(IIIHIWINIWIHIIJHIHIIHHHIWMHHHHHIWWF 03—: (DA yam-A '—1 “I Illlllllllllllllllll This is to certify that the thesis entitled ISOZYME VARIATION AS EVIDENCE OF GENE FLOW AND HYBRIDIZATION BETWEEN RED OAKS FOUND IN AN ISLAND ARCHIPELAGO presented by Stan L. Hokanson has been accepted towards fulfillment of the requirements for M.S. Jegree in Plant Breeding & Genetics Department of Horticulture 4“?“ Major professor Date 9-20-9 1 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution w..— L383...» 4" 1 Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. M. DATE DUE DATE DUE DATE DUE P ab 4" § fi_— . W [\[i MSU Is An Affirmative Action/Equal Opportunity Institution cMcWMH.‘ ISOZYME VARIATION AS EVIDENCE OF GENE FLOW AND HYBRIDIZATION BETWEEN RED OAKS FOUND IN AN ISLAND ARCHIPELAGO BY Stan C. Hokanson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture Plant Breeding and Genetics Program 1991 ABSTRACT ISOZYME VARIATION AS EVIDENCE OF GENE FLOW AND HYBRIDIZATION BETWEEN RED OAKS FOUND IN AN ISLAND ARCHIPELAGO BY Stan C. Hokanson Isozyme variability was examined in the red oak complex, Quercus subg. Erythrobalanus found on an island archipelago and vicinity in northeastern Wisconsin. Dormant leaf bud samples were collected from Quercus rubra L. , Q. ellipsoidalis Hill and their putative hybrids from two peninsula locations and on three islands. Acorns were collected from some of these same trees in three of these locations. Twelve putative loci coding for six enzymes were analyzed. Allele frequency data indicated there was little differentiation between populations. Mean Ffl.values for the adult trees and acorns were 0.042 and 0.020 respectively. Genetic identities according to Nei ranged from .958 to .999. In spite of these high. genetic identities, the jpopulations appeared to be experiencing substantial levels of inbreeding as indicated by positive mean Frr values of 0.183 and 0.373 for the trees and acorns respectively. Estimates of migration rate per generation for the adult trees was 5.70. ACKNOWLEDGMENTS Completing this project was a "peak experience" for me. As is often the case, these experiences arise from quality interactions with, and input from exceptional people. Such is the stuff of which we shall one day measure our lives. It is with extreme gratitude and some pride that I make these acknowledgements. First, I want to thank Dr. Jud Isebrands at the Forestry Sciences Laboratory in Rhinelander, WI, for originally proposing the project, providing substantial funding and considerable logistical support to carry the project. .Also at Rhinelandery I want to thank Gary Gartner and Dave Buckley for their help with collections. At the Apostle Islands National Lakeshore, I want to thank Dr. Robert Brander, Julie Van Stappen and their staff for providing access to the populations and for extending a very cooperative atmosphere in which to conduct the fieldwork. The "friendlies" were not restricted to Wisconsin. At Michigan State both Paco Moore and Mike Kwantes spent some long day/nights doing great things with the computer for me. Dr. Steve Krebs taught.me hOW'tO run isozymes and.moreover was always willing to listen and provide thoughtful input. Everyone in the Hancock lab has helped me to grind samples at one time or another; assistance for which I again say thanks. iii Everyone in the lab strives to maintain a vigorous, cooperative, non threatening atmosphere in which to work. I thank them all past and.present for the unqualified acceptance into such an exceptional group. I want to thank my thesis committee, Drs. Hancock, Iezzoni, Jensen, and Tonsor for, possibly with no intent to do so, teaching me the power and grace of a positive perspective. Thanks to the 11:30 club for being there, Jim, for taking the gamble which made it all possible, and Karen for love and support on all the days, good and bad. iv TABLE OF CONTENTS Page List of Tables.......... ........ ...... ................. vi List of Figures. ............ ........... ..... . .......... vii Introduction........ ................................... 1 Materials and Methods.. ....... . ........ . ............... 