraconfusus colonize host and nonhost pine bolts (pinyon and Coulter pine) presented to populations adjacent to a zone of sympatry; and (2) pioneering L paraconfusus and L confusus males, under the influence of pheromones, are trapped in large numbers at uninfested pinyon and Coulter pine bolts in both habitats. Stridulation can be important for mate recognition in Ips species and other scolytids (Barr, 1969; Alcock, 1982). Ips paraconfusus and L confusus have different densities of striae on the pars stridens of the stridulatory apparatus (Lanier, 1970; Cane et al., 1990a) but produce nearly identical fundamental frequencies of vibration. However, their bisyllabic chirps are temporally distinctive (Lewis and Cane, unpublished). Pheromones may serve as premating barriers for bark beetles. Ips species produce pheromones that elicit behavior, resulting in aggregation of beetles on the new host. This aggregation must be both timely and of sufficient magnitude to exploit the new resource. Pheromones are produced in the insect gut and frass (fecal pellets with phloem and xylem fragments) after prolonged feeding on phloem and have been identified for L paraconfusus and L confusus (sum-

INTERSPECIFIC PAIRING IN

Ips

SPECIES

1423

marized in D. L. Wood, 1982). Differences in response to and production of pheromones has been documented in L pini and may account for some species differentiation (Lanier et al., 1972, 1980; Birch et al., 1980). Lanier and Wood (1975) tested the pheromone specificity of 17 species of Ips and discovered that species within the same species groups are cross-responsive, unlike species in different species groups. Species with broadly sympatric distributions in the same host are from different species groups, while members in the same species group are largely allopatric. Ips paraconfusus and L confusus females were attracted to frass produced by males of the other species when presented individually in the laboratory olfactometer and in simultaneous, paired comparisons with conspecific frass. Cross-responsiveness and interspecific pairing (i.e., females joining male galleries) between I. confusus and I. paraconfusus has been demonstrated in the field, but intermating was not established (Lanier and Wood, 1975). Cane et al. (1990a) reported that for beetles in flight, discrimination of host volatiles and conspecific pheromones were incomplete and asymmetrical because female I. paraconfusus did not have the same degree of discrimination as female L confusus. In the San Bernardino National Forest, heterospecific pairing and natural colonization of nonhosts were not located in zones of sympatry despite persistent search in this and earlier studies (Cane et al., 1990a). Therefore, we further investigated whether this incomplete discrimination of host volatiles and pheromones, coupled with proximal behavioral cues subsequently available to beetles freely walking on the bark, may be sufficient to elicit associations with the native host and conspecific mates. First, we tested the hypothesis that beetles in the field can pair with heterospecifics and colonize nonhosts. Next, this hypothesis was partitioned into specific behavioral elements. In the laboratory, experiments were designed to determine: (1) the likelihood of beetles encountering a heterospecific at a nuptial chamber excavated by a conspecific male; (2) the ability of beetles to discriminate hosts and conspecifics in an atmosphere saturated with pheromones of both species; (3) the influence of host on conspecific discrimination; (4) if heterospecific pairing will occur when populations are limited; and (5) if conspecific-inseminated females will join heterospecific males.

METHODS AND MATERIALS

Field Tests Two test sites were selected during June 1-15, 1986, in the San Bernardino Mountains of southern California. One test site was located near Lake Arrowhead, California, at approximately 1500 m elevation on U.S. Forest Service property along Hook Creek Road 2N26Y, Range 2W, and Township 2N. Here-

