Genetics of geographic variation in pheromone and

Genetics o f geographic variation in pheromone and life history traits of the pine engraver beetle, Ips pini (Coleoptera Scolytidae)

by

Michael J. Domingue

A dissertation submitted in partial fulfillment o f the requirements for the Doctor o f Philosophy Degree State University o f New York College o f Environmental Science and Forestry Syracuse, New York August 2004

Approved: Faculty o f Environmental and Forest Biology

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iphen A.yfeale, Major Professor

Edwin H. W aite , Chair, Examining Com m ittee

Neil H. Ringler, Faculty Chair

Dudley J. Raynal, Dean, Instruction and Graduate Studies

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Acknowledgements I would like to thank Dr. Stephen Teale for introducing me to the subject o f Chemical Ecology, and encouraging my interest in the evolution o f insect pheromone systems. I am also especially grateful to Dr. William T. Starmer, who provided critical assistance in developing the statistical approach for my analyses, and contributed to manuscripts. He has also stimulated my continued interest in population genetics. Members o f my steering and examination committees, including Dr. Roy Norton, Dr. Scott Pitnick, Dr. James Gibbs, and Dr. Charles Maynard are thanked for their unique contributions to my educational and professional development. There are several individuals whose assistance in collecting Ips p in i bark beetles made this work possible. Dr. Anthony Cognato o f Texas A&M University provided specimens from Washington and California. Dr. Mary Reid o f the University o f Calgary granted me access to infested host material from Alberta, which served to initiate a laboratory colony. Emile Begin o f the Invermere Forest District o f British Columbia, assisted in locating field sites in eastern British Columbia. Dave Fomess o f the Cortland branch o f the New York Department of Environmental Conservation helped in the location o f field sites in New York. Additional thanks are given to the SUNY-ESF committee for the LeRoy C. Stegeman Award in Invertebrate Ecology. Funding provided through this award allowed travel to Alberta and British Columbia for field collection o f I. pini and attendance at an annual meeting o f the American Society o f Naturalists.

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Host material was collected primarily from two locations. I’d like to thank Scott Saroff for access to his private land for this purpose. Dick Schwab also facilitated the use o f college property at Heiberg Forest for collecting host material. Dr. Daniel Miller o f the USDA Forest Service allowed me to republish portions o f a copyrighted work in Figure 2-2 o f this document. Special thanks are due to all o f my colleagues whose laboratory cooperation was essential to completing this work. Dr. Dariusz Czokajlo and James Warren helped to introduce me to procedures for rearing bark beetles, as well as gas chromatography and mass spectrometry. Dr. Tomoyo Sakata provided essential assistance with instrument maintenance and operation. Jennifer Lund and Michael Bohne helped to collect lab-reared insects. I’d also like to thank Joseph Francese and Jacob Wickham for assisting in innumerable aspects o f laboratory work that facilitated this research. Also o f importance have been Upstate Freshwater Institute and SHIVA Technologies o f Syracuse. Both organizations were flexible in providing full and part time employment that allowed me to pursue this degree. I’d finally like to express gratitude toward my family for their support and encouragement throughout the process o f completing this dissertation. My parents John and Jane Domingue are responsible for providing the educational experiences, financial support, and personal guidance that led me to pursue this career. Most especially I’d like to thank my wife Joann, who has been an unwavering source o f support, understanding, and inspiration throughout the course o f my graduate studies.

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To my wife, Joann and to my daughters, Lucia and Fiona

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Table of Contents Acknowledgements...........................................................................................i List of Tables................................................................................................... vi List o f Figures................................................................................................. xi A bstract..........................................................................................................xiii Chapter 1: Introduction to geographic variation in pheromone biology and life history o f the pine engraver beetle, Ips pini................. 1 Chapter 2: Major gene control of a male-produced aggregation pheromone that shows a pattern o f character displacement across a hybrid zone..................................................................................15 2-1. Introduction...................................................................................................... 15 2-2. Materials and M ethods....................................................................................19 2-3. R esults...............................................................................................................31 2-4. Discussion......................................................................................................... 38

Chapter 3: Genetic architecture o f pheromone blend in pine engraver beetle populations distant from a hybrid zone...................... 63 3-1. Introduction......................................................................................................63 3-2. Materials and M ethods................................................................................... 67 3-3. R esu lts...............................................................................................................80 3-4. Discussion......................................................................................................... 86

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Chapter 4: Multivariate analysis of life history traits in response to inbreeding in engraver beetles, Ips p in i: Severe inbreeding depression in developmental survival............................ ..................... 111 4-1. Introduction.................................................................................................... I l l 4-2. Materials and M ethods..................................................................................114 4-3. R esults............................................................................................................. 124 4-4. Discussion........................................................................................................129

Chapter 5: General Conclusions....................

147

Literature C ited .............................................................................................155 Curriculum Vitae.......................................................................................... 176

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List of Tables Table

Page

2-1. Number of crosses in all line cross experiments. For BC, the crosses deriving from the independently selected low (+)-ipsdienol lines are separately noted as a and b ............................................................................... 49 2-2. Proportion o f (+)-ipsdienol in the P+, P'a, and P'b lines for the three generations prior to crossing in the BC experiment (mean +/- SD). Data include measurement o f all male siblings in the broods used for further line propagation. P+represents all broods containing individuals from eight lines, while P'a and P'b both represent one line.............................................................................................................................. 50 2-3. Estimates o f parameters for the best model for each line cross experiment. The effects include mean (m), additive (d), dominance (h), X-linked (dx), additive by dominance (j), and dominance by Xlinked (h.dx) ............................................................................................................... 51 2-4. Averaged resampled line means, standard errors o f the line means, sample sizes, and predicted means for the best model for the British Columbia experiment..................................................................................52 2-5. Averaged resampled line means, standard errors o f the line means, sample sizes for each line, and predicted means for the best model for the CA x WA line cross experiment................................................................. 53

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2-6. Averaged resampled line means, standard errors o f the line means, sample sizes, and predicted means for the best model for the AB x NY line crosses.........................................................................................................54 2-7. The X2 values for comparisons o f the ratios o f (+)-ipsdienol: (-)ipsdienol phenotypes to typical Mendelian ratios in British Columbia F I, F2 and backcross progeny..............................................................55 2-8. Castle-Wright estimators o f number o f effective segregating loci for the BC, AB/NY, and CA/WA line cross experiment.......................................... 56 3-1. Number o f crosses and male offspring collected (n) for the CA x NY crosses. P+ and P' were taken directly from the colonies....................................95 3-2. Expected phenotypic ratios (low/high (+)-ipsdienol) arising from segregation models. These models include a simple Mendelian model (Null), and two locus models which feature the lethal effects o f recessive alleles (R1 and R2) or a translocation (T l). Each o f these models are also modified for a NY X-chromosome effect.......................96 3-3. Two locus autosomal models for lethal effects. The gamete and offspring genotypes are given for the F2, B+, and B' generations where NY beetles are ajaibibi, and all CA beetles are a2 a2 b2 b 2 . The first locus (a), controls pheromone blend, while the second (b) is involved only in lethal interactions with the first. Lethal genotypes are indicated for the two lethal recessive models (R1 & R2) and the lethal translocation model (T)................................................................................ 97

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3-4. The expected additive effects (d), dominance effects (h), and X-linked effects (dx) for nine line cross generations............................................................98 3-5. Modifications to the additive effects (d) and dominance effects (h) characteristic o f the three lethality models, R1, R2, T1. These parameters are affected in only the F2, and backcross generations.................. 99 3-6. Mean ( J i), standard deviation, ( a ) and number o f males (n) analyzed for ipsdienol enantiomeric blend and pronotal width....................... 100 3-7. Fraction o f beetles producing greater than 30% (+)-ipsdienol in line crosses. Expected fraction o f high (+)-ipsdienol phenotypes given for six different genetic models. The observed ratios high to low (+)-ipsdienol phenotypes were compared to expectations................................101 3-8. Genetic effects for line means analysis o f pheromone data for null model and lethal models, R l, R2, and T l. The effects include mean (m), additive (d), dominance (h), X-linked (dx), dominance by dominance (1), and dominance by X-linked (h.dx) ....................................... 102 3-9. Line cross parameters and means model predictions for ipsdienol enantiomeric blend. The nine-line models utilize additive, dominance and X-linked effects to predict the line means. Predicted means given for null model and lethal models, R l, R2, and T l .......................................................................................................................103 3-10. Genetic effects for line means analysis o f pronotal width for null model and lethal models, R l, R2, and T l. The effects include


mean (m), additive (d), dominance (h), X-linked (dx), additive by additive (i), and additive by X-linked (d.dx)....................................................... 104 3-11. Line cross parameters and means model predictions for pronotal width. The nine-line models utilize additive, dominance and xlinked effects to predict the line means. Predicted means given for null model and lethal models, R l, R2, and T l ................................................... 105 3-12. Castle-Wright estimators o f number o f effective segregating loci for pheromone blend and pronotal width.................................................................. 106 3-13. Summary o f phenotypic correlations for various groupings o f crosses, and pooled estimates for genetic correlation and heritability o f the two traits................................................................................... 107 3-14. Genetic effects for line means analysis o f AB x NY and BC+x BC‘ experiments with and without translocation assumptions................................ 108 4-1. Summary o f breeding design structure for comparison o f sib to outbred crosses........................................................................................................134 4-2. Regression analysis parameters for four different models. These include 1) inbreeding versus Vadults , 2) pheromone as indicator, 3) time as indicator, 4} specific generations as indicators................................135 4-3. Summary o f descriptive statistics for outbred and sib crosses using eight variables associated with each brood......................................................... 136 4-4. Univariate ANOVA and MANOYA (W ilks’ A.) summarized for eight variables using 38 sib and 38 outbred crosses.................................................... 137

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4-5. Summary o f descriptive statistics for outbred and sib crosses using only the variables gallery length, ^eggs , and Vadults . ) ................................138 4-6. Univariate ANOVA and MANOVA summarized for gallery length, tJ qggs