7 Collections........................ ................ 7 Enzyme Extraction.......................... ........ 9 Electrophoresis ........... . ....... ...... ........... 10 Isozyme Analysis .............................. ..... 11 Statistical Analysis........................ ....... 11 Results............ ...... . ..... .................. ...... 18 Discussion............................................. 27 Identification of species and their hybrids........ 27 Genetic variability in Apostle region oak populations.O..0.000.000000000000000000000000.0...O 28 Levels of gene flow among the Apostle region oakSOOOOOOOO0......OOOOOOOOOOOOOCOOOOOOCOO... ...... 3o ConCIuSiODSOOOOOOOOO0.0000000000000000 OOOOOOOOOOOOOOOOO 36 List.of References........... ....... ....... ............ 39 Table LIST OF TABLES Allele frequencies at 12 putative enzyme loci for five adult and three acorn populations of Quercus Erythrobalanus studied on the Apostle Islands and vicinity........................... Mean sample size per locus, percent polymorphic loci, mean and effective number of alleles per locus, and mean heterozygosity, direct and expected, for five adult and three acorn populations of Quercus in the Apostle Islands and‘vicinity................................... Deviations from Hardy-Weinberg equilibrium among individuals (Fm), among populations (Fm). and total deviation (Fm), with migrants per generation (Nm), according to Wright (1931), for 11 loci from Quercus adults........ Deviations from Hardy-Weinberg equilibrium among individuals (Fm), among populations (Fm), and total deviation (Fm), with migrants per generation (Nm), according to Wright (1931), for 11 loci from.Quercus acorns........ Unbiased genetic identity values according to Nei (1978), for Quercus adults and acorns from the Apostle Islands and vicinity............... vi Page 19 22 23 24 26 Figure 1. LIST OF FIGURES Page Map of the Apostle Islands and adjoining Bayfield Peninsula in northeastern Wisconsin. The five populations studied are initialed, Bayfield Peninsula (BP), Peninsula Perimeter (PPC), Oak Island (0K), Stockton Island (STK), and Outer Island (OI). Underlined initials indicate populations from which acorns were collected. Heavy lines represent the transects sampled..................... ......... 8 Electrophoretic patterns for three of the six enzyme systems used in this study. Numbers to the right of the photographs indicate putative loci. Superscript numbers indicate putative alleles of each locus. PGI was scored as a dimeric enzyme encoded by two loci. Locus one was considered monomorphic. Lane A is heterozygous for alleles three and five of locus two. Lane 8 is heterozygous for alleles one and five of locus two. MDH was scored as a dimeric enzyme encoded by three loci. In this photo all individuals are homozygous for allele one of locus one. Lane A is heterozygous for alleles one and two of locus two. Allele one of locus three has comigrated with allele two of locus two. Lane B is homozygous for allele two of locus two. Allele one of locus three has comigrated with allele two of locus two. SKDH was scored as a monomeric single locus enzyme. Lane A is homozygous for allele two. The second band was thought to be a plastid form of the enzyme and not scored. Lane B shows a heterozygote for alleles three and five. The faint bottom most band was considered a plastid form of allele five and not scored. Lane C is homozygous for allele five.................................... 13 vii Figure LIST OF FIGURES (cont'd) Page Electrophoretic patterns for the remaining three of six enzyme systems used in this study. Labeling remains the same as on the previous page. IDH was scored as a dimeric enzyme encoded by three loci. Lane A is heterozygous for alleles one and three of locus one. Lane B is heterozygous for alleles four and five of locus one. Lane C is heterozygous for alleles one and two of locus two. Lane D is homozygous for allele one of locus three. Lane E is heterozygous for alleles two and three of locus three. 