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after, this site is referred to as Lake Arrowhead. The dominant canopy vegetation was mixed conifer, consisting of mainly Coulter and ponderosa pines with some incense cedar, white fir, and oaks. The nearest pinyon pines were estimated to be 5-7 km distant. An intensive Coulter pine thinning operation provided logging debris heavily infested with I. paraconfusus. The second site was approximately 67 km east of Lake Arrowhead near Baldwin Lake at approximately 2000 m elevation on Highway 38, Range 2E, and Township 3N. Hereafter, this site is referred to as Baldwin Lake. Here the dominant vegetation was primarily pinyon pines, oaks, Joshua trees, and cacti. Pinyon pines weakened by blackstain root disease [Leptographium wageneri (Harrington and Cobb)] (Cobb et al., 1974) were attacked by I. confusus. The nearest ponderosa and Jeffrey pines, potential hosts to L paraconfusus, were estimated to be 2-4 km distant. Coulter pines are not present at this locality. Both areas were monitored for bark beetle activity using multiple-funnel traps (Lindgren, 1983) baited with 1. paraconfusus males tunneling in ponderosa pine. These traps consistently caught approximately 16 beetles/day for three days. Ipsparaconfusus in Coulter pine were collected 5 km north of Lake Arrowhead, and L confusus in pinyon pine were collected at Little Pine Flat, 17 km east of Baldwin Lake. Four living pinyon pines and two Coulter pines, 20-25 cm in diameter, were felled, and 24 hr later, cut into sections each providing 2000 cm 2 surface area. Males of each species were paint-marked on the elytral declivity (which had no apparent effect on beetle behavior) and were singly confined using 2 x 2-cm aluminum screen stapled over artificial entrance tunnels formed in host and nonhost pines. There were 20 logs in both areas, five replicates of each treatment, each containing 25 males: (1) I. confusus on pinyon, (2) L confusus on Coulter, (3) L paraconfusus on pinyon, and (4) L paraconfusus on Coulter pine. After 24 hr, each log had 20-25 beetles producing frass. Logs were assigned randomly over a grid pattern in a fairly uniform area of forest with each position separated from another by at least 25 m. Following placement, the small aluminum screens were removed and the logs were monitored every other day for fresh attacks, termed "volunteers." After five days, the bark and phloem were cut and removed near the nuptial chamber. All adults were identified and galleries were recorded by tracing them onto clear acetate sheets. Test logs not dissected in the field were wrapped in aluminum screen, returned to Berkeley within 48 hr, and placed in cold storage. One hundred ninety-eight heads were examined by SEM (method described in Cane et al., 1990a), approximately 20 from each treatment at each site and 20 beetles captured as emergent adults from infested host trees at each site. Because a randomized block design was used where each block contains four treatments each with five logs at each of the two sites, the data from each treatment were analyzed using an ANOVA for randomized complete blocks (SAS, 1982). When the test indicated significant differences, all pairs of treatments were compared

INTERSPECIFIC PAIRING IN

Ips SPECIES

1425

by using the Studentized range test with Bonferroni's inequality (Bedard et al., 1980). Laboratory tests were analyzed using Bonferroni's inequality. All tests were made at the 5 % level of significance.

Laboratory Behavior Tests Experimental logs were placed in exclusion chambers measuring 1 m in each direction, producing an atmosphere which was assumed to be saturated with pheromones. Pinyon and Coulter pine hosts, stored at 5~ for one month, were cut into sections each providing 2000 cm 2 surface area. Factitious entrance tunnels were made in log sections using a drill, and beetles were confined in tunnels using 2 x 2-cm aluminum screen cages. All beetles used were uniquely paint-marked. At the terminus of the experiments, logs were quick-frozen at - 3 0 ~ and dissected. Beetle identity, presence of an egg gallery, number of eggs and larvae, gallery length, and female and male position in the galleD, were recorded on clear acetate sheets. Displacement of Males in Nuptial Chambers. If female discrimination is important and if females are to reliably recognize a male's species status solely by the pheromone in his frass, which is produced during his excavation of the nuptial chamber, then males should remain associated with their nuptial chambers. If a heterospecific male usurps a nuptial chamber excavated by another male, unproductive pairings can occur. To test the hypothesis that males can displace resident males in nuptial chambers, 25 males of one beetle species were placed in a nonhost and 50 males of another beetle species that normally colonizes that host were released in the same chamber. After six days, these logs were removed from the boxes and external beetles were retrieved. The identity of the beetle occupying the nuptial chamber was observed. Specific Mate Recognition with Host Discrimination. Conspecific pheromones emanating adjacent to heterospecific entrance tunnels may confound discrimination. To test this hypothesis, we examined the ability of beetles to discriminate conspecifics from heterospecifics when they were placed together in a host within an atmosphere saturated with pheromones of both species. Twenty each of L confusus and L paraconfusus were confined in one pinyon pine log and 20 each of both species were placed in one Coulter pine log, so that each male's nearest neighbor was a heterospecific. After 36 hr aluminum screen cages were removed from male entrance tunnels and both logs were placed in a box with 45 female L confusus and 45 female L paraconfusus. After six more days, the logs were removed and dissected. Specific Mate Recognition with No Host Choice. Our field tests indicated that L confusus females had a preference for their host. By eliminating host choice, we hoped to test conspecific discrimination only. We used one pinyon log only and 20 L confusus and 20 L paraconfusus males introduced into arti-