, and Vadults using 42 sib and 42 outbred crosses................................139

4-7. Summary o f discriminate function properties for models selected by stepwise analysis. Both eight and three variable data sets use Vadults and y/eg g s ...............................................................................................140 4-8. Summary o f analysis o f variance for pronotal width and log generation time using maximum likelihood. Significance o f each effect is evaluated by the change in likelihood statistic upon removal o f effect from the complete model........................................................141

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List of Figures Figure ................................................................................................................................. Page 2-1. Distribution o f I. pini pheromone types in North America......................................57 2-2. Summary o f ipsdienol enantiomeric composition in populations o f I. pini at a hybrid zone in southern British Columbia............................................. 58 2-3. Frequency histograms o f the pooled parental hybrid and backcross data from the BC and AB x NY line-cross experiments.....................................59 2-4. Frequency histograms for crosses in the British Columbia experiment: I. pini parental populations (A-C), FI (D-F), backcrosses to P+ (G,H), F2 (I), and backcrosses to P'a (J,K)............................................................60 2-5. Frequency histograms for crosses in the CA x WA experiment: I. pini parental populations (A-B), FI (C, D), backcross to WA (E), F2 (F), and backcrosses to CA (G,H)..........................................................................61 2-6. Frequency histograms for crosses in the AB x NY experiment: I. pini parental populations (A-B), FI (C), backcross to AB (D), F2 (E), and backcrosses to NY (F,G), and B+r crossed to NY (H )..................................62 3-1. Frequency histograms o f all FI, P+, P‘, and F2 lines pooled for pheromone blend (A) and pronotal width........................................................... 109 3-2. Frequency histograms for I. pini parental populations (A-B), FI offspring (C, D), backcrosses to P+ offspring (E, F), backcrosses to P" offspring (G, El), and F2 offspring (I)............................................................. 110

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4-1. Mean and standard deviation o f inbreeding coefficient,/ for the generations o f the British Columbia directional selection experiment that were included in the regression analyses................................ 142 4-2. Pedigree involving uncertainty about which male sibling is mated in the second generation.............................................................................................143 4-3. Residual plots for regression model o f Vadults versus inbreeding coefficient, before (A) and after (B) weighted least-squares modification. Weights (w) are derived by using the reciprocal of variance from four groupings o f the data............................................................144 4-4. Square root o f offspring versus inbreeding coefficient for crosses in the British Columbia pheromone selection experiment. Trend-line and equation represent weighted regression analysis........................................145 4-5. Sex ratio versus inbreeding coefficient for crosses in the British Columbia pheromone selection experiment. Solid trend-line represents regression using all the data. Dotted trend-line removes zero and one observations to achieve normality o f residuals........................... 146

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Domingue, Michael, J. Genetic control of geographic variation in pheromone and life history traits of the Pine Engraver Beetle, Ips pin i (Coleoptera Scolytidae). Word-processed and bound dissertation, 148 pages, 30 tables, 12 figures, 2004. A bstract

North American pine engraver beetles, Ips pini (Say) (Coleoptera: Scolytidae), exhibit two distinct pheromonal types. Western populations utilize predominantly 7N(-)-ipsdienol, while eastern populations utilize higher proportions of S -(+}-ipsdienol and lanierone. A cline between types exists in British Columbia. We examined two aspects o f the biology o f Ips pini related to geographic patterns of genetic variation in the species. First we performed line crosses between populations to assess geographic variation in pheromone blend genetic architecture. We employed line means analysis, and estimated the number o f effective factors. Furthermore, in one set o f crosses (California x New York) we modified the means analysis using lethal effects. Secondly, we examined the impact of inbreeding on brood failure and number o f adult offspring in NY and BC. In NY, we also measured inbreeding effects on egg laying, pronotal width, development time, and sex ratio. Line cross experiments consistently revealed few effective factors separating any two given populations. In line means analysis o f crosses between opposing variants from the hybrid zone, and between CA and NY populations, a model that includes autosomal additive and dominance effects, X-linked effects, and epistasis best explain the results. These results suggest that at least two autosomal loci and one X-linked locus contribute to pheromone blend differences. However, segregation is visibly strong

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and one autosomal locus may explain most o f the variation. Compared to crosses between variants from within the hybrid zone, those between the more distant populations (CA x NY) yielded a more complicated genetic architecture, including the possibility o f a lethal factor. Complementation crosses between W ashington and California, and Alberta and New York populations reveal no transgressive segregation. However there are significant autosomal additive and dominance, and xlinked factors that separate the latter populations. We observed significant inbreeding depression in the adult offspring emergence in the NY and BC populations. The results suggest localized evolution o f genetic architecture for pheromone blend, while effective population sizes are not small enough to purge alleles that cause strong inbreeding depression.

KEY WORDS: Ips pini, ipsdienol, pheromone, evolution, inbreeding, reinforcement, generation means analysis, GC-MS. Author's name

__________________ Michael J. Domingue______________________

Candidate for the degree o f Major Professor

Doctor o f Philosophy

Date

August 2004

______________________ Stephen A. Teale______________________

Faculty

Environmental and Forest Biology______________

State University o f New York College o f Environmental Science and Forestry, Syracuse, New York Signature o f Major Professor

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Chapter 1: Introduction to geographic variation in pheromone biology and life history of the pine engraver beetle, Ips pini.

By their very definition, hybrid zones demonstrate incomplete reproductive isolation (Arnold 1997). The distribution o f organisms along a hybrid zone may be the result o f secondary contact o f isolated species (Anderson 1977; Barton and Hewitt 1989; Harrison 1990). Under such circumstances it has also been proposed that reinforcement o f premating isolation occurs after secondary contact (Dobzhansky 1940, 1951; Mayr 1963). There has been much historical debate about the role of reinforcement in preventing hybridization and completing the speciation process (Patterson 1978, 1993; Carson 1986; Spencer etal. 1986; Butlin 1987a, 1987b, 1989; Giddings et al. 1989; Otte and Endler 1989). Alternatively, shifting balance theory suggests that adaptive traits can develop in different locations in geographically widespread populations (Wright 1932). These adaptive traits may then spread through the overall population until areas o f low population density or geographic discontinuity inhibit further spread, creating a cline for the particular traits (Barton and Hewitt 1989). This framework o f population genetics has been influential in the formation of three major models to explain hybrid zone dynamics. Under the “tension zone model” (Barton 1979a, 1979b; Barton and Hewitt 1985, 1989) hybrid zones are maintained by a balance between selection against hybrids and dispersal into areas o f contact. Assuming this model, clines should be steep, parallel, and marked by significant

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linkage disequilibria (Barton and Gale 1993) and cytonuclear disequilibria (Arnold 1993a). This model has been proposed for the toad species Bombina bombina and B. variegata, for which differences in mating calls, color, preferred habitat, enzymes and mitochondrial DNA have been described (Szymura and Barton 1986, 1991; Szymura 1993). The “bounded hybrid superiority model” assumes that hybrids have higher fitness than their parents do in certain environments (Moore 1977). Such a pattern has been documented in Darwin’s finches (Grant and Grant 1992). Finally, in the “mosaic model”, hybrid zones can be described as a patchy distribution o f genotype frequencies due to genotype/habitat interactions (Howard 1986; Harrison 1990).This model has been postulated for an Iris hybrid zone (Arnold 1993b; Cruzan and Arnold 1993, 1994) and a fire ant hybrid zone (Shoemaker et al. 1996). Sexual selection theory is often relevant to mechanisms for generating and maintaining the levels o f reproductive isolation seen at hybrid zones (Lande 1981; Carson 1986). The coevolution o f female mating preferences and male secondary sexual characteristics can cause the evolution o f divergent signaling systems that facilitate speciation (Fisher 1915, 1930). Male pheromone signals and female pheromone preference could evolve rapidly by means o f such runaway processes (Phelan 1992). All sexual selection models could be relevant to the divergence seen at hybrid zones, including runaway selection (Fisher 1930), indicator (Williams 1966; Hamilton and Zuk 1982), or direct benefits models (Heywood 1989; Hoelzer 1989). Also crucial to understanding the nature o f hybrid zones is the number of genes controlling population differences in reproductive isolating traits. Traditional

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population genetic theory assumed that most meaningful evolution proceeds by the accumulation o f small genetic changes (Dobzhansky 1937; Fisher 1958). This position has been challenged in the past two decades (Kimura 1983; Orr and Coyne 1992; Coyne et al. 1994). With regards to the number o f genes controlling aspects o f pheromone communication, a study o f the European com borer, Ostrinia nubilalis, determined that female-produced pheromone blend is under the control o f one gene, while male preference is controlled by two genes (Roelofs et al. 1987). Recently Roelofs et al. (2002) showed that a pheromone component in Asian Com borer, O. furnacalis, is controlled by an enzyme that is produced by a gene normally inactive in Ostrinia species. Conversely O. furnacalis has an inactive pseudogene for an enzyme used to produce pheromone components o f O. nubilalis. The assumption o f major gene control o f pheromone blend production might not apply to systems where males signal for mates. Generally, female-signaling as observed in moths does not involve the intense social competition that is assumed in most sexual selection models (West-Eberhard 1982; Phelan 1992). Male signaling and response systems may be more likely to involve some form o f sexual selection. Many sexual selection models expect incremental changes at multiple loci, because large changes in pheromone signal would have an overwhelming effect on male fitness. Indeed in Orthopteran systems where males call for female mates, acoustic signals (Butlin and Hewitt 1988; Mousseau and Howard 1998) and response (Shaw 2000) are quantitative traits under polygenic control.