6PGDH was scored as a dimeric enzyme encoded by two loci. Lane A is homozygous for allele one of locus one. Lane B is heterozygous for alleles one and two of locus one. Lane C is heterozygous for alleles one and three of locus one. Lane A is homozygous for allele two of locus two., Lane D is heterozygous for alleles one and two of locus two. LAP was scored as a monomeric, single locus enzyme. Only the uppermost locus was scored for this study. Lane A is homozygous for allele two. Lane B is homozygous for allele three. Lane C is homozygous for allele one. Lane D is heterozygous for alleles two and four......... 15 viii W Quercus is a wide ranging genus of trees and shrubs comprised of up to 500 species worldwide (Nixon, 1989). In eastern North America, the genus is represented by approximately 50 species rather evenly divided into two reproductively isolated subgenera: Quercus subg. Quercus, the white and chestnut oaks, and Quercus subg. Erythrobalanus, the red and black oaks. Within Quercus, a definitive taxonomic structure at the species level has been difficult to achieve. To date, oak classifications have been based almost exclusively on morphological characteristics (Trelease, 1924; Jensen, 1988) . However, the apparent ease of hybridization among species (Palmer, 1948), particularly in Erythrobalanus (e.g., Jensen, 1977a), renders such an approach problematic, especially where the species ranges overlap. In areas of species sympatry, many trees may have morphologies intermediate between species types. These "hybrid" oaks often defy precise identification (Overlease, 1964; Jensen, 1977a). Numerous studies have attempted to find alternative characters suitable for differentiation at the species level. These studies have examined wood anatomy (Muller, 1942) , phenolic compounds in leaves, young twigs, and male flowers (Li and Hsiao, 1973, 1975, 1976a,b), and scanning electron microscopy of pollen (Solomon, 1983a,b). Although these investigations affirmed the subgeneric discrimination, none of the characters investigated allowed consistent distinctions at 1 the species level. Electrophoretic techniques have been employed to establish relationships among the oaks in the eastern United States. Manos and Fairbrothers (1987) evaluated 16 putative isozyme loci among populations of six native red oak species found in New Jersey: Quercus coccinea Muenchh., Q. ilicifolia Wang. , Q. marilandica Muenchh. , Q. palustris Muenchh. , Q. rubra L., and Q. velutina Lam.. The genetic differentiation detected with polymorphic loci did not correlate with previously established taxonomies and, with the exception of Q. palustris, no species specific alleles were detected. Thus, isozymes were of little value in clarifying taxonomic structure in these taxa. Guttman and Weigt (1988) used 18 putative isozyme loci from 12 enzyme systems to study ten species from the subgenus Erythrobalanus and eight from subgenus Quercus. Unlike that of Manos and Fairbrothers (1987), their study of Erythrobalanus did not include Q. coccinea and Q. ilicifolia. The groupings they discovered were discrepant from both traditional taxonomies and recent multivariate comparisons. Jensen (1977b) used a numerical analysis of 36 morphological characters to evaluate taxonomic relationships in the scarlet oak complex in the eastern United States. This inquiry indicated that Quercus ellipsoidalis clustered near Q. palustris and also showed relationship to Q. velutina. Guttman and Weigt's (1988) data did not confirm this relationship with Q. velutina, nor several others proposed by 3 Jensen (1977b) . Guttman and Weigt (1988) attribute these discrepancies to unequal rates of morphological, ecological, and allozymic divergence and the possibility that hybridization between species is preventing the development of a widely accepted, definitive taxonomic structure within the subgenus. Several other authors have mentioned this possibility (Palmer, 1942; Jensen, 1977b; Solomon, 1983b). Thus, determining a taxonomic structure within the subgenus Erythrobalanus becomes partly a matter of identifying hybrids. Because hybridization is a function of the degree of pollen flow between species, understanding the movement of genes within the genus Quercus becomes crucial to elucidating