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ficial entrance tunnels for 36 hr so that each male's nearest neighbor was a heterospecific. Next, the small aluminum screen cages were removed and the log was placed in a chamber with 45 L confusus females. After six days, the log was frozen and dissected. Heterospecific Females Mixed in Galleries. Conspecifics and heterospecifics may segregate when both species are plentiful, but join heterospecific males when populations are limited, because there may be an optimum number of beetles that improves the success of the family gallery (Kirkendall, 1983). To test this hypothesis, 20 L paraconfusus males were introduced into artificial entrance tunnels in a Coulter pine log and placed in a separate chamber. Twenty L confusus males were similarly placed in a pinyon pine log. After 36 hr the confining cages were removed and 15 females of each species were released into each chamber. After nine days the logs were frozen and dissected.

Females Leaving Conspecific Males and Pairing with Heterospecific Males. In the laboratory, interspecific-matings between these two species produce inviable eggs (Lanier, 1970). In the field we observed several males with heterospecific females producing larvae. One explanation for these observations may be that conspecific-inseminated females had joined the heterospecific male. To test this hypothesis, 20 male L paraconfusus and 20 male I. confusus were confined in entrance tunnels on their hosts and placed in separate chambers for 36 hr. Conspecific females were introduced, one per day, into each male gallery until each male had accepted three females. Two days after this, a log containing only heterospecific males in their host was placed into each box. Therefore, females recovered from these nonhost logs containing heterospecific males could only come from the host logs containing conspecific males. To bracket the 14day period that females may leave their egg galleries and possibly join heterospecifics, two other similar nonhost logs containing heterospecific males were added after 10 more days had elapsed. All logs were frozen and dissected. RESULTS

Field Tests Measurements of 10 striae of the pars stridens of 20 females that emerged from Coulter pine collected at Lake Arrowhead ranged from 0.42 to 0.49 #m (X = 0.46). For 19 females emerged from pinyon pine collected at Baldwin Lake, the measurements ranged from 0.56 to 0.69/~m (X" = 0.63). These data generated a size range of the striae for both species (Table 1). Lake Arrowhead appears to be almost exclusively an L paraconfusus population, with only 2 % L eonfusus present. Here only two of 79 females removed from the treatment logs were found with striae widths that did not fit the size ranges of either L confusus or L paraconfusus that had emerged from their respective hosts. We

INTERSPEC1FIC PAIRING IN

[ps SPECIES

1427

TABLE 1. IDENTITIES OF FEMALE

[ps COLLECTED

IN THE SAN BERNARDINO

MOUNTAINS a

SEM Striae Measurements

1. paraconfusus Treatment

L Confusus

(range 0.42-0.49 #m)

Unidentified (range 0.51-0.54 ~zm)

(range 0.56-0.69 ~m)

20

0

0

19

1

0

20

0

0

17

1

2

20

0

0

14

6

0

2

3

14

3

3

13

4

0

14

0

0

19

Lake Arrowhead

L paraconfusus in Coulter pine

L confusus in Coulter pine

L paraconfusus in pinyon pine

1. confusus in pinyon pine

L paraconfusus in Coulter pine/' Baldwin Lake

L paraconfusus in Coulter pine

1. confusus in Coulter pine

L paraconfusus in pinyon pine

L confusus in pinyon pine

L confusus in pinyon pineh

"Identification is based upon the width of 10 striations of the supracephalic stridulatory organ (pars stridens) of the female. Beetles were removed from experimental logs placed in the field or emerged from logging debris collected on site. Beetles within the range 0.51-0.54 #m could not be confidently identified and were designated as unidentified. J'Collected from naturally infested logging debris on site.