Our research employed studies o f the pine engraver beetle, Ips pini (Coleoptera: Scolytidae) to explore the genetics o f pheromone production at a hybrid zone. Ips pini has a number o f characteristics that make it ideal as a model system. Its hybrid zone is characterized by differences in male production o f an aggregation pheromone (Figs. 2-1, 2-2). Within a population outside the hybrid zone, pheromone blend was shown to be correlated with response preference (Hagar and Teale 1996). In the same population, Hager and Teale (1996) showed that the positive correlation between pheromone production and response is broken down after several generations o f forced random mating o f Ips pini, indicating that assortative mating builds this correlation. Sexual selection could be responsible for the genetic correlation between pheromone production and response in this population, and incremental evolution o f pheromone blend diversity seems plausible. At the same time, there may be strong natural selection via interactions with predators and competitors (Raffa and Klepzig 1989). In such case, major pheromone shifts may be more likely if the risks posed by natural enemies are dramatically reduced. Biology o f I. pini Ips pin i (Say) breeds in pines (Pinus spp.) and spruces (Picea spp.) throughout the boreal and montane forests o f North America (Fig 2.1). The biology o f I. pini is typical for the genus (Wood 1982a). Males locate hosts in a manner that is largely unknown, but in lodgepole pine (Pinus contorta var. latifolia) forests in British Columbia, the host monoterpene P-phellandrene has been implicated as a primary attractant (Miller and Borden 1990). Upon locating and boring into a suitable host,

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males release a pheromone, which initiates an aggregation (Anderson 1948). The pheromone is released in the fecal pellets, which are expelled with the boring dust as the males excavate mating chambers in the phloem (Pitman et al. 1965). On average, three to five females enter each male chamber, mate, construct galleries in the phloem, and oviposit. Larvae develop to adulthood within the phloem. One to five generations can occur per year, depending on the length of seasonal temperatures. Beetles over-winter under the bark or in the duff as adults (Shenefelt and Benjamin 1955). Intraspecific variation in I. pini Early taxonomists recognized two species (e.g., Swaine 1918), but the discovery o f clinal variation in morphological characters (Hopping 1964) and an ability to interbreed (Lanier 1972) led to the conclusion that this was a single geographically polymorphic species. Morphological characters (Hopping 1964) and pheromone biology (Lanier et al. 1972, 1980; Birch et al. 1980) distinguish eastern and western populations. More recent investigations into the geographic variation in the pheromone system suggest that there is a contact zone between the eastern and western populations in southern British Columbia (Miller et al. 1989, 1996; Seybold et al. 1995a). The contact zone is characterized by differences in the ratio o f pheromone components produced by males (Miller et al. 1996). However, morphological differences intergrade from east to west and no discontinuity is discemable in and around the hybrid zone (Hopping 1964; Lanier 1972).

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Partial sequences of the mitochondrial cytochrome oxidase 1 (mtDNA C O l) provide the only known potential marker for distinguishing geographic variants of Ips pini (Cognato et al. 1999). Phylogenetic analysis o f 34 haplotypes characterized from populations throughout North America revealed three mitochondrial lineages. One lineage is found in eastern populations, and in some western populations. The other two lineages are found only in western populations. Other markers specific to eastern and western variants have not been discovered. Microsatellite allele variation would provide better diagnostic markers. These hypervariable short tandem nucleotide repeats are scattered throughout the nuclear genome o f most organisms, which provides great potential for many diagnostic loci (Beaumont and Bruford 1999). Indeed, microsatellite loci have been revealed for I. pini. (Cognato, pers. comm. 2003). The potential for many variable loci might increase the ability to diagnose the pheromone races. Pheromone biology The pheromone is a mixture o f the terpene alcohol, ipsdienol (2-methyl-6methylene-2,7-octadien-4-ol) (Birch et al. 1980; Lanier et al. 1980) and lanierone (4,4,6-trimethyl-2-hydroxy-2,5-cyclohexadiene-l-one) (Teale et al. 1991). Gries et al. (1988) reported that A-myrcenol (2-methyl-6-methylene-2,7-octadien-3-ol) is produced by I. p in i in British Columbia, but its function is complex and not fully understood (Miller et al. 1990). Seybold et al. (1995b) reported the production o f amitinol (frara-2-methyl-6-methylene-3,7-octadien-2-ol) by male I. pini in California, but its function is also unknown.

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Lanier et al. (1972) showed that eastern (New York) and western (California and Idaho) populations responded preferentially to their own populations’ pheromone, and this specificity was found to have a heritable basis (Piston and Lanier 1974). This geographic variation in pheromone specificity is due to differences in use of the two enantiomers (optical isomers) o f ipsdienol, S-(+) and R-(-) (Birch et al. 1980; Lanier et al. 1980). Western populations (California and British Columbia) produce and respond to predominantly the (-) enantiomer (Birch et al. 1980; Miller et al. 1989), while eastern populations (New York) produce and respond to roughly equal proportions o f the two enantiomers (Lanier et al. 1980; Miller et al. 1989). Lanierone also plays a role in population specificity. New York populations produce 0.2% to 1% as much o f this component relative to ipsdienol, yet the synergistic action of these amounts o f lanierone results in as much as a 500% increase in attraction (Teale et al. 1991; Miller et al. 1997). In California, lanierone is not known to be produced and has only a minor effect on attraction to ipsdienol (Seybold et al. 1992; Miller etal. 1997). Individual antennal cells appear to be timed to either o f the two enantiomers o f ipsdienol (Mustaparta et al. 1980). Despite differences in proportions o f these receptors in eastern versus western populations and their hybrids (Mustaparta et al. 1985), individuals from all populations show antennal response to both ipsdienol enantiomers (Angst and Lanier 1972). It is thus likely that response preference is controlled at higher levels o f integration in the central nervous system (Mustaparta et al. 1980, 1985).

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Miller et al. (1989) and Seybold et al. (1995a) conducted studies showing that there is relatively little variation in ipsdienol enantiomeric composition within the eastern and western populations, which come together in southern British Columbia (Fig. 2-1, 2-2). Miller et al. (1996) surveyed ipsdienol production for individuals in southern British Columbia (Fig 2-2). They found one population at Kimberley, consisting o f nearly all western-type males, producing > 90% (-)-ipsdienol. North and west o f this site, most males were o f the eastern type, 80% (+)-ipsdienol. However, these population distributions exhibit very high phenotypic variability, often having a secondary peak o f 40-50% (+)-ipsdienol production. Radium, 80 km north o f Kimberley provides the best phenotypic evidence for hybridization. At that location, populations possess greater frequencies o f intermediate phenotypes and include some males with very high ratios o f (-)-ipsdienol (Fig 2-2; Miller et al. 1996). Ipsdienol biosynthesis Many Ips species produce ipsdienol de novo by using the melvonic acid pathway (Ivarsson et al. 1993; Tillman et al. 1999). There is also some evidence that the host-produced compound myrcene can be converted to ipsdienol (Hendry et al. 1980). Little is known o f the precise biochemical mechanisms by which specific blends o f ipsdienol are produced in scolytids. In I paraconfusus enantiomers o f ipsdienol are interconvertible via the intermediate ketone ipsdienone (Fish et al. 1979, 1984). Ipsdienone is related to ipsdienol by dehydration o f the hydroxyl group that determines chirality. The genes controlling ipsdienol blend may code for enzymes that bias this conversion in one direction or the other. Ipsdienol is also produced in

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the floral fragrances o f some plant species (Whitten et al. 1988), but in these cases is not involved in interactions with scolytid bark beetles. Signaling, response, and mitochondrial haplotypes Cognato et al. (1999) demonstrated that eastern 7. pini mitochondrial DNA haplotypes introgress into many western populations. However, there are no western mtDNA haplotypes east o f the hybrid zone. This pattern is consistent with a mating system where western females have more specificity in their pheromone response than eastern females. Eastern females probably hybridize with western males, with the female hybrid offspring backcrossing into the western population. Conversely, western females may be more rigid in their response behavior, very rarely hybridizing with eastern males. Unidirectional gene flow has been documented at other hybrid zones with the aid o f mitochondrial markers (Forbes and A llendorf 1991; Paige et al. 1991). When one looks at the trait o f male pheromone production near the hybrid zone, the opposite pattern emerges. Pheromone blends that are o f the opposing variety, or intermediate are more prevalent in the eastern populations directly north o f the hybrid zone than in the western populations directly south (Fig. 2-2). This pattern suggests that some nuclear genes are introgressing in the opposite direction, also due to matings between western males and eastern females, but with hybrid males backcrossing into the eastern population. Semiochemical interactions with competitors and predators At least 20 ipsdienol-producing scolytid species exist that are potential competitors o f I. pini. These species include members o f the genera Dendroctonus (1)

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(Seybold et al. 1992), Orthotomicus (2), (Giesen et al. 1984; Phillips et al. 1989), Pityokteines (2) (Harring 1978), and Ips (15) (Francke et al. 1980; Borden 1985; and Kohnle et al. 1988). The enantiomeric composition o f ipsdienol production is known for many species (Seybold 1992). Most o f these species have much smaller geographic ranges than I. pini, and have narrower host ranges. Many other members of the genus produce the compound ipsenol (Francke et al. 1980; Borden 1985; Kohnle et al. 1988; Miller et al. 1991). Ipsenol is not produced by Ips pini, and is known to inhibit host colonization by this species (Fumiss and Livingston 1979; Birch et al. 1980). Ipsenol and the ipsdienol enantiomeric composition production by other species may be invoked to explain current geographic variation in Ips p in i pheromone phenotypes. It is possible that the different geographic pheromone systems in I pini evolved to fill information channels available in the two geographic areas. For example, many potential western competitors produce (+) -ipsdienol (e.g. /. paraconfusus and I. plastographus', Seybold 1992). Likewise, one major eastern competitor, I. calligraphus produces (-)-ipsdienol (Seybold 1992). It is o f interest whether these competitors actually provide a selective force at or near the hybrid zone. For example, I. plastographus has a range extending into southern British Columbia (Wood 1982b). It is not known if the northern boundary o f its distribution directly coincides with the Ips pini hybrid zone. There are several arthropod predators that are attracted to the pheromone o f Ips pini. Important among such predators are Thanasimus dubius (Coleoptera:

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Cleridae), Tomicobia tibialis (Hymenoptera: Pteromalidae), Enoclerus sphegeus (Coleoptera: Cleridae), Cylistix cylidrica (Coleoptera: Histeridae), and Temnochila chlorodia (Coleoptera: Trogostidae) (Seybold et al. 1992 and references therein). There is evidence that some o f these predators exhibit preferences for certain pheromone phenotypes o f I. pini (Raffa and Klepzig 1989; Herms et al. 1991; Raffa and Dahlsten 1995). Some have argued that these predator preferences provide strong selective pressures that influence pheromone production in I. pini populations (Raffa and Klepzig 1989; Herms et al. 1991; Raffa 1991). Fitness consequences o f inbreeding and outbreeding Inbreeding depression is common in many outbreeding taxa (see reviews in Thornhill 1993; Husband and Schemske 1996; Lynch and Walsh 1998). In most phloem feeding scolytids such as I. pini, dispersal is a common habit, leading to expectations o f outbreeding (Kirkendall 1993). Inbreeding depression is expected in such large outbreeding populations as a consequence o f the accumulation o f deleterious recessive mutations (Davenport 1908; Bruce 1910; Haldane 1927). Furthermore, genetic variation from outbreeding may be important to phloem feeders to respond to the selective pressures inherent in their exposure to predators, parasites, and competitors. In I. pini, sib and half-sib mating likely occurs before adult emergence. Most dispersing females are inseminated before colonizing new hosts (Lissemore 1997). Their linear egg gallery pattern may allow some pre-dispersal matings to be with unrelated neighboring broods (Kirkendall 1993). At the same time, harem polygamy

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also exposes offspring to half and full sibs, which may result in some inbreeding. In laboratory conditions, females will emerge inseminated from logs containing one full sib brood (personal observation). Most o f these pre-dispersal copulations probably do not result in offspring production. Second male sperm precedence is nearly complete in I. p in i if second males have access to females for more than one week (Lissemore 1997). However, failure o f females to locate mates, or early mortality o f mates, will make occasional use o f sperm from pre-dispersal mating likely. There is some further indirect evidence that pre-dispersal copulations result in offspring production. Egg galleries are often constructed without male presence (Reid and Roitberg 1994). Many o f these cases may represent re-entry into the bark by females inseminated after dispersal, rather than direct colonization after pre-dispersal mating. However, it is clear that females will construct egg galleries without forming pair bonds. Presuming that outbreeding is the typical habit o f I. pini, it is predicted that inbreeding depression will occur in these instances. As such a cost, inbreeding depression may cause selection for ways to further minimize the use o f sperm from pre-dispersal copulations. Alternatively, it is possible that outbreeding is deleterious in particular circumstances. The demonstrated geographic variation in pheromone biology suggests genetic differences that may indicate significant population subdivision. Outbreeding may destroy co-adapted gene complexes where members o f distinct populations interact. Thus, optimal inbreeding in I. p in i may be constrained by the opposing pressures o f genetic load versus localized adaptation.

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Theoretical implications o f pine engraver research The relative evolutionary importance o f Fisherian large population dynamics versus W rightean shifting balance remains a fiercely contended issue (Coyne et al. 1997; Wade and Goodnight 1998). I. pini provides a study system where many predictions o f these models can be tested. Our studies o f inbreeding and variation in pheromone blend in I. pini will be interpreted within the context o f these opposing theoretical positions. While our experiments are not sufficient to clearly distinguish these alternatives, they may provide some insight into which view is more appropriate for this system. The Fisherian view emphasizes large homogenous populations where evolution occurs primarily by incremental genetic changes that are a response to widespread selective pressures (Fisher 1930). If two such populations meet and interact at the I. pin i hybrid zone in British Columbia, we would expect to find evidence o f genetic homogeneity within each population. In this context, higher (+)ipsdienol blends closer to the hybrid zone than in remote areas (Miller et al. 1989, 1996) could be attributed to reinforcement o f mating preferences or natural selection operating at that location. We would also expect this to be a relatively unstable cline with the trait possibly spreading from the contact zone to fixation in the larger populations (Butlin 1989), unless maintained by strong selective pressures. The large population view would also predict strong inbreeding depression within the two parent populations, because substantial genetic load is likely to accrue.

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If reinforcement is firmly assumed, we would expect the genetic system for controlling pheromone blend to be at least as complicated in populations near the hybrid zone as it is in distant populations. This prediction derives from the expectation that any new genetic variation near the hybrid zone would be the result of modifications in alleles common to the whole population, or the appearance of entirely new genes. Conversely, if reinforcement occurs, we would not expect novel genetic variation to arise distant from the hybrid zone. W right’s shifting balance theory emphasizes the importance o f processes such as genetic drift that are active in small populations, but can lead to the spread o f novel genotypes to larger geographic areas (Wright 1931, 1932). Under this view, it is possible that the current population is a mosaic o f different genetic systems for pheromone blend production, which have evolved multiple times. It may be technically incorrect to distinguish two pheromonally distinct eastern and western populations. Rather different genetic architecture for pheromone blend may have evolved in several local environments. In this case, the slight phenotypic differences in eastern and northwestern (+)-ipsdienol producing populations may have radically different genetic causes. Additionally, under shifting balance theory, inbreeding depression may be milder, because effective populations are small and lethal alleles are more effectively purged. However, shifting balance theory may not necessarily require populations to be small enough to trigger purging o f such alleles.

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Chapter 2: Major gene control of a male-produced aggregation pheromone that shows a pattern of character displacement across a hybrid zone. c

2-1. Introduction An important consideration for speciation theory is the number o f genes required for reproductive isolation. Traditional population genetic theory suggests that evolution progresses by the accumulation o f small genetic changes (Fisher 1930; Dobzhansky 1937). In the past two decades, this assumption has been challenged on theoretical and experimental grounds (Kimura 1983; Orr and Coyne 1992; Coyne et al. 1994; Coyne 1996), and the list o f cases where major genes are involved in premating isolation has grown (Ritchie and Phillips 1998). Long-range pheromone communication in insects represents a signal and response system that frequently functions as a reproductive isolating mechanism. The evolution o f changes to such systems is difficult to explain on a theoretical level because such systems are expected to be under strong stabilizing selection (Phelan 1992), meriting further investigation into the genetic control o f such systems (Butlin 1995). Ritchie and Phillips (1998) list 37 major studies o f the genetics o f sexual isolation. Nine o f these studies involved pheromones, and o f those six showed a single segregating element or “major gene” controlling most genetic variation. Single genes possibly controlled the factors involved in the other three cases o f sexual isolation.

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The insect order Lepidoptera has been most intensely studied with respect to the genetics o f pheromone production and response. Within this group, few Mendelian genes generally control differences in female pheromone production and male preference (Lofstedt 1990). Populations o f the European com borer, Ostrinia nubilalis, are marked by geographic variation in the relative proportions o f the E and Z isomers o f 11- tetradecenyl acetate utilized (Klun 1975). In that system, heterozygotes produce and prefer intermediate levels o f the two isomers. Differences in female production o f these two major pheromone components are controlled by two alleles at one locus (Klun and Maini 1979). Hybrids exhibit more variation in the ratio o f isomers produced than either parent population. Hence, there may be a second level o f genetic control o f the trait (Zhu et al. 1996). Male antennal response is also controlled by two alleles at an autosomal locus (Hannson et al. 1987), while behavioral response is controlled by a sex-linked locus (Roelofs et al. 1987). Recently, the activation o f long dormant pseudogenes have been implicated in differences between pheromone components o f Asian versus European corn borers (Roelofs et al. 2002) The present study examines the genetic control o f male pheromone production for the pine engraver beetle, Ips pini (Coleoptera: Scolytidae). The species has eastern and western populations distinguished by morphological characters (Hopping 1964) and pheromone biology (Lanier et al. 1972, 1980; Birch et al. 1980). Geographic variation in the pheromone system o f I. pini suggests that there is a hybrid zone between the eastern and western populations in southern British Columbia, lying on a

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north-south gradient (Miller et al. 1989, 1996; Seybold et al. 1995a). The hybrid zone is characterized by differences in pheromone production by males (Miller et al. 1989; Seybold et al. 1995a). Female response within the hybrid zone has not been well studied. The most intensely investigated and well-understood pheromone components o f I. pini are ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) (Birch et al. 1980; Lanier et al. 1980), and lanierone (4,4,6-trimethyl-2-hydroxy-2,5-cyclohexadiene-lone) (Teale et al. 1991). Much o f the geographic variation in pheromone specificity (Figs. 2-1, 2-2) is due to differences in use o f the two enantiomers (optical isomers) of ipsdienol, S-(+) and R-(-) (Birch et al. 1980; Lanier et al. 1980). Western populations (California to southern British Columbia) are predominantly characterized by relatively greater production o f and preference for the (-) enantiomer (Birch et al. 1980; Miller et al. 1989). Eastern populations (New York to northern British Columbia) produce and respond to blends o f ipsdienol that are racemic or higher in (+)-ipsdienol content (Lanier et al. 1980; Miller et al. 1989; Seybold et al. 1995a). New York and northern British Columbia populations differ little in their pooled (Seybold et al. 1995a) or mean (Miller et al. 1989, 1996) enantiomeric composition o f ipsdienol. However, British Columbia populations have many individual males that produce greater than 80% (+)-ipsdienol (Fig. 2-2), while population profiles o f New York males are characterized by a peak at 70% (+)ipsdienol (Fig. 2-1). Some populations in NY have shown a secondary peak near 50% (+)-ipsdienol (Miller et al. 1989).