the nature of hybrid formation. Inter-specific gene flow is clearly suggested by the wide range of morphologies expressed when two or more red oak species are found in one location (Jensen, 1988). Commensurate with the recognition of the importance of gene flow, much research has been directed toward understanding’ its dynamics in. both. a theoretical and a practical sense (Levin and Kerster, 1974; Moore, 1976; Handel, 1983b; Slatkin, 1981, 1985; Ellstrand, 1988). However, the actual amount of gene flow via pollen or seed is one of the lesser known parameters concerning natural plant populations (Levin, 1984; Hamrick, 1987). Due to a general lack of suitable markers, direct measurement of gene flow is often difficult (Ellstrand and Marshall, 1985). Probably most crucial in designing studies of gene flow is the distinction 4 between potential and actual gene flow (Levin and Kerster, 1974). Potential gene flow is characterized by the movement of pollen whereas actual gene flow does not occur until fertilization. It is generally agreed that the most efficient method for determining actual gene flow is through the use of isozyme "marker genes" detected with electrophoretic techniques. Considerable effort has also been directed towards determining the relationship between life history traits and the genetic diversity and structure within various plant species (Brown, 1979; Hamrick et al., 1979; Gottlieb, 1981; Loveless and Hamrick, 1984; Hamrick and Godt, 1989). Comparisons have been made between plant species with similar ecological and life history attributes to determine whether such species maintain similar levels of genetic variability and/or genetic structure. Hamrick and Godt, (1989) used the following eight ecological and life history traits in making such comparisons: taxonomic status, regional distribution, geographic range, life form, mode of reproduction, breeding system, seed dispersal mechanism, and successional status. This study, along with earlier such studies (Hamrick et al., 1979; Nevo et al., 1984; Loveless and Hamrick, 1984), uncovered significant correlations between ecological and life history traits of species and the patterns of genetic diversity and structure they maintain. These reviews concur that species, such as oaks, which are long-lived, outcrossed, wind pollinated and which prevail in the later stages of 5 succession, generally maintain higher levels of genetic variation within populations than between populations. Schnabel and Hamrick (1990) used electrophoretic techniques to examine population genetic structure in two species of white oak, Quercus macrocarpa Michx. (bur oak) and Q. gambelii Nutt. (Gambel oak). Whether considered as species or as individual populations, both Q. macrocarpa and Q. gambelii were found to maintain levels of allozyme variation which were higher than the overall averages for most other plant species. Quercus macrocarpa had an average of 3.58 alleles per locus while Q. gambelii had 2.67. The overall genetic diversity for Q. macrocarpa was 0.206 with Q. gambelii maintaining a level of 0.215. According to Hamrick and Godt (1989) , such high levels of allozyme variation should not be surprising. The life history traits generally associated with Quercus species, 1.9. broad geographic range, wind pollination, predominantly outcrossing, and a long-lived perennial nature, are generally associated with high levels of allozyme variation. Despite increased efforts to study population genetic structure in woody angiosperms, (Bousquet et al. , 1987, 1988; Surles et al., 1989; Schnabel and Hamrick, 1990), there are still significant gaps in such knowledge. This is particularly true for deciduous trees which make up the northern hardwood forests. Several of these species, quite notable among them Quercus rubra (northern red oak) , are being subjected to extreme logging pressure. Without an 6 understanding of what constitutes a healthy genetic structure in a population of such trees, it will be difficult to determine what level of harvest is sustainable. Coincident with this economic pressure, there is also concern about. the effects of certain environmental perturbations, such as global warming and acid rain, on the genetic health of forest tree species, . To make responsible decisions concerning the possible impacts of such pressures, a more substantive base of knowledge must be built. In particular we need to have a better understanding of the population genetic structure of tree species such as the northern red oak. Populations of oaks on the Apostle Islands in Lake Superior presented us with the opportunity to address the issues noted above. 