classed these f e m a l e s as " u n i d e n t i f i e d . " A m i x e d species population was present at B a l d w i n L a k e with a ratio o f about t w o L confusus to one L paraconfusus and 15 % o f the f e m a l e lps p o p u l a t i o n at B a l d w i n L a k e w e r e in the " u n i d e n t i f i e d " size range. T h e m e a n n u m b e r o f m a l e s that f o r m e d nuptial c h a m b e r s ranged f r o m 2 1 . 4 to 2 4 . 4 for the 25 artificial entrance tunnels at both test sites. A nuptial c h a m b e r was f o r m e d if the e n g r a v i n g on the surface o f the s a p w o o d was similar in size and shape to o t h e r nuptial c h a m b e r s that y i e l d e d a successfully j o i n e d male. At L a k e A r r o w h e a d , f e w e r L confusus in p i n y o n pine p r o d u c e d frass than the o t h e r treatments and at B a l d w i n L a k e f e w e r L paraconfusus p r o d u c e d frass in C o u l t e r

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p i n e t h a n t h e o t h e r t r e a t m e n t s , but t h e s e d i f f e r e n c e s w e r e not significant. A t B a l d w i n L a k e f e w e r I. paraconfusus a n d I. confusus t u n n e l i n g in p i n y o n prod u c e d frass t h a n t h e s e s p e c i e s t u n n e l i n g in C o u l t e r p i n e . T h e s e d i f f e r e n c e s w e r e significant, I n t e r e s t i n g l y , m o r e nuptial c h a m b e r s w e r e a b a n d o n e d in p i n y o n pine t h a n in C o u l t e r p i n e at b o t h sites. A t L a k e A r r o w h e a d , I. paraconfusus f e m a l e s j o i n e d all t r e a t m e n t s . H o w ever, treatments with Coulter pine were significantly different from treatments w i t h p i n y o n p i n e ( T a b l e 2). T h u s I. paraconfusus f e m a l e s a p p e a r to b e a b l e to d i s c r i m i n a t e t h e i r h o s t . A t B a l d w i n L a k e , n o I. confusus f e m a l e s j o i n e d m a l e

TABLE 2. SUMMARY OF OBSERVATIONS" ON FEMALES FLYING TO AND PAIRING WITH 25 MALES OF L paraconfusus AND L conftAsl,IS FACTITIOUSLY INTRODUCED INTO COULTER AND PINYON PINE, SAN BERNARDINO NATIONAL FOREST, JUNE 1986 Species of male factitiously introduced into logs Coulter pine Observations Lake Arrowhead Nuptial chambers produced Nuptial chambers abandoned Frass production after 2 days Available males unjoined Available males paired with females Volunteers Baldwin Lake Nuptial chambers produced Nuptial chambers abandoned Frass production after 2 days Available males unjoined Available males paired with females Volunteers

Pinyon pine

L paraconfusus

1. con[usus

L confusus

L paraconfusus

24.4 ___ 1.3a

24.0 _+ 2.2a

21.8 + 2.9a

23.2 + 2.1a

2.0 + 4.5a

1.4 • 3.1a

5.8 • 4.0a

5.0 • 4.8a

22.4 _+ 1.1a

22.2 _+ 2.1a

19.2 +_ 3.1a

20.4 _+ 2.1a

3.0 _+ 2.6a

2.0 + 1.6a

4.2 + 3.6a

4.0 +_ 2.9a

19.6 • 6.4a 18.4 • 3.7a

19.8 • 5.5a 13.4 • 8.5a

11.2 _+_4. lb 5.0 • 3.5b

13.4 • 7.2b 4.6 • 3.1b

23.4 • 1.3a

23.4 _+ l.la

24.0 + 0.7a

21.4 • 2.7a

3.6 • 2.1a

3.6 __+2.3a

8.4 • 2.8b

8.2 • 1.3b

16.6 • 6.8a

17.6 + 4.4a

21.4 + 2.6b

19.8 • 2.2b

15.6 • 3.7a

i5.2 • 3.8a

9.8 • 2.2b

9.0 • 6.0b

3.6 + 2.3a 2.4 • 2.7a

3.8 • 3.8a 2.2 + 2.3a

6.0 • 2.2a 2.6 • 1.7a

4.0 • 3.4a 3.6 +• 2.7a

"Means followed by different letters in rows are significantly different (P < 0.05) using ANOVA for randomized blocks.