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Lanierone also plays a role in population specificity. New York populations produce 0.2% to 1% as much o f this component relative to ipsdienol, yet the synergistic action o f these amounts o f lanierone results in as much as a 500% increase in trap catches (Teale et al. 1991; Miller et al. 1997). In California, lanierone is rarely produced and has only a minor effect on attraction to ipsdienol (Seybold et al. 1992; Miller et al. 1997). To investigate geographic variation in ipsdienol enantiomeric blend in I. pini, we performed crossbreeding experiments using populations from five locations of geographic interest. The first selected population originated a short distance north of the postulated hybrid zone, where a broad range o f pheromone phenotypes exists and hybridization is likely (Miller et al. 1996). The mean (+)-ipsdienol ratio at this site is similar to that in some New York populations, but less than that for all surrounding northern British Columbia sites (Miller et al. 1989, 1996). We obtained breeding lines that exhibit the extreme pheromone phenotypes within this population, and then performed line crosses between these lines to determine the nature o f the control of ipsdienol blend at the hybrid zone. We are also interested in determining if genetic systems for producing pheromone blend are similar within the broader geographic areas on either side o f the hybrid zone. This hypothesis was tested by crossing populations near to and distant from the hybrid zone, within each side. The inferences we obtained about the genetic control o f ipsdienol are used to discuss the plausibility o f different speciation models that might be applicable to this system.

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2-2. Materials and Methods Source Populations British Columbia.— Ips pini were collected from two sites near the hybrid zone in British Columbia. The first site was 10 km southwest o f Brisco, British Columbia (Fig. 2.2). The site was approximately 30 km from Radium, BC where Miller et al. (1996) observed high population diversity in pheromone blends, suggesting possible hybridization. Forty frass samples were collected for pheromone analysis directly from the surfaces o f lodgepole pine (Pinus contorta var. latifolia) logs. Frass consists o f the boring dust, which contains macerated host tissue and feces. For each pheromone sample, the corresponding males were excavated from the host tree An additional 30 males and 70 females were collected and stored at 5°C in fresh phloem for approximately three months before they were used in establishing a colony on red pine (P. resinosa) logs in Syracuse, NY. At that time, 21 males were each paired to two females in red pine to establish a lab colony for further experiments. Another field site was sampled 5 km northwest o f Parson, BC. From this site, 40 males were excavated from infested lodgepole pine logs. Their frass was sampled for pheromone analyses while they fed on red pine. This site was sampled to assess whether there is any evidence o f hybridization at this location, approximately 60 km north o f Radium BC. We were unsuccessful in locating sites at equivalent distances south of Radium.

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Washington.— From Rosyln, Washington, 10 males and 14 females were collected in lodgepole pine. Ipsdienol enantiomeric blend was measured in the males while they were boring in red pine. Females were assumed to have already mated and were provided individual slabs of red pine, in separate cylindrical containers (60 cm long x 25 cm diameter). All the male progeny o f each female were tested for ipsdienol enantiomeric composition. This population is near (-)- ipsdienol producing populations south o f the hybrid zone that have been previously described (Fig 2-2; Seybold et al. 1995a). California.— Three sites near Lake Tahoe, CA were used to establish a colony o f I. pini. In El Dorado County, at one site, eight males and 20 females were collected from lodgepole pine. At a second El Dorado County site, 29 males and 18 females were collected from ponderosa pine (P. ponderosa var. ponderosa). In Mono County, 14 males and nine females were collected from singleleaf pinyon pine (P. monophylla var. fa lla x ). The ipsdienol enantiomeric ratio was measured in all field-collected males in red pine. Beetles from all three sites were bred randomly on red pine to produce a California colony. Alberta.—A pair o f 1 m long lodgepole pine logs that were colonized by I. pini were collected from Bragg Creek, Alberta. From the debarked logs, eight males and 30 females were collected and transported to Syracuse. All females were secured in individual red pine logs. All male progeny were tested for ipsdienol enantiomeric blend. Before initiating crosses to the New York population we maintained the population from 20 pairs of beetles on separate slabs o f red pine for one generation.

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New York.— Four red pine logs that were colonized by I. pini were collected from two sites Tioga County and Cortland County, NY. The logs were placed in 25 L emergence containers. Approximately 200 emerging beetles were collected and used to initiate a laboratory colony. No males were tested for ipsdienol enantiomeric composition for approximately six months, which corresponds to three to four generations. At that time, males were randomly selected from their colony, their ipsdienol enantiomeric ratios were measured, and line crosses were initiated. Measurement o f Pheromone Component Ratios In most cases pheromone samples were obtained from males that were secured under gelatin capsules (Bioquip, Gardena, CA) into drill holes in the bark of red pine logs. After 48 hours the accumulated frass in the gelatin capsules was transferred to 500 pi glass inserts in 1.5 ml autosampler vials with 200 pi o f chromatography-grade pentane. Field sampling o f the Brisco, BC population was the only exception to this procedure, where frass samples were obtained directly from the freshly colonized logs. We stored pheromone extracts at -30°C for up to six months before analysis. To measure pheromone component ratios, a 1 pi aliquot o f each sample was injected into a Hewlett Packard gas chromatograph/mass spectrometer (GC-MS) (model 5890 series II/ model 5971), equipped with a chiral GC column (0.25 mm internal diameter x 50 m) (Cydex-B, SGE Inc., Austin, TX). A Hewlett Packard Automatic Liquid Sampler (model 7673) was used to increase the throughput o f the GC-MS. The mass spectrometer was set to selective ion-monitoring mode to

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minimize signal from host volatiles and maximize signal from pheromones (m/z = 85 for ipsdienol, and m/z = 109, 124, 137, 152 for lanierone). The expected locations of the peaks were verified with synthetic standards of racemic ipsdienol at 10 ng/pl. Approximately one standard was run for every 50 frass samples. While we screened for lanierone in all samples, we did not include lanierone composition in any analyses. Because the percentage o f lanierone is at most 1 % o f the total pheromone blend (Teale et al. 1991), detection in individual samples can be difficult. To ensure detection of lanierone, more than half o f the samples that were satisfactory for ipsdienol enantiomeric blend analysis would be discarded. Our purpose in measuring lanierone was simply to determine whether it can be detected in all o f the populations sampled. Genetic Crosses Initiating crosses.— Before performing crosses, pheromone composition was measured in all males as described above. Potential female mates were tested for virginity by securing them in drill holes in bark for five to seven days and observing the resulting gallery pattern. Virgins make a gallery that is usually less than 2 cm long, with an irregular n oblong shape. Mated females exhibit a long straight gallery that is the same width as the insect. Several egg niches generally mark non-virgin galleries. To initiate crosses, males were individually secured in drill holes under wire screen in the bark o f separate red pine slabs (~15 x ~25 cm). For approximately 48 hours they were allowed to establish nuptial galleries. Ordinarily, males reside in this

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oblong nuptial gallery as females sequentially enter, mate, and begin constructing individual linear egg galleries. For each male in our crosses, a single female was introduced under the wire screen to initiate mating. The slabs o f wood containing each pair were held in metal cans (60 cm long x 25 cm diameter). Emergent offspring were collected from a glass vial that extended downward from a hole in the bottom of each rearing container. Divergent selection o f BC population For the population originating near the presumed hybrid zone in Brisco, BC, we divergently selected lines for extremes ratios o f (+)- to (-)-ipsdienol production. Initially, 189 male I. p in i were randomly sampled for their individual ipsdienol enantiomeric compositions. Males producing greater than 75% (+)-ipsdienol, or less than 30% (+)-ipsdienol were retained and mated to one random virgin female from the colony. These males represent the 46% o f the colony producing the highest proportion o f (H-)-ipsdienol and the 34% producing the lowest proportion o f (+)ipsdienol. In succeeding generations, males and females were selected from crosses where more than half o f male offspring produced greater than 70% or less than 30% (+)-ipsdienol. Males o f either o f these two desired phenotypes were preferentially crossed with non-sib virgin females from broods with similar phenotypic distributions. When non-sib females from the appropriate broods were unavailable, males were mated to virgin sisters or to random virgin females from the colony. In a few cases, males were paired with non-virgin sibs. However, it is not likely that such

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broods exhibited mixed paternity. In I. pini, sperm competition nearly exclusively favors second males when they remain with females for more than seven days (Lissemore 1997). A total o f five generations were used to obtain lines for crossbreeding. The high (+)-ipsdienol-producing beetles were from eight highly related lines that bred true for three successive generations. Beetles from these lines will be referred to as P+. Males o f two unrelated lines were used for the opposite parental type. These lines produced consistently low proportions of (+)-ipsdienol in the last generation, but were derived from some lines that had many high (+)-ipsdienol producers in the third and fourth generations. Beetles from these broods will be referred to as P 'a and P 'b. We proceeded with the line crosses at this time because we feared that inbreeding depression might reduce fecundity to the risk o f extinction of the P’a and P 'b lines. We observed a general trend o f decrease in brood size with inbreeding coefficient (Chapter 4), and the inbreeding coefficients for the possible crosses within these lines were among the highest for the entire experiment (f = 0.3) Line Crosses British Columbia lines.— O f the 20 FI crosses, 15 were between the P‘a and P+ lines (F la). Six F l a were between P+ males and P 'a females. Nine F l ar were P'a males crossed to P+ females. (Table 2-1). In general we use the subscript r to indicate that X-chromosome inheritance is different despite similar autosomal inheritance. The remaining five crosses (F lb) were between P 'b males and P+ females. There were no virgin P'b females available for crossing to P+ males. Ipsdienol enantiomeric