'The islands and adjacent Bayfield Peninsula are inhabited by natural populations of two species of subgenus Erythrobalanus. Leaf and acorn morphological characters indicate that. Quercus.rubra L. (northern red oak) predominates on the outer most island of the archipelago (Outer Island), while Q. ellipsoidalis Hill (northern pin oak) predominates in the peninsula interior. Between these distal populations exist populations which can be characterized as being intermediate or* hybrid in nature (Jensen et al., manuscript in review). This pattern of morphological variability presented us with a system to answer the following questions: 1) Do species specific alleles exist for Quercus rubra or Q. ellipsoidalis which would allow for the 7 unambiguous identification of these species and their hybrids? 2) What levels of genetic variation are contained within these small populations of adult trees and their acorns? 3) How much and how far do genes move among these island populations of oak? HAIEBIAL§_AND_MEIHQQ§ M We studied five populations of oaks in the Bayfield, Apostle Islands National Lakeshore region in northeastern Wisconsin (Figure 1). Samples were collected on five separate trips to the region. Two trips were made in June of 1989, and one each in October 1989, March 1990, and May of 1990. Bayfield Peninsula collections (BP) were made along roads which defined the east and.north perimeters of the Chequamegon National Forest. At approximately 0.4 km intervals, the first mature oak sighted was sampled. Peninsula perimeter collections (PPC) were made on the public roads which skirted the perimeter of the Bayfield peninsula. As in the national forest, we sampled the first mature oak sighted at 0.4 km intervals along the roads. Collections on Oak Island (OK) were made on a north to south trail which bisected the island. After five minutes of vigorous walking we sampled the first.mature oak sighted.within 20 yards of the trail. A similar sampling strategy was used for the other island collections. The Stockton Island (STK) and Outer Island (OI) collections were done on a trail which ran from southwest to northeast the length of the island. Figure 1. Map of the Apostle Islands and adjoining Bayfield Peninsula in northeastern Wisconsin“ The five populations studied are initialed, Bayfield Peninsula (BP), Peninsula Perimeter (PPC), Oak Island (OK), Stockton Island (STK), and Outer Island (01). Underlined initials indicate populations from which acorns were collected. Heavy lines represent the transects sampled. 9 Samples collected in June, 1989 consisted of rapidly expanding leaves 15-75mm in length. On the subsequent trips dormant buds and acorns were collected. In all cases, pencil sized branches were removed from individual trees. Buds were left on small twigs while acorns and leaves were removed. Samples were placed in labeled zip-lock bags and stored within three hours in a cooler. On return to East Lansing, MI, the plant material was placed in a cooler at 2.5° C until enzymes were extracted. Leaves rapidly lost their enzyme activity and had to be prepared immediately for electrophoresis, while dormant buds could be stored up to two months before extraction with no appreciable loss in activity. Acorns ground 11 months after harvest also had high enzyme activity. W All samples were macerated with pestles in chilled mortars using the Soltis phosphate grinding buffer (Soltis et al., 1983). Prior to grinding, one spatula tip of insoluble PVPP, (Sigma # P-6755) completely hydrated with the grinding buffer was added. Approximately 10 mg of dormant bud was ground per specimen. For acorns, 35 to 