INTERSPECIFIC PAIRING IN Ips SPECIES

1429

L paraconfusus in Coulter pine (Table 1). However, L paraconfusus females clearly preferred conspecific males in their host Coulter pine. The remainder of I. paraconfusus and a small number of "unidentified" females joining males were distributed almost equally among the other three treatments. At Baldwin Lake, Ips confusus females appeared to be capable of discrimination of the combination of L paraconfusus in Coulter pine from all other host-species combinations. There were no significant differences in mean number of females recovered per male (Table 3). At Lake Arrowhead, the mean number of egg galleries in pinyon pine was different (i.e., lower) from that in Coulter pine, due to the reduced female joining observed in pinyon pine (Table 2). Egg gallery length in pinyon pine with male L confusus and L paraconfusus also was reduced compared to other treatments in Coulter pine. At Baldwin Lake, mean egg gallery length in the treatment with Coulter pine and L paraconfusus females was reduced. There were significant differences between abandoned nuptial chambers in both pine species at Baldwin Lake. More than twice as many nuptial TABLE 3. SUMMARY OF OBSERVATIONSa ON EGG GALLERIES FORMED BY FEMALES OF L

paFacOt'lfblSblS AND I. COI'lfUSI~SIN COULTER PINE AND PINYON PINE, SAN BERNARDINO NATIONAL FOREST, JUNE 1986

Coulter pine Observations Lake Arrowhead Males paired with females Eggs Larvae Females Egg galleries Gallery length (cm) Volunteers Eggs Baldwin Lake Males paired with females Eggs Larvae Females Egg galleries Gallery length (cm) Volunteers Eggs

L paraconfitsus 19.6 17,6 8.6 2.1 2.5 5.8 18.4 2.2

Pinyon pine

L confusus

L confusus

1. paraconfusus

• 6.4a • 5.2a • 8.3a _+ 1.0a • 0.7a • 2.8a _+ 3,7a • 0.9a

19,8 17.4 3,80 1.2 2.4 4.6 13.4 2.3

• 5.5a • 6.5a • 5.0ab _+ 0,% • 0.8a _+ 1,5a • 8.5a • 0.7a

11.2 • 4. lb 10.8 + 3.7a 1,6 • 1.5b 1.8 __ 1.5a 1.4 • 0.8b 3.6 • 1.5b 5.0 • 3,5b 1.6 + 1,2b

13,4 • 7.2b 11.0 -4- 7.7a 3.8 • 4.2ab 1.2 + 0.9a 2.2 _+ 0.7b 3.6 • 1.6b 4.6 • 3.1b 1,3 _+ 0.9b

3.6 _+ 2.3a 1.4 • 1.5a 0a 1,4 • 0.7a 1,5 • 0.8a 2,3 • 0.% 2.4 _+ 2.7a 0a

3.8 2.8 1.0 1,3 1.3 5.0 2.2 0.6

• 3.8a • 3.0a • 1.0ab • 0.5a • 0,5a _+ 4.0b • 2.3a _+ 0.7a

6.0 4.2 1.8 1.2 1,5 5,3 2.6 0.8

4.0 3.0 0.20 1.2 1,3 4.7 3.6 0,3

• 2.2a _% 3.1a • 2.5b • 0.7a • 0,7a • 2.7b • 1.7a + l.la

• 3.4a • 4,2a ___ 0.5a • 0.7a + 0.7a + 2.6b _+ 2,7a • 0.7a

"Means followed by different letters in rows are significantly different (P < 0.05) using ANOVA for randomized blocks.

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Fox E'r AL.

chambers were abandoned in pinyon pine than in Coulter pine. These differences were not observed at Lake Arrowhead. Male "volunteer" attacks were significantly different between sites (Table 2). There were no significant differences between volunteers on Coulter and pinyon pine at Baldwin Lake. At Lake Arrowhead, there were significant differences between Coulter and pinyon pine, where Coulter pine was more frequently attacked and more eggs were produced (Table 3).