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composition was measured in all male FI progeny. Likewise, all female FI progeny were tested for virginity in anticipation o f F2 and backcrosses. To propagate the parental lines, twelve crosses were performed within and between the P+ broods. Twelve crosses were also performed within and between the P"a and P’b lines, but only two crosses within the P 'a line were used for further breeding. After the F l b results suggested that the P'b line was heterozygous, only beetles derived from the P 'a line were used in F2 crosses and backcrosses. A total o f 30 F2 crosses were made from the F l a and F l ar lines. The frequencies o f the different crosses ranged from five to twelve for the four possible types o f F2 crosses (Table 21). This bias was due to a shortage o f females from the F l ar crosses. Twenty backcrosses, referred to as B+, were made from F l a and F l ar to the high (+)-ipsdienol producing line, P+. The relative proportions o f the directions o f these crosses ranged from four to six (Table 2-1). Because P'a males and females were scarce, only 10 backcrosses from F l a and F l arto P'a could be performed. These backcrosses are referred to as B \ Each possible type o f backcross was represented two or three times (Table 2-1). Ipsdienol enantiomeric composition was measured in all F2 and backcross male offspring. California by Washington crosses.— For the California and Washington line crosses, the parental lines are represented as P' and P+, respectively (Table 2-1). All parental type beetles were taken from colonies established one generation after collection. For the FI generation, eleven crosses were performed in both directions with respect to sexes o f parents. There were 20 F2 crosses performed, five for each of

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the specific types o f F2 that relate to the male-female directionality o f crosses. Similarly, there were 20 backcrosses to both California and Washington lines (B \ and B+ respectively) with five crosses dedicated to each o f the four possible types o f crosses. Alberta by New York crosses.— We had hoped to obtain a population originating near the hybrid zone that was uniform in producing high ratios o f (+)ipsdienol. All o f the populations north o f the hybrid zone, described by Miller et al. (1996) had a few males producing lower ratios o f (+)-ipsdienol. The Bragg Creek, Alberta population we sampled unfortunately possessed similar variation in male pheromone blend (Fig 2-2). We did not wish to select for this high (+)-ipsdienol phenotype as we did for the BC crosses, because in this case we did not want to inadvertently remove epistatic genetic variation that might influence the ipsdienol blend in the line crosses. To avoid these difficulties, we used only males that produced > 80% (+)ipsdienol. Because only males produce pheromone that can be tested, no AB females were used. Thus, all 18 FI crosses were between AB males (P+) and NY females (P') (Table 2-1). These FI offspring were used to perform 19 F2 crosses. There were ten backcrosses o f FI females to AB males producing greater than 80% (+)-ipsdienol (B+). FI offspring were also backcrossed to NY beetles, ten times in the two possible directions (B‘, B 'r). Overall, the AB x NY crosses were skewed toward having greater effects from the NY X-chromosome. We thus performed an additional cross between the B+

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females and NY males to increase our power to detect X-linked genetic effects. This additional cross matches a high proportion of Alberta X-chromosomes (3/4) with a nearly hybrid additive background (3/8 P+). The cross is similar in this respect to the reciprocal FI cross between NY males and AB females that could not be performed. Means Analysis A genetic model based on the means o f the line crosses was used to evaluate composite additive [d\, dominance [h], and X-linked [dx], effects, and all possible digenic epistatic effects. The model derives from groupings o f the parental, F I, F2 and backcrosses that share the same possible autosomal and x-linked genetic effects (Table 2-1), and included up to 9 lines. The epistatic factors considered included additive by additive [z], additive by dominance [/], dominance by dominance [/], additive by X-linked [d.dx], and dominance by X-linked [h.dx]. The mean effect m is included in all models. The notation follows Mather and Jinks (1982). The line cross data o f each experiment did not generally meet the assumption of normality, due in many cases to pronounced bimodality in the phenotypic distribution o f ipsdienol blend. Therefore, a re-sampling procedure was performed to estimate mean pheromone production o f each line. For 1000 replicates, data from each line mean were sampled with re p la c e m e n ttim e s. Here n, is the number o f observations for the ith line. The average re-sampled line means were used to estimate the parameters for each genetic effect contained in vector y, and the variances S from the diagonal of

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their variance covariance matrix (Mather and Jinks 1982; Lynch and Walsh 1998). The estimates o f y and S are:

y = (CTV '1C )'1CTV 1x,

and

s^cV'c)-', where C is the coefficient matrix for the contributions o f genetic effects to each line mean, V is the diagonal matrix o f the variances o f the re-sampled line means, and x is the vector o f the average re-sampled line means. For any model, represented by C, ■y

the goodness o f fit was tested using a % , where

x 2 = x Y 'x - x V 1Cy (Hayman 1958).

The degrees o f freedom for this x2 are the number o f line means minus the number o f model parameters. The stepwise procedure began with only the mean effect. Then we tested models with either the additive or X-linked parameters, since dominance and interaction effects are not possible without addition o f these effects. We select the effect that provides the best fit to the model, as demonstrated by the %2 statistic. At

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each successive step we tested all the models that added an appropriate genetic effect to the existing model. For example, if the additive effect is added first, then in the next step we try models including dominance, x-linked, or additive by additive interaction. We continued with this process until a model was accepted that showed no significant improvement in the %2 statistic if additional factors are added. This significance o f improvement was evaluated by the joint-scaling tests (Lynch and Walsh 1998) whereby the difference in x2 statistic between steps in the addition procedure is itself evaluated as a %2 statistic with 1 d.f. Significance o f individual parameters within the models was determined by comparing the two-tailed (95%), ordered interval o f the 1000 re-sampled estimates with the null hypothesis o f zero. The data also allowed us to expand the analyses to consider maternal and paternal effects. When we consider these effects we must break the F2 and backcross lines up into four lines that have different maternal and paternal inheritance. This is seen for example in Table 2-1 by noting that the four entries o f F2 have different combinations o f paternal grandfathers and maternal grandmothers. Thus, up to 16 lines were considered for this model. We performed such analyses for the BC experiment and the CA x WA experiment (analysis was not possible for AB x NY). In both cases these maternal and paternal effects are not included in the best model, and the results are similar to that for the reduced model. We therefore do not present the results o f these analyses.

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Mendelian analysis

Pronounced bimodality appeared in the outcomes of many o f the British Columbia crosses. Furthermore, examination o f the pooled distribution o f phenotypes in all the parental hybrid and backcross lines reveals a bimodal distribution with a sharp trough at 70% (+)-ipsdienol (Fig. 2-3). This observation led us to categorize all the male beetles in this experiment into high or low (+)-ipsdienol categories. We employed y f statistics to test the categorical data for deviations from patterns predicted by Mendelian inheritance. This analysis was not performed for the other experiments. For the California and Washington populations there is no significant difference in the phenotypic distributions o f pheromone blends. The pooled frequency histogram o f the AB x NY line crosses illustrates the difficulty in classifying the offspring into paternal groups (Fig. 2-3). Two peaks in the distribution o f phenotypes, which center near the means of the parental populations, overlap substantially. Number o f Effective Factors For all three line cross experiments we calculated the Castle-Wright estimator (Castle 1921; Wright 1968) for number o f effective factors that cause differences in pheromone blend production between the divergently selected lab lines or the field populations. We used the following expressions for deriving the estimator.

4 { a - ( P +) 2 + q - ( p - ) 2} | 70% (+)-ipsdienol. The ratio o f offspring in the two categories for F I b does not significantly differ from 1:1, but does differ from 3:1 or 1:3 (Table 2-7). In the F2 generation there were 41 high (+)-ipsdienol males to 116 low (+)ipsdienol males. These results do not significantly differ from a 1:3 phenotypic ratio. The ratio was significantly different from 1:1 or 3:1 (Table 2-7). The backcrosses to the P+ lines (B+) exhibited a 52:58 ratio o f the high (+)-ipsdienol to low (-)-ipsdienol phenotypes. The B+ offspring showed no significant difference from a 1:1 phenotypic ratio, but there were significant differences from 3:1 and 1:3 phenotypic ratios (Table 2-7). All 23 offspring o f backcrosses to P'a (B‘) were assigned the low (+)-ipsdienol phenotype, which significantly differs from 1:3, 1:1, and 3:1 phenotypic ratios. An Xlinked single gene model shows significant difference from expected ratios in each generation (comparisons not shown.)

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Number o f Effective Factors Significance o f the estimates for number of effective factors cannot be estimated because o f non-normality and non-additive effects. The estimates were low for each o f the three experiments (Table 2-8). Both the least squares and 2F2 - B+- B' estimates for the segregation variance in the BC line crosses yielded estimates o f ne close to one. The AB x NY crosses led to estimates o f ne being less than one. The Castle-Wright estimator for the CA x WA crosses was near zero for both estimates.

2-4. Discussion This study represents the first instance where the genetic control o f a longrange pheromone signal has been investigated at a natural hybrid zone. Miller et al. (1989) speculated that there was quantitative variation in ipsdienol blend production because o f the variety o f phenotypes found in his samples. Our analyses o f the BC experiment suggest that most o f the variation in ipsdienol enantiomeric ratio at the hybrid zone is determined by a single locus that exhibits much dominance. Quantitative variation may exist at a lower level, but a single locus or linkage group is responsible for the broad differences between the eastern and western populations. The Mendelian analysis o f the British Columbia crosses implicates a recessive allele at a single locus. The ratios o f phenotypes in the line cross generations are consistent with a model where a recessive gene causes expression o f the P+ phenotype. The F l b line is the only line that does not fit this model, because half of the beetles show the phenotype associated with P+. This result can easily be explained

38


if all the P"b parents o f this line were heterozygous carriers o f the recessive allele. It is thus quite fortunate that all the P'a beetles were apparently homozygous for the dominant allele. Otherwise, the line-cross experiment would not have been possible. While the Mendelian analysis is useful for identifying the action o f this important locus, it cannot explain the substantial variation seen within all the beetles to which we assigned the low (+)-ipsdienol phenotype in the line crosses. The line means analysis o f the BC data confirms the importance o f a strong dominance effect, but also suggests that interacting genetic factors are needed to fully explain variation in ipsdienol composition. The negative sign o f the dominance parameter [h\ in the BC experiment is consistent with the action o f a recessive gene. Although the magnitude of [h\ alone is small, a large portion o f the composite additive effect [d\ could result from the same gene that causes [h\. More importantly, both o f the significant interactions [/'] and [h.dx] involve dominance and are o f large magnitude. These interactions suggest that the expression of additive and X-linked genes at additional loci is heavily dependent on the major gene locus. It is most likely that these secondary loci are able to control ipsdienol blend when it falls between 0% and 70% (+)-ipsdienol, but their activity is masked if there are two copies o f the recessive allele that cause a narrow range o f pheromone blends near 80%(+)-ipsdienol. The Castle-Wright estimator for the BC experiment is close to one and supports our general conclusion that few genetic factors control the differences in pheromone blend at the hybrid zone. While this system violates assumptions o f the Castle-Wright model, the two methods o f estimating segregation variance provide