40 mg of cotyledon tissue removed from the cap end of the acorn was ground per sample. After grinding, the resulting slurry was absorbed through nylon mesh onto 3 x 4 x 11 mm wicks cut from Whatman chromatography paper. Wicks sufficient for four electrophoretic examinations were prepared at this time. These wicks were placed in Corning 96 well disposable Elisa plates which were double wrapped in cellophane, bagged in 10 ziplock bags and stored at -80°C until electrophoresed. Only enough wicks for one analysis were placed in a plate, thus the wicks were never taken out of the freezer until they were to be analyzed. Well resolved isozymes were obtained from wicks stored up to 14 months under these conditions. Elegtzgphgzgggs Experiments with a number of gel/electrode buffer systems revealed that the pH 8.3 lithium borate, tris- citrate system (Scandalios, 1969) and the morpholine-citrate pH 6.1 system (Clayton.and.Tretiak, 1972) resolved.the largest number of enzymes clearlyu Ten millimeter thick 6.1 gels were typically run for seven hours at 55-65 milliamps, and approximately 250 volts. Six millimeter thick 8.3 gels were run for six hours at 50 milliamps or until 300 volts was reached. Slices from the 8.3 Scandalios system were stained for phosphoglucose isomerase (PGI) and leucine amino peptidase (LAP). Slices from the 6.1 morpholine-citrate system were stained for 6-phosphogluconate dehydrogenase (6-PGDH) (Conkle, 1982), isocitrate dehydrogenase (IDH) (Soltis et al., 1983), malate dehydrogenase (MDH) (Vallejos, 1983), and shikimate dehydrogenase (SKDH) (Soltis et al., 1983). All staining assays were conducted as cited except for LAP. The substrate used was L-leucine-B-napthylamide HCL (Sigma # L0376) which was dissolved directly in the buffer solution. Once the bands stained clearly, the slices were rinsed in 1% acetic acid solution and then fixed in a 50% ethanol solution. Slices were then bagged in zip lock bags and refrigerated at 4°C for later analysis. 11 During our initial experiments, we found that resolution became increasingly poor as leaves expanded, while dormant buds gave us consistently'good.resultsw 'Thus, we discontinued using leaves, and all results reported herein were obtained using dormant bud or acorn samples. The acorns analyzed in this study were collected from adult trees which were also analyzed electrophoretically. Five acorns were electrophoresed from each of these parent trees. Isgzymg_Analy§i§ Bands were read in the conventional manner with those loci migrating farthest from the origin being designated as number one, the next farthest number two, etc. Within a locus the fastest allele was named one, the next two etc. (Figures 2,3). Because we made no controlled crosses to analyze segregation of the isozyme banding patterns, all allele and loci designations are putative. Statistical___finaly§i§ Allele frequencies, average heterozygosities (direct and estimated), percent loci polymorphic, mean alleles per locus, F-statistics and genetic identity according to Nei (1978b) were calculated using the BIOSYS-l program, release 1.7, which is adapted for the PC (Swofford and Selander, 1989). Direct or observed average heterozygosities were calculated by adding the number of heterozygous individuals at each locus in the population, dividing this number by the total number of individuals in the population and averaging this value over loci. Calculations of average estimated 12 Figure 2. Electrophoretic patterns for three of the six enzyme systems used in this study. Numbers to the right of the photographs indicate putative loci. Superscript numbers indicate putative alleles of each locus. PGI was scored.as.a dimeric enzyme encoded.by two loci. Locus one was considered monomorphic. Lane A is heterozygous for alleles three and five of locus two. Lane B is heterozygous for alleles one and five of locus two. MDH was scored as a dimeric enzyme encoded by three loci. In this photo all individuals are homozygous for allele one of locus one. Lane A is heterozygous for alleles one and two of locus two. Allele one of locus three has comigrated with allele two of locus two. Lane B