Laboratory Tests Displacement of Males in Nuptial Chambers. Heterospecific displacement was observed in only two nuptial chambers. These were in artificial entrance tunnels that displayed no frass after 24 hr. Generally males of one species do not displace males of another species from nuptial chambers. Specific Mate Recognition with Host Discrimination. The mean number of nuptial chambers for each species was 16.25 (range 14-18). An average of 11.25 females (range 7-20) were distributed among these nuptial chambers. There were no significant differences between species of females joining males and pines in each treatment. However, there was a significant difference for females joining host, i.e., more females joined males in Coulter than males in pinyon pine. In pinyon pine, I. confusus males were not joined by I. paraconfusus females. Five L paraconfusus males in pinyon were joined by L confusus females but they produced no larvae. In Coulter pine, two males of each species were joined by heterospecific females, but these crosses yielded no larvae. Specific Mate Recognition with No Host Choice. Six female I. confusus joined male I. confusus and six female L confusus joined male L paraconfusus when both male species were placed in pinyon pine. Four egg galleries with eggs and larvae were formed by female I. confusus paired with I. confusus males. Two egg galleries were formed, one with larvae, when L confusus females joined L paraconfusus males in pinyon pine. Heterospecific Females in Mixed Galleries. Two of 11 galleries fonaaed by L confusus males in pinyon pine contained only I. paraconfusus females but no galleries contained L confusus and L paraconfusus females together. The remaining male galleries contained conspecific females. For male L paraconfusus in Coulter pine, there were no families with I. confusus females only. Four male galleries contained L confusus and L paraconfusus females together. One male gallery had only conspecific females. There were no differences in numbers of single versus multiple gallery systems found in pinyon pine treatments; five galleries had single females and six galleries had two or more females. In Coulter pine eight male galleries contained multiple females and two contained single females. Galleries more often contained heterospecifics than conspecifics.

INTERSPECIFIC PAIRING IN

Ips SPECIES

1431

Females Leaving Conspecific Males and Pairing with Heterospecific Males. In all situations, we found that females left conspecifics in their host and readily paired with or were admitted by heterospecific males in the nonhosts. Seven L con~sus females joined L paraconfusus males in Coulter pine and 10 L paraconfusus females joined L confusus males in pinyon pine. These females produced eggs and larvae in gallery systems with the conspecific males, but only L paraconfusus females produced eggs and larvae in heterospecific galleries in pinyon pine.

DISCUSSION

Speciation in engraver bark beetles must be accompanied by barriers to gene flow. For synchronistic and sympatric populations of two or more species, such barriers can exist as premating mate recognition systems or reproductive isolation mechanisms. One premating mate recognition system may involve the density of striae on the pars stridens of the female stridulatory apparatus, which are different between I. paraconfusus and I. confusus. The female of either species stridulates at the entrance tunnel to the male nuptial chamber, and this may provide an opportunity to discriminate conspecifics. However, Lewis and Cane (unpublished) recently demonstrated that for these species: (1) fundamental stridulatory frequencies are not different, (2) significant differences appeared in their stridulatory tempos, but (3) males in freshly established nuptial chambers accepted stridulating heterospecific females walking on the bark surface. In 1986, SEM examinations revealed that the test area at Baldwin Lake contained a mixed population of these sibling species together with a sizable number of "unidentified" individuals. Species were indiscriminately joining with heterospecifics at both Baldwin Lake and Lake Arrowhead, except that L confusus females did not join I. paraconfusus males in Coulter pine at Baldwin Lake. The preferences of joining females in the field follow the asymmetrical prelanding patterns of beetles found by Cane et al. (1990a). Heterospecific females joined males in all of our laboratory tests, which supports our field observations; however, few gallery systems contained females of both species during these laboratory tests. Finally, larval production for heterospecific crosses can be explained by prior mating of the females (Anderson et al., 1979). From these results we concluded that there were incomplete premating reproductive barriers between these populations of I. paraconfusus and I. confusus. In the laboratory, heterospecific males do not displace other males in nuptial chambers and do not use a nuptial chamber that has been excavated by a male of another species. In the field, males likely abandon a nuptial chamber if not joined by a female. At Baldwin Lake, where pinyon pine is the only