39


similar estimates o f ne. The line means analysis confirms the importance o f genetic factors beyond additive variation. Also, because o f the strong interactions involving dominance, the magnitudes o f genetic effects are not equivalent across loci. These violations might downwardly bias the estimate o f ne and limit our ability to interpret its significance (Lynch and Walsh 1998). However, strong segregation is visibly apparent and obviously causes the departures from normality seen in many o f the distributions (Fig. 2-4). Thus, the autosomal locus described by the Mendelian analysis may constitute a single effective factor in practice, while an undetermined number o f interacting loci determine ipsdienol enantiomeric composition at a much smaller scale. The experiment that involved crosses between beetles from California and Washington confirm that there is little difference between the genetic control o f pheromone blend production in these areas distant from and near the hybrid zone. In the parent populations and all the line crosses derivatives most beetles produced close to 0% (+)-ipsdienol. The very small, but significant additive and X-linked effects uncovered in line means analysis were likely due to rare alleles that cause slightly higher ratios o f (+)-ipsdienol. It is unlikely that these effects accurately indicate any real population differences between the two geographic areas. The Castle-Wright estimator is close to zero, which further indicates the similarity o f these populations. Our results confirm that many I. pini males near the hybrid zone produce proportions o f (+)-ipsdienol that are higher than ever seen in eastern populations like New York (Miller et al. 1989, 1996). The use o f the same host material and lab

40


conditions ensures that these differences are not due to environmental conditions as previously speculated (Miller et al. 1989; Seybold et al. 1995a). This study also explains several aspects o f the genetic nature of this difference. Our failure to observe transgressive segregation in the Alberta x New York crosses shows that the NY population may possess a gene similar to that distinguishing the P+ and P' lines of the BC experiment. All the AB x NY line cross means are within the range o f the two parental line means (Table 2-7), and none o f the line cross distributions shows concentrations o f phenotypes outside this range (Fig 2-6). Furthermore, there were no complicated genetic interactions in the AB x NY crosses that suggest radically different genetic control o f pheromone blend. However, our results do not completely rule out different genetic architectures for ipsdienol enantiomeric composition in the Alberta and New York populations. More precisely, the recessive nature o f the 80% (+)-ipsdienol blend from the Pacific Northwest may only be relevant to genetic variation from western populations. The AB x NY crosses exhibit additive, dominance, and X-linked effects, suggesting that at least one autosomal locus and one X-chromosome locus differ between the populations. The Castle-Wright estimator for the AB x NY crosses is less than one using both methods o f estimating segregation variance, and thus predicts that few loci are responsible for population differences. Because Castle-Wright model assumptions are again violated by departures from normality and non-additive effects, we cannot have strong confidence in this estimate.

41


Evolution o f the Pheromonally Distinct Populations The separation o f eastern and western forests during Pleistocene glaciation is the likely cause o f the divergence in pheromone biology between the corresponding populations o f I. pini (Seybold et al. 1992, 1995a). On the basis o f mitochondrial DNA lineages o f eastern and western populations, Cognato et al. (1999) confirmed the plausibility o f Pleistocene divergence, and hypothesized that I. pini has a western origin. They also found a unidirectional introgression o f mtDNA lineages from east to west. At the hybrid zone, the opposite pattern exists for the trait o f male pheromone production. Intermediate and opposing pheromone blends are more prevalent in the “eastern” populations directly north o f the hybrid zone than in the “western” populations directly south. This pattern suggests a mating system where western females have more specificity in their pheromone response than eastern females, or that there is strong post-zygotic isolation that prevents such mating. Wolbachia infection has been reported in the same family (Vega et al. 2002) and has been implicated in unidirectional mating incompatibility at other insect hybrid zones (Giordano et al. 1997). However, there is no such evidence o f strong post-zygotic isolation in I. pini. Lanier (1972) found high egg hatchability in hybrid crosses between Ontario and California populations o f I. pini in both directions. Also, during the course o f our divergent selection experiment we observed many broods with high phenotypic variation, suggesting that hybrids can produce viable offspring for several generations.

42


Previously studied aspects of the New York population suggest the importance of coevolution of pheromone production and response preference that could also apply to premating isolation at the hybrid zone. In that population, assortative mating occurs based upon male pheromone production and female preference (Teale et al. 1994). There is also a genetic correlation between pheromone production and response in New York, which was shown not to be a result o f pleiotropy or close chromosomal linkage (Hager and Teale 1996). While directly examining the behavioral interactions and underlying genetics o f pheromone preference will be crucial to fully understanding this system, the genetic system o f pheromone production helps assess the plausibility o f different speciation models. For example, the reinforcement model is often considered problematic for both multilocus and single locus models. For multilocus models, assortative mating is difficult to maintain in the face o f any gene flow, but for single locus models narrow clines leave little genetic variation for traits used in reinforcement (Butlin 1989). A two-level model o f control o f pheromone production (Zhu et al. 1996) could potentially circumvent these problems with the reinforcement scenario. First, in one allopatric population pheromone production is altered by minor mutations that are neutral or environmentally selected. A new response preference might evolve in the presence o f the new variant. Runaway sexual selection (Fisher 1930; Lande 1981) may follow which is incremental in nature and thus is likely to result in polygenic control o f pheromone production and response. A major gene may arise that causes

43


more extreme divergence in the pheromone phenotype or response preference. If such major genes are neutral or only very weakly selected, they may remain at very low frequencies in the diverging population. When the populations meet again, assortative mating may intensify and greatly increase the fitness o f bearers o f major gene, because their hybrid descendents are more likely to possess the extreme pheromone production or response phenotypes that are favored on one side o f the hybrid zone. An increase in the frequency o f a major gene in such a hybrid zone meets the definition o f reinforcement (Butlin 1989). A rare allele for an extreme trait becomes fixed at the point of secondary contact due to hybridization interactions. Taken alone, the results from crosses between the high and low (+)-ipsdienol blend lines from British Columbia are consistent with such a model for the evolution of high (+)-ipsdienol blend. However, the AB x NY cross results argue against reinforcement. There is no indication that the approximately 80% (+)-ipsdienol phenotype is linked to a single recessive factor as in the hybrid zone crosses. These results suggest that the major gene may be fixed in all the eastern populations, with relatively minor genetic effects causing the higher (+)-ipsdienol blends near the hybrid zone. Interpreted as such, the major gene seems to have evolved before any of these modifiers. Alternatively, a different major gene may exist in New York that does not have strong interactions with that from Alberta. These scenarios do not absolutely preclude reinforcement, but makes it less likely, particularly with respect to the two-level model suggested by Zhu et al. (1996).

44


The evolution o f genetic control o f ipsdienol enantiomeric composition is also compatible with simpler evolutionary models. Strong selection in local environments, rather that reinforcement at the hybrid zone could explain the origin o f the major gene after the initial divergence of pheromone systems in eastern populations o f I. pini. Under this model, strong selective pressures may also have caused the secondary difference between the New York and Alberta/British Columbia populations. Under this model, strong and specific selective pressures on the pheromone communication system o f I. pini are predicted throughout its geographic range. I. pini has many interactions with its assemblages o f competitors and predators that could cause selection for the use o f certain pheromone blends. For example, resource partitioning among Scolytids is one factor that may lead to selection for divergence o f pheromone systems among competing species in a geographic area. A western species, I. paraconfusus, is inhibited by (-)-ipsdienol (Light and Birch 1979). Conversely, this species produces (+)-ipsdienol and (-)ipsenol which interrupt aggregation by I. pini (Birch et al. 1977, 1980). An eastern species, I. calligraphus, that utilizes the same hosts as I. pini, produces only 24% (+)ipsdienol (Seybold 1992). However the selective pressures from such competitors are often more complicated. There are also cases where heterospecific pheromones synergistically increase attraction, presumably because they aid in host selection. The response o f I. avulsus to its own pheromone is increased when that o f the sympatric species I. grandicollis is added (Hedden et al. 1976). Likewise, I. pini, I. perroti, and I.