is homozygous for allele two of locus two. Allele one of locus three has comigrated with allele two of locus two. SKDH was scored as a monomeric single locus enzyme. Lane A is homozygous for allele two. The second band was thought to be a plastid form of the enzyme and not scored. Lane B shows a heterozygote for alleles three and five. The faint bottom most band was considered a plastid form of allele five and not scored. Lane C is homozygous for allele five. 13 A B C 14 Figure 3. Electrophoretic patterns for the remaining three of six enzyme systems used in this study. Labeling remains the same as on the previous page. IDH was scored as a dimeric enzyme encoded by three loci. Lane A is heterozygous for alleles one and three of locus one. ILane B is heterozygous for alleles four and five of locus one. Lane C is heterozygous for alleles one and two of locus two. Lane D is homozygous for allele one of locus three. Lane E is heterozygous for alleles two and three of locus three. 6PGDH was scored as a dimeric enzyme encoded by two loci. Lane A is homozygous for allele one of locus one. Lane B is heterozygous for alleles one and two of locus one. Lane C is heterozygous for alleles one and three of locus one. Lane A is homozygous for allele two of locus two. Lane D is heterozygous for alleles one and two of locus two. LAP was scored as a monomeric, single locus enzyme. Only the uppermost locus was scored for this study. Lane A is homozygous for allele two. Lane B is homozygous for allele three. Lane C is homozygous for allele one. Lane D is heterozygous for alleles two and four. _‘ «a- 6PGDH A C B D Figure 3 . 16 heterozygosity were based on Hardy-Weinberg expectations. For each locus, allele frequencies were inserted into a Hardy- Weinberg equation derived for the number of alleles at that locus. These values were then averaged over the number of loci within the population. The percentage of polymorphic loci was calculated using a 95% criterion, iJh a locus was considered polymorphic only if the most common allele occurred at a frequency of 0.95 or less in the population. The number of loci in a population which fit this criterion was divided by the total number of loci in the population to generate this percentage. Mean alleles per locus were calculated by summing all the alleles across loci in.a population.and.dividing by the number of loci in the population. F-statistics (fixation indices; Wright 1951, 1965b) were calculated according to Nei (1977c). This procedure measures the deviation of genotype frequencies from Hardy-Weinberg expected frequencies in a subdivided population. Deviation in heterozygosity from the level expected under Hardy-Weinberg equilibrium is partitioned into three components, Fm, Fm” and F”. Fls describes the inbreeding in individuals relative to the subpopulations to which they belong. F18 = (h,-ln)/h“ where ho is equal to the frequency of heterozygous individuals in an island population and h, is equal to the expected frequency of heterozygous individuals in an equivalent random mating island population. F“, the proportion of the deviation from equilibrium contained within subpopulations, can be 17 utilized as a measure of the deviation between populations. F31- : (hT - h,)/h-,, where hT = the expected frequency of heterozygous individuals in an equivalent random mating total population. Frr is a measure of the reduction in heterozygosity of an individual in relation to the whole population. F,-r can be viewed as the total heterozygote deviation from Hardy-Weinberg equilibrium. It is comprised of both the deviation due to nonrandom mating within island populations (F13) , and to the heterozygote deviation due to the subdivision of the population (F51); F“- = (hT - ho)/hT. Effective number of alleles per locus (AC?) was calculated according to Weir (1989), where A“, = 1/(1 - Hep). B the .9, genetic diversity per locus is equal to 1 - 2p}. Here pi equals the frequency of the 1" allele in each population. This analysis was done to "weight" the alleles present in the population. Because the calculation is based on the frequency of the allele in the population rather than mere presence, more "weight" is given to alleles with a higher frequency. The number of migrants exchanged per generation (Nm) , was also estimated where N equals the effective population size and m equals the proportion of migrants exchanged between populations per generation. Nm was calculated using the FST value described previously. According to Wright (1931) , FST = 1/(1 + 4Nm). 