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conifer present, L confusus males abandoned their host more frequently than the nonhost, Coulter pine. At both sites, a number of nuptial chambers initiated by experimentally introduced males were occupied by females only, or by no beetles; however, larvae or eggs were present. Adults in some bark beetle species are known to leave after oviposition is completed (Coulson et al., 1978; Schultz and Bedard, 1987). There were some beetles that exhibited strial density measurements between the size ranges of both sibling species taken from their respective hosts (Table 1). If these "unidentified" individuals represent errors in the SEM measurements, we would expect to find them distributed in all treatments. Instead, we find them mainly clustered in treatments at Baldwin Lake. At either site "unidentified" individuals did not join endemic, conspecific males in their hosts, i.e.I, paraconfusus in Coulter pine at Lake Arrowhead and L confusus in pinyon at Baldwin Lake. The unusual combinations of beetles in pines provided by our experiments may favor beetles that have intermediate strial density measurements. These beetles may be of limited number in the population. Possibly, they may be hybrids of L paraconfusus and I. confusus. A zone of sympatry for beetles but not for hosts has formed at Baldwin Lake. Blackstain root disease has weakened pinyon pines in this area, making them available for bark beetle colonization for decades (Ernest Del Rio, San Bernardino National Forest, personal communication). Surprisingly, we have not found these "unidentified" individuals or heterospecifics emerging from infested logs collected in (Table 1) or near (Cane et al., 1990a) either locations. Ancestors common to both I. confusus and I. paraconfusus may have been allopatric and different in degree of polyphagy than these species are today. These ancestors may have had their diet restricted due to the reduction in the distribution of the more mesic pines or altered due to the invasion of these areas by the xeric pinyons. Isozyme studies suggest that these beetle species diverged hundreds of thousands of years ago (Cane et al., 1990b). Why haven't effective premating barriers arisen and spread from the regions of host sympatry, given the reproductive costs incurred by heterospecific parents in "fatal matings" (Kirkendall, 1983)? Perhaps the ecotones shared by the beetles' hosts have always been geographically limited (Wells and Berger, 1967). If discriminating premating behaviors were of some selective disadvantage outside of sympatry, such as mistaken rejection of conspecific mates, and there was limited gene flow into areas of sympatry (likely for these vagile beetles), then premating barriers may not be manifest. Relative to mortality caused by other factors during a generation (e.g., predators, parasites, disease, host defenses, and poor food quality), reduced fecundity due to a "fatal mating" may be rare, and of little reproductive consequence, especially if the female mates more than once (Lanier and Oliver 1966), or mates at least once with a conspecific male during her lifetime.

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The preference for the host and conspecific pheromone displayed by L

confusus may further reduce the chance of a mating mistake (Cane et al., 1990a). Ips paraconfusus females displayed little olfactory preference among conspecific and heterospecific pheromones at Lake Arrowhead; however, this species exhibited considerable preference for conspecific males in Coulter pine at Baldwin Lake. Females can avoid a nonproductive mating by rejecting the heterospecific pheromone. Yet females may encounter a conspecific mate at a resource where pheromones are produced by heterospecifics, as our tests demonstrate. When mated females join heterospecific males, they procure resources for their progeny. Although these males have not contributed genes to these offspring, they may gain a female, which contributes to the overall success of the gallery system by diminishing individual risks due to predation and parasitism (Kirkendall, 1983). If this is so, then this could be why ancestral semiochemical recognition has persisted. Genes leading to the cytoplasmic incompatibility accounting for the postmating reproductive barriers may be more flexible than genes for premating barriers and set by genetic drift (Paterson, 1981; Wiley, 1978). For engraver beetles in southern California, reinforcement of postmating isolation by premating isolation has not played a significant role in the production of separate species (Sanderson, 1989), Acknowledgments--We wish to dedicate this manuscript to the memory of Gerry N. Lanier who pioneered this research. We are appreciative of the dedicated field and laboratory work of Dr. Margaret E. Silliker, Dr. Stepbanie Meyers, Lester Casher, and Linda Kervin. We thank Laura A. Merrill lbr her review of the manuscript. This research was made possible through NSF grant BSR8415910 (D.L.W., J.H.C., Isao Kubo, University of California Berkeley; Molly Stock, University of Idaho) and USDA/SEA Regional Research Project W-110.

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