45


grandicollis in Wisconsin are attracted to logs baited with conspecific pheromones (Ayres et al. 2001). Underscoring the complexity o f such interactions, Ayres et al. (2001) also found that baiting logs with males o f multiple species usually inhibited attraction of the targeted species. Another potentially strong selective factor includes bark beetle predators that often exhibit preferences for certain pheromone phenotypes o f I. pini (Raffa and Klepzig 1989; Herms et al. 1991; Raffa and Dahlsten 1995). These preferences may result in selective feeding on beetles that produce certain pheromone blends. There is some evidence that these predators cause changes in pheromone response o f eastern /. pini populations (Raffa and Klepzig 1989; Herms et al. 1991; Raffa 1991) presumably through selection upon linked genes for pheromone blend characteristics, as demonstrated in NY (Hagar and Teale 1996). The addition o f lanierone to ipsdienol increases trap captures o f some predator species in western populations, but not in eastern populations where I. pini normally produces lanierone (Miller et al. 1997). It is possible that such predator response led to selection against lanierone production by Ips pini in western populations. A similar study has not been performed to examine ipsdienol enantiomeric blend preferences for multiple eastern and western predator species. While examples o f selective pressures from predators and competitors are insightful, current knowledge cannot explain the apparent differences between the Alberta/British Columbia and New York populations. Much more detailed information about all the pheromone interactions in these areas would be required to

46


suggest a reason for the slightly higher (+)-ipsdienol blends in northern BC at the hybrid zone. Indeed, it is difficult to imagine that such selection could be strong enough to cause such a minor difference in ipsdienol blend. Finally, it is possible that the differences between Alberta/British Columbia and New York populations reflect historical separation o f these populations. Differences between the populations may have evolved due to genetic drift or different genetic responses to similar selective pressures. Considering our AB x NY crosses, genetic drift seems more likely than convergent evolution, because both populations appear to be fixed for the same major gene at the same locus. Also supporting the possibility o f three historic allopatric populations, Cognato et al. (1999) found three mitochondrial lineages o f I. pini. The distributions o f these lineages roughly coincide with the eastern, Rocky Mountain, and southwestern populations o f I. pini and thus could signal the geographical origin o f three distinct pheromone systems. Both lodgepole and jack pine, the primary host species o f /. pini in Canada and Alaska may have had multiple refuges at the last glacial maximum (Critchfield 1985). Study o f female mate preference may support this theory and explain why these mitochondrial haplotypes appear to be introgressing only westward (Cognato et al. 1999). If western females discriminate very strongly against eastern males, most natural hybrids will be between western males and eastern females, which carry eastern mitochondria. As these hybrids cross back into the parent populations, introgression of autosomal loci will occur in both directions. However, mtDNA

47


introgression will only be from east to west. Introgression of western autosomal genes into populations north and east o f the hybrid zone is also predicted, and consistent with ipsdienol enantiomeric composition profiles in Fig 2-2. However, natural selection or weak assortative mating must ultimately limit this introgression. While much o f the proposed evolutionary model for Ips p in i pheromone production remains speculative, this study clearly demonstrates the importance o f a single autosomal gene or linkage group to the canalization o f the Ips pini pheromone system. Major genes control differences in pheromone blend and response between several closely related species o f moths (Lofstedt 1990). Similarly, different populations o f the European com borer, Ostrinia nubilalis, that have been introduced into North America exhibit major gene control o f pheromone production and response (Klun and Maini 1979; Hannson et al. 1987; Roelofs et al. 1987). While further research into the genetic control o f pheromone preference in I. pini is needed to complete these comparisons, this study further supports the importance o f major genes in the evolution o f pheromone communication systems. Despite the implications o f discovering this major gene, understanding the reasons for minor differences between (+)-ipsdienol producing populations near and distant to the hybrid zone also remain important for understanding the evolution o f the system.

48


T a b le

2-1. Number o f crosses in all experiments. For BC, the crosses deriving from

the independently selected low (+)-ipsdienol lines are separately noted as a and b. Line Name P+

Paternal Grandparents Male Female + +

Maternal Grandparents Female Male + +

BC Crosses

CA/WA Crosses

AB/NY Crosses

8

C

C

P'

—

—

■ —

—-

la, lb

C

C

FI

+

+

—

—

6a, Ob

11

18

F lr

—

—

+

+

9a, 5b

11

0

F2

+

—

+

—

12a

5

19

F2

—

+

—

+

5a

5

0

F2

+

—

■ —

+

5a

5

0

F2

—

+

+

—

8a

5

0

B+

+

+

+

—

6a

5

10

B+

+

+

—

+

4a

5

0

B+r

+

—

+

+

5a

5

0

B+r

—

+

+

+

5a

5

0

B’

—

—

+

—

3a

5

10

B'

—

—

—

+

2a

5

0

Br

+

—

—

—

3a

5

10

Br

—

+

—

—

2a

5

0

P‘ x B+

---

—

+

FI

0

0

16

49


T a b le

2-2. Proportion o f (+)-ipsdienol in the P+, P 'a, and P'b lines for the three

generations prior to crossing in the BC experiment (mean +/- SD). Data include measurement o f all male siblings in the broods used for further line propagation. P+ represents all broods containing individuals from eight lines, while P 'a and P 'b both represent one line.

Generation

P+

p-a

p -b

3

80.2 ± 1.0

53.4 ± 30.9

43.1 ± 34.8

4

84.1 ± 1.6

31.2 ± 27.3

28.4 ± 15.4

5

81.3 ± 1.6

9.6 ±

4.6

14.1 ±

5.9

50


T able 2-3. Estimates of parameters for the best model for each line cross experiment. For all the parameters 95% confidence intervals (not shown) do not overlap zero. The effects include mean (m), additive (d), dominance (h), X-linked (dx), additive by dominance (j), and dominance by X-linked (h.dx).

Effect

BC+/BC'

AB/NY

WA/CA

m

45.061

73.938

1.978

d

72.086

6.960

0.425

h

-19.756

-2.437

dx

-35.853

-1.655

j

-35.470

h.dx

31.081

-0.235

51


T a b le 2-4. Averaged resampled line means, standard errors o f the line means

(determined from the variance o f the resampled means), sample sizes, and predicted means for the best model for the British Columbia experiment (p = 0.446).

Line

Mean

S.E

n

Predicted % (+)-ipsdienol

% (+)-ipsdienol BC:P+

81.309

0.310

24

81.295

BC:P'a

8.990

1.056

19

8.827

F la

30.360

1.393

120

30.076

F l ar

21.601

2.702

31

20.533

F2

36.397

2.348

157

35.183

B+

60.162

3.714

50

62.358

B+r

38.370

3.886

60

42.047

B*

7.686

1.420

7

8.007

Br

24.704

3.853

16

28.319

52


T a b le

2-5. Averaged resampled line means, standard errors o f the line means

(determined from the variance of the resampled means), sample sizes for each line, and predicted means for the best model for the CA x WA line cross experiment (p = 0.132).

Line

Mean

S.E

n


% (+)-ipsdienol WA:P+

2.097

0.213

51

1.434

CA:P'

2.149

0.335

55

2.252

FI

2.214

0.239

88

2.211

F lr

1.730

0.087

53

0.621

F2

2.653

0.411

69

3.115

B+

3.374

0.642

42

3.935

B'

1.765

0.099

33

0.562

B"r

1.834

0.159

26

0.751

53


T a b le

2-6. Averaged resampled line means, standard errors o f the line means

(determined from the variance o f the resampled means), sample sizes, and predicted means for the best model for the AB x NY line crosses (p = 0.1392).

Line

Mean

S.E

n


% (+)-ipsdienol AB:P+

80.131

0.231

77

80.102

NY:P'

67.921

0.485

79

68.156

FI

73.846

1.626

30

73.672

B+ x P’

68.515

1.092

42

69.728

F2

72.834

1.224

67

73.045

B+

74.367

1.556

30

76.887

B'

70.724

0.976

54

69.204

B 'r

71.289

0.619

101

70.914

54


T a b le 2-7. The X 2 values for comparisons o f the ratios o f (+)-ipsdienol: (-)-ipsdienol

phenotypes to typical Mendelian ratios in British Columbia F I, F2 and backcross progeny (*p < 0.01, ** p < 0.005).

Cross

Ratio

3:1

1:1 144.1 **

1:3

F la

2: 150

440.1 **

F2

41: 116

200.1 **

B+

52: 58

45.10 **

0.3273

29.10**

B'

0: 23

69.00 **

23.00 **

7.667*

F lb

22: 24

18.12**

0.0870

12.78**

35.83 **

45.47** 0.1040

55


T a b le

2-8. Castle-Wright estimators of number of effective segregating loci for the

BC, AB/NY, and CA/WA line cross experiment.

V(S) estimate

BC crosses

AB/NY crosses

CA/WA crosses

Least Squares

0.662 ± 0.072

0.340 ± 0.071

-0.002 ±0.040

2F2 - B+ - B'

1.383 ±0.565

0.228 ± 0.086

-0.004 ±0.064

56


Inset Fig. 2-2

50% 100%

California N=28

25%

50%

0%

0%

New York N=56

.. 50 100 % (+) - ipsdienol

_

100

% (+) - ipsdienol

Fig. 2-1. Distribution o f I. pini pheromone types in North America. Shaded area indicates range o f I. pini (Lanier 1972). Sample populations derive from fourth generation o f New York colony and first generation o f California colony.

57


40%

40% Williams Lake N=38

40% 20%

_

Parson N®22

' ■

20% -

40%r

1

Brisco N=39

20%

0% '

■

'

% (+) - ipsdienol

0%

SO % (+) - ipsdienol

0%

)

50 % (+) - ipsdienol

100

100 0


100

40%i O sprey Lake N=35

Bragg Creek, AB N=33

20% -

_

-

. 0


100

J . 1 50 100 %(+) - ipsdienol

0

Radium N=87

40% Princeton N=344

20%

0 50 % (+) - ipsdienol

100

100%

50%

Rosyin, WA N=80

-

0%

0

50 100 %(+) - ipsdienol

W inthrop, W A

Tonasket, WA

3%

4%

(+) - ipsdienol

(+) - ipsdienol


100

Kimberley

0

50 % (+) ■ipsdienol

100

Fig. 2-2. Summary o f ipsdienol enantiomeric composition in populations o f I. p in i at a hybrid zone in southern British Columbia. Figure is modified from Miller et al. 1996, including frequency distributions from Williams Lake, Pemberton, Princeton, Kimberly, Radium, and Osprey Lake populations. Brisco, Parson, Rosyln, and Bragg Creek frequency distributions are from populations described in the text. Winthrop and Tanasket data are from pooled samples by Seybold et al. (1995a).

58


BC -o 52 0

(/) £ £ 26 CO

ft 0

50

100

% (+)-ipsdienol

ABxNY "O 64

4CD

- —'

CO CD

i 32 co 50

100

% (+)-ipsdienol

Fig. 2-3. Frequency histograms of the pooled parental hybrid and backcross data from the BC and AB x NY line-cross experiments. Mean ± standard deviation o f the two parental lines shown above the histograms. In the BC histogram a vertical dotted line indicates the decision point for separating phenotypes for Mendelian analysis.

59


=8: 0

100

o

100

G)

-O 18

BH

O 03 12 & (/) O6

H)

B+

l2