18 BE§QLI§ Twelve putative enzyme loci were consistently scoreable and subsequently employed in this study. Eleven of the 12 loci were polymorphic, however no alleles were found to be unique to any of the populations of adult trees or acorns (Table 1). All populations, both adult and acorn, had 63.6 percent polymorphic loci except for the STK adults which were polymorphic for 54.5 percent of their loci (Table 2). Mean number of alleles per locus ranged from 2.4 for OI adults to 3.2 for the PPC adults (Table 2). The effective number of alleles per locus ranged from 1.60 in the Bayfield Peninsula adult population to 1.79 in the Peninsula Perimeter adult trees (Table 2). Both adult and acorn populations exhibited an overall deficiency in heterozygotes. Direct mean heterozygosities were less than the expected values for all adult and acorn populations except among the OI adult trees (Table 2). On an individual locus basis, 6 of the 11 loci showed some deficiency of heterozygotes in the adult population (Table 3) . Of the remaining five, only two of .these differed substantially from equilibrium, 6-PGDH1 with a value of -0.057 and MDH2 with a value of -0.065 (Table 3). In the acorn populations five loci showed slight excesses in heterozygotes, however none deviated substantially from equilibrium (Table 4). The overall deficiency in heterozygotes was reflected by 19 m2. 53. - new. 03. SN. mum. «KN. - one. m E. E. - as. 2:. E. 1.8. m8. - as. a .5. 2K. - OS. SS. :6. as. MS. - so... a 50% ea. 8a. - as. as. as. we. as. - es. I. one 3.. . E. 0%. es. SN. one - as. m 3.. one - 8m. own. an. as. as. - 8m. N 3a. SN. - 5. .5. as. R... at. - 8.... a 35 ea. 8m. - an. 9;. 2m. 9:. Ga. - at. n So. we. - as. so. as. :o. 98. - cs. 1. so. as. - one. as. on. as. so. - as. M So. o8. - o8. Re. So. so. So. - cs. N So. So. - o8. Bo. 8e. 8o. «8. - 8s. a $2 . 2: - o3 - 8." - 2: . 2: I Zoo 82 =3. 33. =3 .82. =3 82.. =3. .8... =3. 22?. 253 so M5 M0 PE mm 53s.... as 933 seeds 3. .8 Best. 43.23% was. go Egon «I83 ES: as: as Se: as»: sfié .3 we?» tween 2 3 Egg asst ._ 03¢ 20 OOO. OOO. - NNO. NNO. OOO. ONO. ONO. - ONO. N OOO. OO._ - NNO. ONO. NNO. NS. ONO. - ONO. N OOO. OOO. - NNO. OOO. OOO. O2. NNO. - ONO. _ Nina OOO. ONO. - 2O. NNO. NNO. NON. OOO. - OO._ N NE. OOO. - NO. N2. ONO. NN_. OOO. - OOO. N NNO. ONO. - OOO. OOO. ONO. OOO. OOO. - OOO. I Nina NNN. NNN. - NON. RN. OON. OOO. NON. - NON. N OOO. OOO. - OOO. OOO. OS. OOO. ONO. - OOO. O OOO. SN. . SN. ONO. NO. :N. NNN. - ONN. N 3O. OOO. - NNO. OOO. OOO. OOO. OOO. - OOO. N NNN. OON. - NON. 2N. NON. NON. SN. - NOO. _ 7:9 OOO. NNO. - OOO. OOO. OOO. OOO. OOO. - OOO. N OOO. NNO. - OOO. NNO. OOO. NNO. ONO. - O3 N OOO. OOO. - OOO. OOO. ONO. NNO. OOO. - OOO. a 80% .82. :3. O8< OOO. .8< OOO. c8< =3 ._8< =3. 20:... 984 No New so 0.: mm $3.5 .N 03¢ 21 OOO. OOO. - OOO. OOO. ONO. OOO. NOO. - ONO. O NON. NNN. - OON. OON. OON. Oz. NNN. - OOO. O ONO. ONO. - NNO. OOO. ONO. ONO. NNO. - ONO. O ONN. OOO. - NON. NON. NNO. ONO. N2. - ONO. N ONO. ONO. - OOO. NON. ONO. OE. NNO. - OON. N OOO. OOO. - OOO. OOO. OS. OOO. ONO. - NOO. _ Eco—N NNO. OOO. - OOO. :O. ONO. :O. NO. - ONO. N ONO. OO._ - OO._ NNO. ONO. ONO. NNO. - ONO. N 352 OOO. E. - O3 OOO OS ONO. ONO. - OOO N OOO. ONN. - OOO. . OOO. OOO. :O. ONO. - OOO. _ NOE: ONO. OOO. - OS. OOO. ONO. :O. ONO. - ONO. N ONO. OOO - OOO. OOO. ONO. ONO. ONO. - ONO. N SE: .8... :3. O8... =3. 83. =3. O8< =3 O8< =3. OE? N83 8 EN mo PE mm #835530 A 03oF dome—bob asses... Boom—cow Nae—museum E 838ml E AONOO AOOOO g8 :NOO O.NO AOONO NON. NNN O: N O.NO N.NN 252 GNOO GOOO ANOO szoo< NNN. NON. NS O.N O.NO ANNO NON ONES AONOO AOOOO ANOO azim— NNN. ONN. OS ON O.NO 88 OOH No.50 AOOOO AOOOO ANOO 9242 ON. OON. OS N.N NON AOOO O.NO 20.5505 EOO ANNOO ANOO OOe NO. ON ON O.NO O.NO N.NO szoo<.o_